WO2024256530A1 - Improved method for generating kidney organoids - Google Patents
Improved method for generating kidney organoids Download PDFInfo
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- WO2024256530A1 WO2024256530A1 PCT/EP2024/066348 EP2024066348W WO2024256530A1 WO 2024256530 A1 WO2024256530 A1 WO 2024256530A1 EP 2024066348 W EP2024066348 W EP 2024066348W WO 2024256530 A1 WO2024256530 A1 WO 2024256530A1
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Classifications
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0686—Kidney cells
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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- C12N2513/00—3D culture
Definitions
- the invention describes a method for culturing kidney organoids using a prolonged exposure to FGF9, FGF20, or an activator of FGFR1 and/or FGFR3.
- the method results in improved kidney morphology in the resulting organoids, in particular the organoids have reduced cartilage induction.
- the invention further relates to organoids obtained by the method and uses thereof.
- Chronic kidney disease is characterized by a gradual loss of kidney function and is linked to a diminished glomerular filtration rate. Its prevalence varies from 3.3 to 17.3% and has risen in the last decades.
- kidney replacement is the most reliable therapeutic option for CKD.
- 9.7 million people need kidney replacement therapy, but due the shortage of donor organs and healthcare costs, only 2.6 million will receive it.
- the numbers of kidney transplants needed are expected to double within the next 10 years, increasing the pressure to find alternative solutions.
- Recent developments to generate organoids in vitro have opened the possibility for a regenerative medicine-based approach with the potential to provide a functional substitute to the failing kidney.
- hiPSCs human induced pluripotent stem cells
- Organoids present advantages compared to 2D-culture as they are isolated multicellular systems. This 3D-culture model is more physiologic and allows the study of interactions between different cell types.
- kidney organoids can recapitulate renal structures as well as the cellular complexity of human kidney, and have demonstrated the potential to restore glomerular filtration upon transplantation.
- renin production responsive to regulation by parathyroid hormone, in iPSC-derived kidney organoids under cyclic AMP stimulation, reflecting their physiological functionality in the endocrine system.
- kidney organoids might be a good model to study kidney diseases as well as a good therapeutic approach to treat kidney deficiency.
- kidney diseases commonly involve different cell types interactions, and the multicellular aspect of kidney organoids allows their use as in vitro kidney diseases models.
- kidney organoids are already used to model diseases, as ciliopathies or polycystic kidney disease, and to study the mechanistic of renal pathologies as well as potential treatments. More than modelling diseases, the physiologic potential of kidney organoids makes them good candidates for transplant therapeutic approaches.
- kidney organoids Despite their great therapeutic potential, kidney organoids also present several drawbacks, including a lack of maturation and vascularization, as well as the appearance of 10-20% off-target cell populations, such as neurons, myocytes and chondrocytes. Indeed, when performing single cell-RNA sequencing it was shown that neural markers are expressed. Inhibition of these markers via a pharmacological approach reduced the neuron population without affecting kidney structures, showing the possibility to improve kidney organoids using a small molecule approach. The appearance of cartilage in kidney organoids is observed after in vivo engraftment of the organoid under the renal capsule of mice. The off-target tissue appears 4 weeks after the graft and expands over time.
- the present invention aims to overcome these problems, among others.
- the invention in a first aspect, relates to a method for differentiating stem cells to kidney organoids, the method comprising a first differentiation stage and a second differentiation stage, wherein the first differentiation stage comprises culturing the stem cells under conditions to allow differentiation of the stem cells to a cell culture comprising cells in the intermediate mesoderm stage, wherein the second differentiation stage comprises transferring the cell culture comprising cells in the intermediate mesoderm stage to a 3D culture environment and culturing the cells under conditions that allow the formation of a kidney organoid, wherein the second differentiation step is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture, followed by culturing in growth factor free medium.
- the invention relates to an organoid obtained or obtainable by the method according to the first aspect of the invention.
- the invention relates to an organoid according the second aspect of the invention for use as a medicament.
- the invention relates to the in vitro or ex vivo use of an organoid according to claim 12 for one or more of:
- the invention in a fifth aspect relates to a method for differentiating intermediate mesoderm cells under conditions that allow for the formation of an immature kidney organoid expressing ECAD, LTL, and NPHS1 , wherein the cells are cultured in a 3D culture environment, and wherein the method is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture, followed by culturing in growth factor free medium.
- the invention relates to an immature kidney organoid obtained or obtainable by the method according to the fifth aspect of the invention.
- Figure 1 (Reference protocol (Takasato protocol according to Leiden adaptation - top part of the figure) Differentiation of iPSCs into kidney organoids is done in two phases. First differentiation in 2D using CHIR99021 during 4 days and FGF9/heparin cocktail for 3 days. Then a second phase of culture after aggregation of the cells at the air liquid interface to form the 3D structured organoids. During this second phase organoids are maintained in presence of FGF9/heparin cocktail for 5 days and then all growth factors are removed until day 7+25.
- the method of the invention differs in that FGF9 or FGF20 is provided longer in the air liquid interface culture stage.
- FGF9 is provided together with heparin from day 7 until day 7+5, followed by FGF9 only from day 7+5 until day 7+12.
- the protocol is otherwise identical to the reference protocol.
- Figure 2 (Reference protocol (Takasato protocol - top part of the figure) Differentiation of iPSCs into kidney organoids is done in two phases. First differentiation in 2D using CHIR99021 during 4 days and FGF9/heparin cocktail for 3 days. Then a second phase of culture after aggregation of the cells at the air liquid interface to form the 3D structured organoids. During this second phase organoids are maintained in presence of FGF9 for 5 days and then all growth factors are removed until day 7+25.
- FGF9 is provided longer in the air liquid interface culture stage.
- FGF9 is provided from day 7 until day 7+12.
- the protocol is otherwise identical to the reference protocol.
- Figure 3 (Reference protocol (Morizane protocol - top part of the figure) Differentiation of iPSCs into kidney organoids is done in two phases. First differentiation in 2D using CHIR99021 and Noggin during 4 days and activin for 3 days followed by FGF9 for 2 days. Then a second phase of culture after aggregation of the cells at low adherent wells to form the 3D structured organoids. During this second phase organoids are maintained in presence of FGF9 for 5 days (until day 14) and then all growth factors are removed until day 26. (exemplary protocol of the invention - bottom part of the figure) The method of the invention differs in that FGF9 is provided longer in the 3D culture stage. In the provided example, FGF9 is provided from day 9 until day 21. The protocol is otherwise identical to the reference protocol.
- FIG. 4 Off-target cell population develops in iPSC-derived kidney organoids and disrupts the renal structures’ development.
- A Schematic of kidney organoid culture. iPSCs were stimulated with CHIR99021 for 3 days and a FGF9/heparin cocktail for 4 days. After a 1 h pulse of CHIR99021 , cells were aggregated and cultured at the air-liquid interface for 5 days in the presence of the FGF9/heparin cocktail. Organoids were then cultured until day 7+25 without growth factors. A spherical kidney organoid shape was observed at day 7+18 (B) and 7+25 (C) by brightfield microscopy.
- D-G Renal structures assessed by immunofluorescence in cryosections show (D, F) glomeruli (NPHS1), proximal tubules (LTL), distal tubules (ECAD); (E, G) loops of Henle (SLC12A1) and a stromal population (MEIS1/2/3). Nuclei are stained with DAPI. Asterisks indicate an off-target cell population at day 7+25 (F, G). Scale bars represent 1000 pm (B-C) and 50 pm (D-G).
- Figure 5 The off-target population was identified as cartilage developing between days 7+18 and 7+25.
- A-D Alcian blue staining revealed the presence of cartilage at days 7+18 and 7+25 in the whole organoid (left images, scale bars represent 1000 pm) and cryosections (right images; scale bars represent 50 pm).
- J Western blotting of SOX9 and COL2A1 protein levels in kidney organoids from day 7+18 to 7+25.
- GAPDH levels are shown as loading controls.
- Figure 6 FGF9 treatment abrogates cartilage formation at day 7+25.
- A Schematic of the FGF9 treatment, which was extended after aggregation from day 7+5 to day 7+12.
- B-E Cartilage stained with Alcian blue in whole organoids (left images, scale bars represent 1000 pm) and on cryosections (right images, scale bars represent 50 pm) was strongly reduced with FGF9 treatment (bottom row).
- FIG. 7 FGF9 treatment reduces expression of cartilage markers at day 7+25.
- F Western blotting of SOX9 and COL2A1 protein levels in kidney organoids at day 7+25 showed decreased expression in FGF9-treated organoids compared to untreated controls. GADPH levels are shown as loading controls.
- FIG. 8 FGF9-treated organoids show renal and vessel-like structures.
- A-D Renal structures assessed by immunofluorescence in cryosections show (A, C) glomeruli (NPHS1), proximal tubules (LTL), distal tubules (ECAD); (B, D) loops of Henle (SLC12A1) and stromal population (MEIS 1/2/3). Nuclei are stained with DAPI. The off-target cell population (asterisks) observed in day 7+25 untreated organoids (top row) was not observed with FGF9 treatment (bottom row). Scale bars represent 50 pm.
- E-F Toluidine blue staining showed the appearance of vessel-like structures (#) in FGF9-treated organoids (F) that were absent in control organoids (E) at day 7+25. Scale bars represent 50 pm; inset represents 20 pm.
- G-H Immunostaining of cryosections showed an increase of the endothelial marker CD31 in FGF9-treated organoids (H) compared to controls (G). Scale bars represent 50 pm.
- FIG. 9 FGF9-treated organoids express lower levels of EMT markers at day 7+25.
- A Western immunoblotting showed lower levels of EMT markers vimentin and a-SMA in FGF9-treated (+) organoids compared to control (-) organoids at day 7+25. Notably, vimentin and a-SMA progressively increased from day 7+5, 7+10, 7+14, 7+18 and 7+25 in control organoids. GADPH levels are shown as loading controls.
- B-C Quantification of vimentin (B) and a-SMA (C) levels at day 7+5, 7+10, 7+14, 7+18 and 7+25 showed significant increases in untreated organoids, which were ameliorated by FGF9 treatment (7+25+FGF9). Protein levels are expressed as -fold change relative to day 7+5. *** p ⁇ 0.001 ; **** p ⁇ 0.0001 from 3 samples each.
- FIG. 10 FGF9 treatment does not negatively affect renal structures.
- A-D Gene expression of markers of renal structures, CUBN, NPHS1, SLC12A 1 and stromal population MEIS1 were assessed by qPCR and shown as -fold change compared to expression at day 7+18. *p ⁇ 0.05; **p ⁇ 0.01 ; from 3-4 samples.
- E FGF9 treatment (+) upregulates PECAM1 expression compared to control (-) organoids at day 7+25. PECAM1 expression assessed using qPCR shown as -fold change compared to expression in untreated organoids at day 7+18. *p ⁇ 0.05 from 3 samples.
- FIG. 11 FGF9 treatment delays the appearance of cartilage in iPSC-derived kidney organoids.
- A-D Cartilage (asterisks) stained with Alcian blue in whole organoids (left images, scale bars represent 1000 pm) and on cryosections (right images, scale bars represent 50 pm) was less abundant with FGF9 treatment but the appearance of small islands of cartilage were visible (bottom row).
- E-l FGF9 treatment (+) significantly decreased four of five markers of chondrogenesis in kidney organoids at day 7+32 compared to control (-) organoids. Gene expression assessed by qPCR and shown as -fold change compared to expression in untreated organoids at day 7+18. ***p ⁇ 0.001 from 4 samples.
- J Western blotting COL2A1 protein in control (-) kidney organoids showed increased expression over time which was abrogated with FGF9 treatment (+). GADPH levels shown as loading controls.
- FIG 12 FGF20 treatment abrogates cartilage formation at day 7+25. Cartilage stained with Alcian blue in whole organoids was strongly reduced with FGF20 treatment (middle and right panels).
- FIG. 13 FGF20-treated organoids show renal structures. Renal structures assessed by immunofluorescence in cryosections show glomeruli (NPHS1), proximal tubules (LTL), and distal tubules (ECAD). Nuclei are stained with DAPI. The off-target cell population observed in day 7+25 untreated organoids (left panel) was not observed with FGF20 treatment (middle and right panels). Scale bars represent 50 pm.
- a portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.).
- copyright protection such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.
- the copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.
- the term "at least" a particular value means that particular value or more.
- “at least 2" is understood to be the same as “2 or more” i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... , etc.
- the term “at most " a particular value means that particular value or less.
- “at most 5” is understood to be the same as “5 or less” i.e., 5, 4, 3, ... .-10, -11 , etc.
- Antagonist refers to a compound or agent having the ability to initiate or enhance a biological function of a target protein or polypeptide, such as increasing the activity or expression of the target protein or polypeptide.
- agonist is defined in the context of the biological role of the target protein or polypeptide. While some agonists herein specifically interact with (e.g., bind to) the target, compounds and/or agents that initiate or enhance a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target polypeptide is a member are also specifically included within this definition.
- Antagonist and “inhibitor” are used interchangeably, and they refer to a compound or agent having the ability to reduce or inhibit a biological function of a target protein or polypeptide, such as by reducing or inhibiting the activity or expression of the target protein or polypeptide. Accordingly, the terms “antagonist” and “inhibitor” are defined in the context of the biological role of the target protein or polypeptide. An inhibitor need not completely abrogate the biological function of a target protein or polypeptide, and in some embodiments reduces the activity by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
- While some antagonists herein specifically interact with (e.g., bind to) the target, compounds that inhibit a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target protein or polypeptide are also specifically included within this definition.
- Non-limiting examples of biological activity inhibited by an antagonist include those associated with the development, growth, or spread of a tumor, or an undesired immune response as manifested in autoimmune disease.
- Cell line this term refers to continuously growing or immortalized cells. Sometimes also referred to as “immortalized cell line”, a cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division.
- a “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with; differentiation is the process of progression.
- Human pluripotent stem cells can differentiate into lineage-restricted progenitor cells (cells that, like a stem cell, have a tendency to differentiate into a specific type of cell, but are already more differentiated than a stem cell and are pushed to eventually differentiate into its end-stage cell; e.g.
- endoderm mesoderm and ectoderm
- further restricted cells e.g., cardiomyocyte progenitors, neuronal cell progenitors
- terminally differentiated cells e.g., cardiomyocytes or neurons
- Differentiation is controlled by the interaction of a cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.
- “differentiation” is the biological process whereby an unspecialized human pluripotent stem cell (population) acquires the features of a specialized cell such as a cardiomyocyte under controlled conditions in in vitro culture.
- Embryonic stem cells abbreviated as ‘ES cells’ or ESC (or if of human origin ‘hES cells’ or ‘hESCs’) refers to stem cells that are derived from the inner cell mass of a blastocyst. The skilled person understands how to obtain such embryonic stem cells, for example as described by Chung (Chung et al (2008) Stem Cell Lines, Vol 2(2): 113- 117), which employs a technique that does not cause the destruction of the donor embryo(s). Various ESC lines are listed in the NIH Human Embryonic Stem Cell Registry.
- In vivo This term refers to an event that takes place in a subject's body
- in vitro This term refers to an event that takes places outside of a subject's body.
- an in vitro assay encompasses any assay conducted outside of a subject.
- In vitro assays encompass cell-based assays in which cells, alive or dead, are employed.
- In vitro assays also encompass a cell-free assay in which no intact cells are employed.
- Induced pluripotent stem cell or “iPSC”: These terms refer to pluripotent stem cells that are derived from a cell that is not a pluripotent stem cell (i.e. , from a cell this is differentiated relative to a pluripotent stem cell). Induced pluripotent stem cell can be derived from multiple different cell types, including terminally differentiated cells. Induced pluripotent stem cell generally have an ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei.
- induced pluripotent stem cell may express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181 , TDGF 1 , Dnmt3b, FoxD3, GDF3, Cyp26a1 , TERT, and zfp42.
- key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181 , TDGF 1 , Dnmt3b, FoxD3, GDF3, Cyp26a1 , TERT, and zfp42.
- Examples of methods of generating and characterizing induced pluripotent stem cells may be found in, for example, U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191
- somatic cells may be provided with reprogramming factors (e.g. Oct4, SOX2. KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells( see, for example, Takahashi et. al, Cell. 2007 Nov. 30; 131 (5):861-72; Takahashi et. al, Nat Protoc. 2007; 2(12):3081-9; Yu et. al, Science. 2007 Dec. 21:318(5858): 1917-20. Epub 2007 Nov. 20).
- reprogramming factors e.g. Oct4, SOX2.
- Markers or “lineage-specific markers”: these terms refer to a characteristic specifically associated with the phenotype of cells of a lineage and can be used to assess the differentiation of cells.
- the terms may refer to nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. The detectable level of the marker is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
- Media This term refers to an aqueous solution, including buffers, suitable for maintaining human or animal cells for a sufficient period.
- a media is suitable if it allows the treatment of cells for a period required to obtain the effect intended by the treatment.
- the term “media” also, and preferably, includes growth media that are suitable for the in vitro cell culture of human or animal cells.
- a “defined media” refers to a (growth) media suitable for the in vitro cell culture of human or animal cells and in which all of the chemical components are known. Such defined media does not or essentially not comprise any ill-defined source of nutrients and/or other ill-defined factors.
- the defined media used may still contain defined amounts of products such as (purified) albumin, growth factors, and hormones, but is essential free of serum (i.e. less than 1 % w/w, preferably less than 0.5% w/w. even more preferably less than 0.1 % w/w, even more preferably less than 0.05% w/w of the medium ready for use, most preferably the medium is free of serum (i.e. 0% w/w serum; albeit it might contain defined amount of specified compounds like (recombinant) albumin.
- serum has many limitations. It contains high levels of numerous and unknown proteins and compounds which interfere dramatically with the small quantities of the desired proteins produced by the cells. The presence of serum may also affect in vitro testing results with the cells obtained since some compounds may bind up to 99% to serum proteins. Another limitation is the serum batch-to-batch inconsistencies, resulting in serious regulatory concern about various serum protein contaminations in the product.
- “Pluripotency” This term is generally understood by the skilled person and refers to an attribute of a (stem) cell that has the potential to differentiate into all cells constituting one or more tissues or organs, for example, any of the three germ layers: endoderm (e.g. interior stomach lining, gastrointestinal tract, the lungs), mesoderm (e.g. heart, muscle, bone, blood, urogenital tract), or ectoderm (e.g. epidermal tissues and nervous system).
- endoderm e.g. interior stomach lining, gastrointestinal tract, the lungs
- mesoderm e.g. heart, muscle, bone, blood, urogenital tract
- ectoderm e.g. epidermal tissues and nervous system
- PSC Preferred stem cell
- Pluripotent stem cell This is a stem cell capable of producing all cell types of the organism and can produce cells of the germ layers, e.g. endoderm, mesoderm, and ectoderm, of a mammal and encompasses at least pluripotent embryonic stem cells and induced pluripotent stem cells.
- Pluripotent stem cells can be obtained in different ways.
- Pluripotent embryonic stem cells may, for example, be obtained from the inner cell mass of an embryo.
- Induced pluripotent stem cells (iPSCs) may be derived from somatic cells.
- Pluripotent stem cells may also be in the form of an established cell line.
- Times used for indicating organoid age when used herein the age of cells or organoids is indicated in hours or days. When done so, the age refers to the time since initiating the first differentiation stage. For example, “3 day” or “day 3” refers to cells cultured for three days in the first differentiation stage (in a 2D culture environment) and “day 10” or “10 day” refers to organoids where the cumulative time culturing in the first and the second differentiation stage is 10 days (e.g. 7 days in the first differentiation stage and 3 days in the second differentiation stage).
- age can be annotated as “day A+B” or “A+B day”, here A indicates the number of days cultured in the first differentiation stage (in 2D culture) and B indicates the number of days cultured in the second differentiation stage.
- day 7+12 indicates organoids which are grown for 7 days in the first differentiation stage (2D culture) and 12 days in the second differentiation stage (3D culture environment), and thus are cultured for an accumulative of 19 days in the first and second differentiation stage together.
- the term “after onset”, when for example used in the context of “from onset of the 3D culture” intends to indicate immediately after the start thereof (e.g. the 3D culture), but allows also to include shortly after onset of the intended event (e.g. 3D culture), such as 1 , 2, 3, 4, 6, 12, 24, 36 or 48 hours after initiating the referred event.
- the phrase providing FGF9, FGF20, or an activator of FGFR1 and/or FGFR3 is provided for 8 to 16 days after onset of the 3D culture implies that FGF9 or FGF20 is provided immediately after the 3D culture starts or within 1 , 2, 3, 4, 6, 12, 24, 36 or 48 hours of starting the 3D culture.
- the time period e.g. 8 to 16 days
- is calculated accordingly e.g. starting immediately after or within 1 , 2, 3, 4, 6, 12, 24, 36 or 48 hours of starting the 3D culture, whichever applies).
- any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein.
- Embodiments discussed in the context of methods, use and/or compositions of the invention may be employed with respect to any other method, use or composition described herein.
- an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.'
- kidney disease affects 11-13% of the global population. Worldwide, 9.7 million people need kidney replacement therapy but only 2.6 million will receive it.
- One regenerative medicine alternative comprises the use of iPSC-derived kidney organoids as a therapeutic engraftment to the dysfunctional kidney.
- several drawbacks must be overcome before clinical translation, among which is the presence of non-renal cell populations such as cartilage in the organoids.
- the inventors herein describe a modification of the culture protocol and maintained kidney organoids in medium containing FGF9 or FGF20 for one additional week compared to the control protocol (Takasato). In comparison to control, the FGF9 (or FGF20) treated kidney organoids had no cartilage at day 7+25 and diminished chondrocyte marker expression.
- the renal structures assessed by immunofluorescence were unaffected by the FGF9 or FGF20 treatment. This reduction of cartilage produces a higher quality kidney organoid that can be maintained longer in culture to improve their maturation for further in vivo work.
- the present invention relates to decreasing formation of cartilage in kidney organoids.
- Cartilage formation involves the condensation of mesenchyme tissue, which differentiates into chondrocytes and produces the extracellular matrix protein collagen 2 (COL2A1).
- the pathways leading to chondrocyte differentiation mainly involve the SOX protein family, particularly SOX9.
- EMT epithelial to mesenchymal transition
- the protocols have been schematically described in Figures 1 , 2 and 3.
- the protocols have in common that initially stem cells (e.g. induced pluripotent stem cells (iPSCs)) are cultured as a monolayer to allow differentiation, and then transferred to an environment that allows the formation of organoids (e.g. air-liquid interface or low adherence wells, also referred herein as the second differentiation phase or 3D culture stage), while being provided FGF9 for defined time period.
- stem cells e.g. induced pluripotent stem cells (iPSCs)
- iPSCs induced pluripotent stem cells
- the inventors added a step in the differentiation protocol and incubated 3D kidney organoids with FGF9 or FGF20 from day 5 until day 12 (counting from onset of the 3D culture) as described in the examples below.
- the inventors demonstrate a clear reduction of the appearance of cartilage at the latter timepoint correlated with a reduction of EMT and cartilage markers.
- the inventors hereby disclose an improved protocol to generate kidney organoids at the air-liquid interface, using growth factor FGF9 or FGF20.
- the invention relates to method for differentiating stem cells to kidney organoids, the method comprising a first differentiation stage and a second differentiation stage, wherein the first differentiation stage comprises culturing the stem cells under conditions to allow differentiation of the stem cells to a cell culture comprising cells in the intermediate mesoderm stage, wherein the second differentiation stage comprises transferring the cell culture comprising cells in the intermediate mesoderm stage to a 3D culture environment and culturing the cells under conditions that allow the formation of a kidney organoid, wherein the second differentiation step is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture, followed by culturing in growth factor free medium.
- FGF9 also plays an important role in the formation of renal structures, therefore completely removing FGF9 from the culture medium is not an option.
- the stepwise differentiation of pluripotent stem cells to kidney begins with the induction of the primitive streak which is the -progenitor population for both endoderm and mesoderm. While the anterior primitive streak gives rise to the endoderm, the posterior primitive streak has potential to develop into the mesoderm, including the axial, paraxial, intermediate and lateral plate mesoderm.
- the intermediate mesoderm differentiates to the ureteric epithelium and the metanephric mesenchyme, which are two key kidney progenitor populations subsequently undergoing a reciprocal interaction to form the kidney (Takasato et al. Nat Cell Biol 16, 118-126 (2014)).
- kidney organoids it is attempted to recreated these developmental steps in vitro.
- the most used protocols to develop kidney organoids at present are the Takasato protocol (and the Leiden adaptation thereof) and the Morizane protocol. Schematic overviews of these protocols are provided in Figures 1-3. All the protocols have in common a 2D culture phase where stem cells are differentiated to intermediate mesoderm followed by a 3D culture phase where the cells are transferred to a 3D culturing system to allow the formation of organoids.
- the 3D culture phase in all protocols is characterized in that FGF9 or FGF20 is provided for about 5 days starting from the onset of the 3D culture phase.
- days from onset of the 3D culture phase preferably means starting from the 3D culture phase, meaning that when referring to the number of days FGF9, FGF20 (or and activator of FGFR1 and/or FGFR3) is provided is also the first day of the 3D culture phase, however it is possible to start providing FGF9, FGF20 (or and activator of FGFR1 and/or FGFR3) close to the start of the 3D culture phase (e.g. after 12, 24 or 48 hours) and start the numbering of days FGF9, FGF20 (or and activator of FGFR1 and/or FGFR3) is provided accordingly.
- first differentiation stage refers to culturing stem cells in a 2D (monolayer) culture under conditions to allow differentiation of the stem cells to a cell culture comprising cells in the intermediate mesoderm stage.
- the first differentiation stage may comprise several distinct steps. Generally as a first step the stem cells are cultured in the presence of a GSK3 inhibitor or a WNT activator.
- a GSK3 inhibitor or a WNT activator Such compounds are known to the skilled person, an exemplary compound that may be used is for example CHIR99021 which is a GSK3 inhibitor.
- the ureteric epithelium is characterized by expression of the markers PAX2, GATA3 and CDH1.
- the metanephric mesenchyme is characterized by the expression of PAX2, Wnt4 and BMP7.
- the cells in the intermediate mesoderm stage comprise at least one of: ureteric epithelium, metanephric mesenchyme, progenitors of renal interstitium or endothelium.
- the first differentiation stage comprises a step (A) wherein the stem cells are cultured under conditions that allow induction of both the ureteric epithelium and the metanephric mesenchyme.
- the first differentiation stage comprises a step (A) wherein the stem cells are cultured under conditions that allow induction of both cells expressing PAX2, GATA3 and CDH1 and cells expressing PAX2, Wnt4 and BMP7.
- the first differentiation stage comprises a step (A) which step comprises culturing the stem cells in the presence of a GSK3 inhibitor or a WNT activator.
- the GSK3 inhibitor is CHIR99021.
- step (A) is performed for 2 to 6 days, preferably 3 to 5 days more preferably about 4 days.
- the metanephric mesenchyme is further differentiated to nephron progenitor cells, while further differentiating the ureteric epithelium.
- This step is performed by culturing the partially differentiated cells at least in the presence of FGF9, FGF20 or an activator of FGFR1 and/or FGFR3.
- the nephron progenitor cells are characterized by the expression of the markers SIX2, HOXD11 , WT1 and PAX2.
- the ureteric epithelium is at this stage also characterized by expression of the markers PAX2, GATA3 and CDH1.
- the first differentiation stage comprises a step (B) which step comprises culturing the partially differentiated cells under conditions that allow differentiation of the metanephric mesenchyme into nephron progenitor cells while further differentiating the ureteric epithelium.
- the first differentiation stage comprises a step (B) wherein the stem cells are cultured under conditions that allow induction of both cells expressing PAX2, GATA3 and CDH1 and cells expressing SIX2, HOXD11 , WT1 and PAX2.
- the first differentiation stage comprises a step (B) which step comprises culturing the stem cells in the presence of FGF9, FGF20 or an activator of FGFR1 and/or FGFR3.
- step (B) is performed for 1 to 4 days, preferably 2 or 3 days.
- the cells may be cultured in the presence of additional factors besides FGF or an activator of FGFR1 and/or FGFR3.
- additional factors besides FGF or an activator of FGFR1 and/or FGFR3.
- heparin may also be included in the culture medium.
- Step (B) may directly follow step (A), or there may be an intermediate step between step (A) and step (B).
- a step (A1) may be included in the method wherein the cells are cultured in the presence of activin.
- step (A1) is performed for 1 to 5 days, preferably 2 to 4 days, more preferably about 3 days.
- the term second differentiation stage refers to culturing differentiated cells in a 3D culture environment under conditions to allow formation of an organoid and further differentiation of the cells to a kidney organoid.
- the second differentiation stage may comprise several distinct steps.
- a first step (C) the cells are cultured in the presence of FGF9, FGF20 or an activator of FGFR1 and/or FGFR3.
- immature organoids are formed which demonstrate renal structures and start to express renal factors such as LTL, ECAD and NPHS1.
- the second differentiation stage comprises a step (C) wherein the cells are cultured under conditions that allow the formation of immature organoids.
- the second differentiation stage comprises a step (C) wherein the cells are cultured under conditions that allow induction of cells expressing LTL, ECAD and NPHS1.
- the second differentiation stage comprises a step (C) which step comprises culturing the cells in the presence of FGF9, FGF20 or an activator of FGFR1 and/or FGFR3. This is the characterizing step of the protocol and differs from existing protocols in that step (C) is performed for 8 to 16 days, preferably 9-15 days, preferably 10-14 days, more preferably 11-13 days.
- Existing protocols like the Morizane protocol or the Takasato protocol teach that FGF9 is provided in the 3D culture environment for 5 days only.
- the cells may further be cultured in presence of additional factors besides FGF9, FGF20 or an activator of FGFR1 and/or FGFR3.
- a pulse treatment or short treatment of a GSK3 inhibitor or a WNT activator such as for example CHIR99021 may be provided.
- the pulse treatment may for example be provided for 15 minutes to 4 hours, preferably 30 minutes to 2 hours, more preferably about 1 hour, or for a short period such as 12 hours to 4 days, preferably 1 to 3 days, more preferably about 3 days.
- Exemplary concentrations when using CHIR99021 are between 1 to 10 micromolar, where the concentration is preferably lower when using short treatment compared to when using pulse treatment, e.g. 5 micromolar for pulse treatment and 3 micromolar for short treatment.
- the culture medium may further be supplemented with heparin for the first days.
- heparin may be supplemented for the first 3 to 7 days, preferably the first 4 to 6 days more preferably for about the first 5 days after onset of the 3D culture.
- Heparin may be supplemented in a concentration of 1 microgram per millilitre culture medium.
- 3D culture environment should be interpreted as any culture method that allows the formation of organoids from the harvested partially differentiated cells obtained in step (B), provided the appropriate culture conditions are provided.
- the Takasato protocol describes a trans-well membrane system to culture cells on a liquid air interface
- the Morizane protocol describes the use of ultra-low attachment plates.
- Other culture methods that allow the formation of organoids are however known to the skilled person and should be construed as included under the term 3D culture environment.
- Non-limiting examples are hydrogels, scaffolds or suspension culture.
- the 3D culture environment is selected from culturing on a trans-well membrane, culturing in a low attachment well, culturing in a hydrogel, culturing on an air-liquid interface culturing, culturing on a scaffold, or suspension culture.
- the immature organoids are further differentiated to kidney organoids.
- This step is performed by culturing the immature organoids in the absence of any growth factors.
- the kidney organoids are expressed by an increased expression of LTL, ECAD and NPHS1 compared to the immature organoids and further express SLC12A1 .
- the second differentiation stage comprises a step (D) which step comprises culturing the immature organoids under conditions that allow differentiation to kidney organoids.
- the second differentiation stage comprises a step (D) wherein the immature organoids are cultured under conditions that allow induction of the organoids to express LTL, ECAD, NPHS1 and SLC12A1.
- the second differentiation stage comprises a step (D) which step comprises culturing the stem cells in the absence of growth factors.
- step (D) is performed for at least 5 days, preferably at least 6, 7, 8, 9, 10, 11 or at least 12 days.
- Step (D) preferably directly follows step (C), although there may be an optional intermediate step between step (C) and step (D).
- the second differentiation step is performed for at least 20 days, preferably at least 21 , 22, 23, 24, or at least 25 days.
- combined steps (C) and (D) are performed for at least 20 days, preferably at least 21 , 22, 23, 24, or at least 25 days.
- the second differentiation stage comprises transferring the cell culture comprising cells in the intermediate mesoderm stage to a 3D culture environment and culturing the cells under conditions that allow the formation of a kidney organoid, wherein the second differentiation stage is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture, followed by culturing in growth factor free medium, wherein the second differentiation stage is performed for at least 20 days, preferably at least 21 , 22, 23, 24, or at least 25 days.
- step (D) is performed for at least 4, for example 4, 5, 6, 7, 8 , 9, 10, 11 , 12, or 13 days or even more.
- the second differentiation step comprises:
- C culturing the cells under conditions that allow the formation of an immature kidney organoid in the presence of FGF9, FGF20 or the FGFR1 and/or FGFR3 activator, wherein the immature kidney organoid expresses ECAD, LTL, and NPHS1 , and
- kidney organoid (D) culturing the immature kidney organoid in the absence of growth factors to obtain a kidney organoid, preferably wherein the kidney organoid differs from the immature kidney organoid in the increased expression of ECAD, LTL, NPHS1 , and SLC12A1.
- step (C) further comprises culturing the cell in the presence of Heparin for the first 3-7, preferably 4-6 days after onset of the 3D culture.
- the differentiated cells are harvested and transferred to the 3D culture environment to start step (C) of the second differentiation phase (the 3D culture phase).
- Suitable method for harvesting the differentiated cells are known to the skilled person, for example non limiting examples are dissociating cells with Trypsin-EDTA or Accutase.
- FGF9, FGF20 or an activator of FGFR1 and/or FGFR3 is intended to refer to FGF9, FGF20, a functional homolog of FGF9 or FGF20 or an alternative of FGF9 or FGF20 that can be used to replace FGF9 or FGF20 in the presented method, wherein the alternative activates FGFR1 and/or FGFR3.
- FGF9 is a member of the fibroblast growth factor (FGF) family. FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. FGF9 was isolated as a secreted factor that exhibits a growth-stimulating effect on cultured glial cells.
- the peptide sequence of FGF9 is represented by SEQ ID NO: 1 below.
- the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 1 , is provided for 8 to 16 days after onset of the 3D culture.
- the protein is provided in the culture medium, thus allowing contact of the cells with the protein.
- functional homologs of FGF9 (the protein defined by SEQ ID NO: 1) can be used in the context of the invention. Therefore in an embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 1 or a functional homolog thereof, is provided for 8 to 16 days after onset of the 3D culture, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 1.
- the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 1 which is able to activate FGFR1 and/or FGFR3
- FGF20 is also member of the fibroblast growth factor (FGF) family.
- the peptide sequence of FGF20 is represented by SEQ ID NO: 2 below.
- the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 2, is provided for 8 to 16 days after onset of the 3D culture.
- the protein is provided in the culture medium, thus allowing contact of the cells with the protein.
- the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 2 or a functional homolog thereof, is provided for 8 to 16 days after onset of the 3D culture, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 2.
- the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 2 which is able to activate FGFR1 and/or FGFR3
- Fibroblast growth factor receptor 1 also known as basic fibroblast growth factor receptor 1 , fms-related tyrosine kinase-2 I Pfeiffer syndrome, and CD331 , is a receptor tyrosine kinase whose ligands are specific members of the fibroblast growth factor family.
- Fibroblast growth factor receptor 3 is a protein that in humans is encoded by the FGFR3 gene.
- FGFR3 has also been designated as CD333 (cluster of differentiation 333). The gene, which is located on chromosome 4, location q16.3, is expressed in tissues such as the cartilage, brain, intestine, and kidneys.
- FGFR1 and FGFR3 are cell surface membrane receptors that possess tyrosine kinase activity.
- a full-length representative of these four receptors consists of an extracellular region composed of three immunoglobulin-like domains which bind their proper ligands, the fibroblast growth factors (FGFs), a single hydrophobic stretch which passes through the cell's surface membrane, and a cytoplasmic tyrosine kinase domain.
- FGFs fibroblast growth factors
- these receptors form dimers with any one of the four other FGFRs and then cross-phosphorylate key tyrosine residues on their dimer partners.
- FGFR1 receptor is in humans encoded by the gene annotated as
- the FGFR3 receptor is in humans encoded by the gene annotated as
- ENSG00000068078 (Ensembl) or HGNC:3690.
- the second differentiation step is characterized in that an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture.
- the activator of FGFR1 and/or FGFR3 may be a small molecule or a protein.
- Activation of FGFR1 and/or FGFR3 by a compound or protein can easily be verified by the skilled person by for example phosphorylation specific antibodies targeting the FGFR1 or FGFR3 receptor.
- Non limiting examples of activators of the FGFR1 and/or FGFR3 receptors are FGF20, FGF2, or Smoothened agonist (SAG). Therefore in an embodiment of the method of the invention the second differentiation step is characterized in that FGF20, FGF2, or Smoothened agonist (SAG), is provided for 8 to 16 days after onset of the 3D culture.
- FGF20 is a member of the fibroblast growth factor (FGF) family and capable of activating both FGFR1 and FGFR3.
- the peptide sequence of FGF20 is represented by SEQ ID NO: 2 below.
- the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 2, is provided for 8 to 16 days after onset of the 3D culture.
- the protein is provided in the culture medium, thus allowing contact of the cells with the protein.
- the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 2 or a functional homolog thereof, is provided for 8 to 16 days after onset of the 3D culture, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 2.
- the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 2 which able to activate FGFR1 and/or FGFR3.
- FGF2 is a member of the fibroblast growth factor (FGF) family and capable of activating both FGFR1.
- the peptide sequence of FGF2 is represented by SEQ ID NO: 3 below.
- the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 3, is provided for 8 to 16 days after onset of the 3D culture.
- the protein is provided in the culture medium, thus allowing contact of the cells with the protein.
- the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 3 or a functional homolog thereof, is provided for 8 to 16 days after onset of the 3D culture, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 3.
- the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 3 which able to activate FGFR1 and/or FGFR3.
- SAG Smoothened agonist
- SAG was one of the first small-molecule agonists developed for the protein Smoothened, a key part of the hedgehog signalling pathway, which is involved in brain development as well as having a number of other functions in the body. It is shown that SAG increases expression of FGF9 and thus indirectly stimulates FGFR1 and FGFR3 by increasing expression of FGF9.
- SAG is represented by formula 1 below and also known as 3-Chloro-/V-[trans-4-(methylamino)cyclohexyl]-/ ⁇ /-[[3-(4- pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide with CAS registry number 912545-86-9
- the second differentiation step is characterized in that Smoothened Agonist (SAG) is provided for 8 to 16 days after onset of the 3D culture.
- SAG Smoothened Agonist
- SAG can be provided in addition to FGF9 or an activator of FGFR1 and/or FGFR3. Without wishing to be bound be theory, it is theorized that SAG increases expression of the FGF receptors and therefore renders the cells more susceptible to activation with FGF9 or other activators of FGFR1 and/or FGFR3.
- SAG is preferably provided for 2 to 4 days, more preferably from the start of the second differentiation stage.
- the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 1 or a functional homolog thereof, a protein having or comprising a peptide sequence as defined in SEQ I D NO 2 or a functional homolog thereof, a protein having or comprising a peptide sequence as defined in SEQ ID NO 3 or a functional homolog thereof, or a small molecular compound such as Smoothened Agonist is provided for 8 to 16 days after onset of the 3D culture.
- the main advantage of the protocol of the invention is that, when compared to protocols known in the art, the protocols described herein result in reduced expression of EMT and cartilage markers.
- reduced expression of EMT and cartilage markers should be interpreted as either a reduced number of cells in the organoid show expression of EMT and cartilage markers, or the expression levels of the of EMT and cartilage markers is significantly lower in kidney organoids grown according to the invention when compared to kidney organoids grown according to the reference protocols (e.g. the Takasato protocol or the Morizano protocol).
- the kidney organoids grown according to the method of the invention have a lower expression of EMT and/or cartilage markers compared to kidney organoids cultured under conditions wherein the second differentiation step comprises providing FGF9, FGF20 or the FGFR1 and/or FGFR3 activator for fewer than 8 days after onset of the 3D culture.
- Typical EMT and cartilage markers are COL2A1 , SOX9, Aggrecan, COL1A1 COL10, Vimentin and/or ACTA2, although other markers are known in the field.
- the EMT and/or cartilage markers are selected from COL2A1 , SOX9, Aggrecan, COL1A1 , COL10, Vimentin and/or ACTA2.
- markers in an organoid e.g. immunohistochemistry staining of markers can be used to determine number of cells within the organoid expressing a certain marker, while qPCR may be used to determine overall expression levels in the organoid or isolated cells to compare expression levels of the markers.
- the EMT or cartilage markers are preferably determined on day 20 or later after onset of the 3D culture stage, e.g. day 21 , 22, 23, 24, 25 or later after onset of the 3D culture stage.
- the invention relates to an organoid obtained or obtainable by the method according to the first aspect of the invention.
- Organoids produced according to the method of the invention differ from organoids grown according to the reference protocols in that they display reduced expression of EMT and/or cartilage markers. Therefore in an embodiment the organoids according to the second aspect of the invention has reduced expression of EMT and/or cartilage markers.
- the organoid has reduced expression of a EMT and/or cartilage marker selected from COL2A1 , SOX9, Aggrecan, COL1A1 , COL10, Vimentin and/or ACTA2.
- the invention relates to an organoid according to the second aspect of the invention for use as a medicament.
- the invention relates to a method of treatment, the method comprising providing an organoid as defined herein to a subject in need thereof.
- the invention relates to an organoid according to the second aspect of the invention for use in the treatment of a kidney disease.
- the kidney disease is chronic kidney disease.
- the invention relates to a method of treating a kidney disease in a subject, the method comprising providing an organoid as defined herein to the subject.
- the invention relates to the in vitro or ex vivo use of an organoid according to the second aspect of the invention for one or more of:
- the invention in a fifth aspect relates to a method for differentiating intermediate mesoderm cells under conditions that allow for the formation of an immature kidney organoid expressing ECAD, LTL, and NPHS1 , wherein the cells are cultured in a 3D culture environment, and wherein the method is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture.
- the invention relates to an immature kidney organoid obtained or obtainable by the method according to the fifth aspect of the invention. Sequences described in the application:
- SEQ ID NO: 1 maplgevgny fgvqdavpfg nvpvlpvdsp vllsdhlgqs eagglprgpa vtdldhlkgi Irrrqlycrt gfhleifpng tiqgtrkdhs rfgilefisi avglvsirgv dsglylgmne kgelygsekl tqecvfreqf eenwyntyss nlykhvdtgr ryyvalnkdg tpregtrtkr hqkfthflpr pvdpdkvpel ykdilsqs
- SEQ ID NO: 3 (FGF2) mvgvgggdve dvtprpggcq isgrgargcn gipgaaawea alprrrprrh psvnprsraa gsprtrgrrt eerpsgsrlg drgrgralpg grlggrgrgr apervggrgr grgtaapraa hindergsrpgp agtmaagsit tlpalpedgg sgafppghfk dpkrlyckng gfflrihpdg rvdgvreksd phiklqlqae ergvvsikgv canrylamke dgrllaskcv tdecffferl esnnyntyrs rkytswyval krtgqyklgs ktgp
- Kidney organoids were generated at the air liquid interface using previously described protocol (Takasato, M. et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564-568 (2015)). Briefly, 10 5 cells were seeded in a 6-well plate and cultured in Stemdiff APEL2 medium (StemCell Technologies), supplemented with 1 % Protein-Free Hybridoma Medium II (ThermoFischer Scientific) and 1% antibiotic-antimycotic, and treated with CHIR99021 (8pM GSK-3 inhibitor, R&D system) and FGF9 (fibroblast growth factor 9, 200ng/ml, R&D system)/heparin (1 pg/ml, Sigma-Aldrich) cocktail.
- Stemdiff APEL2 medium Stemdiff APEL2 medium
- CHIR99021 8pM GSK-3 inhibitor, R&D system
- FGF9 fibroblast growth factor 9, 200ng/ml, R&D system
- CHIR99021 4 days resulted in the induction of both the ureteric epithelium and the metanephric mesenchyme in monolayer culture and is followed by 3 days of FGF9/heparin. Maximal nephron number per organoid required a pulse of CHIR99021 for one hour. The continued presence of FGF9 after this CHIR99021 pulse was essential for nephrogenesis.
- hiPSCs human induced pluripotent stem cells
- the hiPSC line LUMC0072iCTRL01 was provided by the hiPSC core facility at the U.M.C. Leiden (the Netherlands). Cells are cultured in 6-well plates coated with vitronectin (Fisher Scientific) using E8 medium (Thermo Fisher Scientific) supplemented with 1% penicillin-streptomycin (Gibco). The medium was refreshed daily and the cells were passaged twice weekly using TrypLE dissociation reagent (Invitrogen). After passaging, cells were seeded in the presence of RevitaCell (Fisher Scientific) overnight. Pluripotency and karyotype of the iPSCs were verified. iPSC-derived kidney organoid generation and treatment
- Kidney organoids were generated at the air-liquid interface using a the Takasato protocol. Briefly, 100,000 cells were seeded in a vitronectin-coated 6-well plate. After 24 h, regular E8 medium was replaced by STEMdiff APEL2 medium (STEMCELL Technologies) supplemented with 1% protein-free hybridoma medium II (PFHMII) (Fisher Scientific), 1% antibiotic-antimycotic (AA), and 8 pM CHIR99021 for 4 days to induce differentiation. This step was followed by an incubation with 200 ng/mL fibroblast growth factor 9 (FGF9, R&D Systems), and 1 pg/ml heparin (Sigma-Aldrich) for 3 days.
- PFHMII protein-free hybridoma medium II
- AA antibiotic-antimycotic
- FGF9 fibroblast growth factor 9
- Sigma-Aldrich 1 pg/ml heparin
- kidney organoids were fixed on days 7+18, 7+25 or 7+32 on the cell culture inserts in 70% ethanol overnight (ON) at 4°C. They were then incubated at room temperature (RT) for 1 h in 95% ethanol followed by acetone overnight. Alcian blue (0.03% (w/v)) diluted in 20% acetic acid was then added to the organoids for 4 h at RT followed by a rinse in 1% potassium hydroxide (KOH) (w/v) until the tissue was transparent.
- KOH potassium hydroxide
- the organoids were stored in glycerol at 4°C before imaging on a Nikon SMZ25 automated stereomicroscope equipped with a 1 * objective and a PHOTONIC LED-Set Ringlight stereomicroscope (SMZ25, Nikon). Z stack images were taken every 15 pm, images were analyzed using NIS-Elements software (version 5.30.06, Nikon).
- kidney organoids were incubated for 20 min in 2% (v/v) paraformaldehyde diluted in phosphate-buffered saline (PBS) at 4°C. After a rinse in PBS, organoids were incubated in 15% (w/v) sucrose solution (in 0.1 M phosphate buffer pH 7.4) at 4°C for 24 h under agitation. The organoids are then transferred for 48 h into a 30% (w/v) sucrose solution at 4°C and set in a cryomold with the freezing buffer (7.5% (w/v) gelatin and 15% (w/v) sucrose in 0.1 M phosphate buffer).
- PBS phosphate-buffered saline
- cryosections were cut 16 m thick on a CM3050S cryostat (Leica), mounted on Superfrost slides (Fisher Scientific), and used for immunostaining or Alcian blue staining. To remove the gelatin/sucrose embedding before any staining, cryosections were placed in PBS for 15 min at 37°C.
- Imaging was performed on an inverted Nikon Ti-S/L100 microscope, equipped with a Nikon DS-Ri2 camera using a CFI Plan Apochromat K 20xobjective (NA: 0.75, WD: 1.0). Images were analyzed using NIS- Elements software (version 5.30.06, Nikon).
- the slides were incubated for 20 min at RT in a blocking buffer containing 10% bovine serum albumin (BSA), 0.1 M glycine and 0.2% Tween-20 in PBS.
- BSA bovine serum albumin
- Primary antibodies (Table 1), diluted in PBS with 1% BSA, 0.1 M glycine and 0.2% Tween-20, were incubated overnight at 4°C.
- secondary antibodies diluted in PBS with 1% BSA, 0.1 M glycine and 0.2% Tween-20 for 1 h at RT.
- Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI, 0.1 pg/ml) in PBS with 0.2% Tween-20 for 5 min. Slides were mounted using Dako fluorescence mounting medium (Agilent Technologies). Imaging was performed on an automated inverted Nikon Ti-E microscope, equipped with a Lumencor Spectra light source, an Ador Zyla 5.5 sCMOS camera, and an MCL NANO Z200-N Tl z-stage. The objective used was CFI PLAN APO LBDA 10* 0.45/4 mm. Images were analyzed using NIS-Elements software (version 5.30.06, Nikon). Western blot
- Proteins were extracted from the kidney organoids using TRIzol (Invitrogen) as described by the manufacturer. After performing a bicinchoninic acid protein assay (Pierce), 15 pg of protein was loaded per well of an 8% or 10% acrylamide gel. Migration was performed at 120 V in a migration buffer (pH 8.3) consisting of 25 mM Tris-Base, 192 mM glycine, and 0.1 % sodium dodecylsulfate (SDS) (Bio-Rad). Transfer to nitrocellulose membrane was performed at 4 C for 90 min in a transfer buffer consisting of 50 mM Tris, 40 mM glycine, and 1.5 mM SDS at a constant amperage of 350 mA.
- a migration buffer pH 8.3
- SDS sodium dodecylsulfate
- the membranes were then incubated in a blocking solution consisting of Tris-buffered saline (TBS) added with 5% BSA and 0.1% Tween-20 for 1 h at RT.
- Primary antibodies diluted in blocking solution were incubated overnight at 4°C.
- Primary antibodies used were the following: anti-SOX-9 (1/1000, Cell Signaling Technology, 82630), anti-GAPDH (1/10000, Cell Signaling Technology, 2118), anti- vimentin (1/500, Thermo Fisher Scientific, MA5-16409), anti-a-SMA (1/1000, Cell Signaling, 19245S) and anti-COL2 (1/1000; Abeam, ab34712).
- Membranes were rinsed twice in TBS with 0.1 % Tween and incubated for 1 h with secondary antibodies coupled to a horseradish peroxidase (HRP) (1/3000; BioRad) at RT. After two additional washes, the membranes were developed using a chemiluminescence substrate (Clarity Western ECL Substrate, Bio-Rad) and detected for 10 s to 5 min on CL-Xposure film (Thermo Fisher Scientific) or using a Chemidoc (Bio-Rad). Films were scanned and the protein bands were quantified by measuring density via Imaged software (National Institutes of Health, Imaged 1.53e). GAPDH was used as the reference protein for the normalization of the proteins of interest.
- HRP horseradish peroxidase
- Kidney organoids were manually crushed and lysed in TRIzol (Invitrogen) and mixed with chloroform (200 pl/ml Trizol). After homogenization, the samples were incubated for 15 min at RT followed by a centrifugation at 18000 RCF for 15 min at 4°C. The clear supernatant was collected, and isopropanol (500 pl/ml Trizol) was added to precipitate the RNA. The samples were homogenized by vortexing and incubated for 15-20 min at RT and centrifuged again at 4°C and 18000 RCF for 15 min. The RNA pellets were then washed several times with ethanol. After centrifugation at 4°C and 6800 RCF for 5 min, the ethanol was removed, and the pellet was air-dried before being eluted in RNAse-free water (Qiagen).
- Reverse transcription was performed using 500 ng of RNA mixed with water, reverse transcription enzyme and iScript buffer (BioRad) in a final volume of 20 pl. The samples were heated for 5 min at 25°C for priming followed by 20 min at 46°C for reverse transcription and 1 min at 95°C for enzyme inactivation. The obtained cDNA was diluted five times in RNAse-free water, and 2 pl were prepared for qPCR with a reaction mix comprising 250 nM of each primer (Table 2), 4 pl of water and 10 pl SYBR (Bio-Rad) Green Master Mix.
- the real-time PCR CFX96 (Bio-Rad) was programmed to perform 40 cycles of 2-steps amplification consisting of 10 s of denaturation at 95°C, and 60 s of combined annealing and extension at 60°C. Experiments were performed in technical duplicate and normalized to GAPDH as a housekeeping gene.
- Non-renal cell populations appeared between day 7+18 and 7+25 of kidney organoid culture
- kidney organoids comprised glomeruli (NPHS1), tubules (ECAD), proximal tubules (LTL) (Fig. 4D) as well as loops of Henle (SLC12A1) and the expected stromal population (MEIS) (Fig. 4E).
- NPHS1 glomeruli
- ECAD tubules
- LTL proximal tubules
- MEIS expected stromal population
- the non-renal cell population was identified as cartilage progressively developing over time in culture
- FGF9 without heparin was added from day 7+5 until day 7+12 (Fig. 6A).
- Organoids that underwent this treatment are referred to as FGF9-treated organoids (day 7+25+FGF9), while the organoids cultured in regular conditions are referred to as control organoids (day 7+25).
- Fig. 6B and 6C the cartilage present in control organoids (Fig. 6B and 6C) was absent in the FGF9-treated organoids at day 7+25 (Fig. 6D and 6E).
- Fig. 8A- D NPHS1 (glomeruli), ECAD (distal tubules), LTL (proximal tubules) and SLC12A1 (loops of Henle).
- NPHS1 glomeruli
- ECAD distal tubules
- LTL proximal tubules
- SLC12A1 loops of Henle
- FIG. 8E While control organoids do not contain vessels at day 7+25 (Fig. 8E), FGF9-treated organoids developed vessel-like structures, as observed by toluidine blue staining (Fig. 8F). This observation was correlated with an increased level of the vascularization marker CD31 (PECAM1) in immunofluorescence (Fig. 8G and 8H) and PECAM1 expression measured by qPCR (Fig. 11) in FGF9-treated organoids compared to control organoids.
- PECAM1 vascularization marker CD31
- FGF20-treated organoids day 7+25+FGF20 or day 7+25+FGF20+heparin
- control organoids day 7+25
- NPHS1 glomeruli
- ECAD distal tubules
- LTL proximal tubules
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Abstract
The invention describes a method for culturing kidney organoids using a prolonged exposure to FGF9, FGF20 or an activator of FGFR1 and/or FGFR3. The method results in improved kidney morphology in the resulting organoids, in particular the organoids have reduced cartilage induction. The invention further relates to organoids obtained by the method and uses thereof.
Description
Title: Improved method for generating Kidney organoids
Field of the Invention
The invention describes a method for culturing kidney organoids using a prolonged exposure to FGF9, FGF20, or an activator of FGFR1 and/or FGFR3. The method results in improved kidney morphology in the resulting organoids, in particular the organoids have reduced cartilage induction. The invention further relates to organoids obtained by the method and uses thereof.
Introduction
Chronic kidney disease (CKD) is characterized by a gradual loss of kidney function and is linked to a diminished glomerular filtration rate. Its prevalence varies from 3.3 to 17.3% and has risen in the last decades. Currently, kidney replacement is the most reliable therapeutic option for CKD. Worldwide, 9.7 million people need kidney replacement therapy, but due the shortage of donor organs and healthcare costs, only 2.6 million will receive it. The numbers of kidney transplants needed are expected to double within the next 10 years, increasing the pressure to find alternative solutions. Recent developments to generate organoids in vitro have opened the possibility for a regenerative medicine-based approach with the potential to provide a functional substitute to the failing kidney. Indeed, using the knowledge of developmental biology, human induced pluripotent stem cells (hiPSCs) are widely used to produced organoids mimicking various organ systems, including kidney organoids. Organoids present advantages compared to 2D-culture as they are isolated multicellular systems. This 3D-culture model is more physiologic and allows the study of interactions between different cell types. Indeed, kidney organoids can recapitulate renal structures as well as the cellular complexity of human kidney, and have demonstrated the potential to restore glomerular filtration upon transplantation. Moreover, it was shown that renin production, responsive to regulation by parathyroid hormone, in iPSC-derived kidney organoids under cyclic AMP stimulation, reflecting their physiological functionality in the endocrine system.
This physiologic functionality in vitro demonstrates that kidney organoids might be a good model to study kidney diseases as well as a good therapeutic approach to treat kidney deficiency. Indeed, kidney diseases commonly involve different cell types
interactions, and the multicellular aspect of kidney organoids allows their use as in vitro kidney diseases models. Actually, kidney organoids are already used to model diseases, as ciliopathies or polycystic kidney disease, and to study the mechanistic of renal pathologies as well as potential treatments. More than modelling diseases, the physiologic potential of kidney organoids makes them good candidates for transplant therapeutic approaches.
Despite their great therapeutic potential, kidney organoids also present several drawbacks, including a lack of maturation and vascularization, as well as the appearance of 10-20% off-target cell populations, such as neurons, myocytes and chondrocytes. Indeed, when performing single cell-RNA sequencing it was shown that neural markers are expressed. Inhibition of these markers via a pharmacological approach reduced the neuron population without affecting kidney structures, showing the possibility to improve kidney organoids using a small molecule approach. The appearance of cartilage in kidney organoids is observed after in vivo engraftment of the organoid under the renal capsule of mice. The off-target tissue appears 4 weeks after the graft and expands over time. Transplantation of kidney organoids with kidney decellularized extracellular matrix (ECM) resulted in a reduction of cartilage appearance in vivo and emphasizes the importance of microenvironment in kidney organoid development, however improved method further reducing cartilage appearance are needed for clinical application of kidney organoids in kidney diseases such as CKD.
The present invention aims to overcome these problems, among others.
Summary of the invention
In a first aspect, the invention relates to a method for differentiating stem cells to kidney organoids, the method comprising a first differentiation stage and a second differentiation stage, wherein the first differentiation stage comprises culturing the stem cells under conditions to allow differentiation of the stem cells to a cell culture comprising cells in the intermediate mesoderm stage, wherein the second differentiation stage comprises transferring the cell culture comprising cells in the intermediate mesoderm stage to a 3D culture environment and culturing the cells under conditions that allow the formation of a kidney organoid, wherein the second differentiation step is characterized in that FGF9, FGF20, or an activator of FGFR1
and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture, followed by culturing in growth factor free medium.
In a second aspect, the invention relates to an organoid obtained or obtainable by the method according to the first aspect of the invention.
In a third aspect the invention relates to an organoid according the second aspect of the invention for use as a medicament.
In a fourth aspect the invention relates to the in vitro or ex vivo use of an organoid according to claim 12 for one or more of:
- target identification for a drug;
- target validating for a drug;
- performing safety studies for a drug;
- performing pharmacodynamics studies for a drug;
- drug development;
- drug discovery;
- performing stratification studies for a drug;
- predicting an individual patient’s response to a drug;
- modelling a disease;
- modelling development; or
- studying kidney biology.
In a fifth aspect the invention relates to a method for differentiating intermediate mesoderm cells under conditions that allow for the formation of an immature kidney organoid expressing ECAD, LTL, and NPHS1 , wherein the cells are cultured in a 3D culture environment, and wherein the method is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture, followed by culturing in growth factor free medium.
In a sixth aspect the invention relates to an immature kidney organoid obtained or obtainable by the method according to the fifth aspect of the invention.
Brief description of the figures
Figure 1 : (Reference protocol (Takasato protocol according to Leiden adaptation - top part of the figure) Differentiation of iPSCs into kidney organoids is done in two phases. First differentiation in 2D using CHIR99021 during 4 days and FGF9/heparin cocktail for 3 days. Then a second phase of culture after aggregation of the cells at the air
liquid interface to form the 3D structured organoids. During this second phase organoids are maintained in presence of FGF9/heparin cocktail for 5 days and then all growth factors are removed until day 7+25. Between day 7+18 and day 7+25, development of the organoid (including maturation) reaches a plateau and cartilage appears during this period, (exemplary protocol of the invention - bottom part of the figure) The method of the invention differs in that FGF9 or FGF20 is provided longer in the air liquid interface culture stage. In the provided example, FGF9 is provided together with heparin from day 7 until day 7+5, followed by FGF9 only from day 7+5 until day 7+12. The protocol is otherwise identical to the reference protocol.
Figure 2: (Reference protocol (Takasato protocol - top part of the figure) Differentiation of iPSCs into kidney organoids is done in two phases. First differentiation in 2D using CHIR99021 during 4 days and FGF9/heparin cocktail for 3 days. Then a second phase of culture after aggregation of the cells at the air liquid interface to form the 3D structured organoids. During this second phase organoids are maintained in presence of FGF9 for 5 days and then all growth factors are removed until day 7+25. Between day 7+18 and day 7+25, development of the organoid (including maturation) reaches a plateau and cartilage appears during this period, (exemplary protocol of the invention - bottom part of the figure) The method of the invention differs in that FGF9 is provided longer in the air liquid interface culture stage. In the provided example, FGF9 is provided from day 7 until day 7+12. The protocol is otherwise identical to the reference protocol.
Figure 3: (Reference protocol (Morizane protocol - top part of the figure) Differentiation of iPSCs into kidney organoids is done in two phases. First differentiation in 2D using CHIR99021 and Noggin during 4 days and activin for 3 days followed by FGF9 for 2 days. Then a second phase of culture after aggregation of the cells at low adherent wells to form the 3D structured organoids. During this second phase organoids are maintained in presence of FGF9 for 5 days (until day 14) and then all growth factors are removed until day 26. (exemplary protocol of the invention - bottom part of the figure) The method of the invention differs in that FGF9 is provided longer in the 3D culture stage. In the provided example, FGF9 is provided from day 9 until day 21. The protocol is otherwise identical to the reference protocol.
Figure 4: Off-target cell population develops in iPSC-derived kidney organoids and disrupts the renal structures’ development. (A) Schematic of kidney organoid
culture. iPSCs were stimulated with CHIR99021 for 3 days and a FGF9/heparin cocktail for 4 days. After a 1 h pulse of CHIR99021 , cells were aggregated and cultured at the air-liquid interface for 5 days in the presence of the FGF9/heparin cocktail. Organoids were then cultured until day 7+25 without growth factors. A spherical kidney organoid shape was observed at day 7+18 (B) and 7+25 (C) by brightfield microscopy. (D-G) Renal structures assessed by immunofluorescence in cryosections show (D, F) glomeruli (NPHS1), proximal tubules (LTL), distal tubules (ECAD); (E, G) loops of Henle (SLC12A1) and a stromal population (MEIS1/2/3). Nuclei are stained with DAPI. Asterisks indicate an off-target cell population at day 7+25 (F, G). Scale bars represent 1000 pm (B-C) and 50 pm (D-G).
Figure 5: The off-target population was identified as cartilage developing between days 7+18 and 7+25. (A-D) Alcian blue staining revealed the presence of cartilage at days 7+18 and 7+25 in the whole organoid (left images, scale bars represent 1000 pm) and cryosections (right images; scale bars represent 50 pm). (E- I, N=5) Progressively and significantly increased expression of five different markers of chondrogenesis was detected by qPCR at day 7+18 and 7+25. *p < 0.05; **p < 0,01 ; ***p < 0.001 based on -fold change relative to day 7+18. (J) Western blotting of SOX9 and COL2A1 protein levels in kidney organoids from day 7+18 to 7+25. GAPDH levels are shown as loading controls. (K-L) Quantification of protein levels using Imaged normalized to GAPDH showed a significant increase (***p < 0.001 ; N=5) of COL2A1 (L) from day 7+18 to 7+25. SOX9 levels (K; N=3) did not show significant differences. Figure 6: FGF9 treatment abrogates cartilage formation at day 7+25. (A) Schematic of the FGF9 treatment, which was extended after aggregation from day 7+5 to day 7+12. (B-E) Cartilage stained with Alcian blue in whole organoids (left images, scale bars represent 1000 pm) and on cryosections (right images, scale bars represent 50 pm) was strongly reduced with FGF9 treatment (bottom row).
Figure 7: FGF9 treatment reduces expression of cartilage markers at day 7+25. (A-E) FGF9 treatment significantly decreased five markers of chondrogenesis in kidney organoids at day 7+25 compared to control organoids. Gene expression was assessed by qPCR and shown as -fold change compared to expression at day 7+18. **p < 0.01 ; ***p < 0.001 ; N=5. (F) Western blotting of SOX9 and COL2A1 protein levels in kidney organoids at day 7+25 showed decreased expression in FGF9-treated organoids compared to untreated controls. GADPH levels are shown as loading
controls. (G-H) Quantification of protein levels confirmed significantly decreased expression of SOX9 (G) and COL2A1 (H) at day 7+25 with FGF9 treatment (+) compared to nontreated (-) organoids. **p < 0.01 from 3-4 samples. Relative expression was assessed as the -fold change compared to day 7+18.
Figure 8: FGF9-treated organoids show renal and vessel-like structures. (A-D) Renal structures assessed by immunofluorescence in cryosections show (A, C) glomeruli (NPHS1), proximal tubules (LTL), distal tubules (ECAD); (B, D) loops of Henle (SLC12A1) and stromal population (MEIS 1/2/3). Nuclei are stained with DAPI. The off-target cell population (asterisks) observed in day 7+25 untreated organoids (top row) was not observed with FGF9 treatment (bottom row). Scale bars represent 50 pm. (E-F) Toluidine blue staining showed the appearance of vessel-like structures (#) in FGF9-treated organoids (F) that were absent in control organoids (E) at day 7+25. Scale bars represent 50 pm; inset represents 20 pm. (G-H) Immunostaining of cryosections showed an increase of the endothelial marker CD31 in FGF9-treated organoids (H) compared to controls (G). Scale bars represent 50 pm.
Figure 9: FGF9-treated organoids express lower levels of EMT markers at day 7+25. (A) Western immunoblotting showed lower levels of EMT markers vimentin and a-SMA in FGF9-treated (+) organoids compared to control (-) organoids at day 7+25. Notably, vimentin and a-SMA progressively increased from day 7+5, 7+10, 7+14, 7+18 and 7+25 in control organoids. GADPH levels are shown as loading controls. (B-C) Quantification of vimentin (B) and a-SMA (C) levels at day 7+5, 7+10, 7+14, 7+18 and 7+25 showed significant increases in untreated organoids, which were ameliorated by FGF9 treatment (7+25+FGF9). Protein levels are expressed as -fold change relative to day 7+5. *** p < 0.001 ; **** p < 0.0001 from 3 samples each.
Figure 10: FGF9 treatment does not negatively affect renal structures. (A-D) Gene expression of markers of renal structures, CUBN, NPHS1, SLC12A 1 and stromal population MEIS1 were assessed by qPCR and shown as -fold change compared to expression at day 7+18. *p < 0.05; **p < 0.01 ; from 3-4 samples. (E) FGF9 treatment (+) upregulates PECAM1 expression compared to control (-) organoids at day 7+25. PECAM1 expression assessed using qPCR shown as -fold change compared to expression in untreated organoids at day 7+18. *p < 0.05 from 3 samples.
Figure 11 : FGF9 treatment delays the appearance of cartilage in iPSC-derived kidney organoids. (A-D) Cartilage (asterisks) stained with Alcian blue in whole
organoids (left images, scale bars represent 1000 pm) and on cryosections (right images, scale bars represent 50 pm) was less abundant with FGF9 treatment but the appearance of small islands of cartilage were visible (bottom row). (E-l) FGF9 treatment (+) significantly decreased four of five markers of chondrogenesis in kidney organoids at day 7+32 compared to control (-) organoids. Gene expression assessed by qPCR and shown as -fold change compared to expression in untreated organoids at day 7+18. ***p < 0.001 from 4 samples. (J) Western blotting COL2A1 protein in control (-) kidney organoids showed increased expression over time which was abrogated with FGF9 treatment (+). GADPH levels shown as loading controls.
Figure 12: FGF20 treatment abrogates cartilage formation at day 7+25. Cartilage stained with Alcian blue in whole organoids was strongly reduced with FGF20 treatment (middle and right panels).
Figure 13: FGF20-treated organoids show renal structures. Renal structures assessed by immunofluorescence in cryosections show glomeruli (NPHS1), proximal tubules (LTL), and distal tubules (ECAD). Nuclei are stained with DAPI. The off-target cell population observed in day 7+25 untreated organoids (left panel) was not observed with FGF20 treatment (middle and right panels). Scale bars represent 50 pm.
Definitions
A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.
Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.
“A,” “an,” and “the”: these singular form terms include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
“About” and “approximately": these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... , etc. As used herein, the term "at most " a particular value means that particular value or less. For example, "at most 5 " is understood to be the same as "5 or less" i.e., 5, 4, 3, ... .-10, -11 , etc.
"Agonist": This term, as used herein refers to a compound or agent having the ability to initiate or enhance a biological function of a target protein or polypeptide, such as increasing the activity or expression of the target protein or polypeptide. Accordingly, the term "agonist" is defined in the context of the biological role of the target protein or polypeptide. While some agonists herein specifically interact with (e.g., bind to) the target, compounds and/or agents that initiate or enhance a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target polypeptide is a member are also specifically included within this definition.
"Antagonist" and "inhibitor": These terms are used interchangeably, and they refer to a compound or agent having the ability to reduce or inhibit a biological function of a target protein or polypeptide, such as by reducing or inhibiting the activity or expression of the target protein or polypeptide. Accordingly, the terms "antagonist" and "inhibitor" are defined in the context of the biological role of the target protein or polypeptide. An inhibitor need not completely abrogate the biological function of a target protein or polypeptide, and in some embodiments reduces the activity by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. While some antagonists herein specifically interact with (e.g., bind to) the target, compounds that inhibit a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target protein or polypeptide are also
specifically included within this definition. Non-limiting examples of biological activity inhibited by an antagonist include those associated with the development, growth, or spread of a tumor, or an undesired immune response as manifested in autoimmune disease.
“And/or”: The term “and/or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
“Conventional techniques” or “methods known to the skilled person”: These terms refer to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology and related fields are well- known to those of skill in the art and are discussed, for example, in the following literature references: Human Embryonic Stem Cell: The Practical Handbook. Publisher: John Wiley & Sons, LTD, Editors (Sullivan, S., Cowan, C. A., Eggan, K.) Harvard University, Cambridge, MA, USA (2007); Human Stem Cell, a Laboratory Guide (2nd Edition) by Peterson, S., and Loring, J. F. (2012).
“Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.
“Cell line”: this term refers to continuously growing or immortalized cells. Sometimes also referred to as “immortalized cell line”, a cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division.
“Differentiating” and “differentiation”: these terms, in the context of living cells, relate to progression of a cell further down the developmental pathway. A “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with; differentiation is the process of progression. Human pluripotent stem cells can differentiate into lineage-restricted progenitor cells (cells that, like a stem cell, have a tendency to differentiate into a specific type of cell, but are already more differentiated than a stem cell and are
pushed to eventually differentiate into its end-stage cell; e.g. endoderm, mesoderm and ectoderm), which in turn can differentiate into further restricted cells (e.g., cardiomyocyte progenitors, neuronal cell progenitors), which can differentiate into terminally differentiated cells (e.g., cardiomyocytes or neurons). Differentiation is controlled by the interaction of a cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface. In the present invention, “differentiation” is the biological process whereby an unspecialized human pluripotent stem cell (population) acquires the features of a specialized cell such as a cardiomyocyte under controlled conditions in in vitro culture.
“Embryonic stem cells”: abbreviated as ‘ES cells’ or ESC (or if of human origin ‘hES cells’ or ‘hESCs’) refers to stem cells that are derived from the inner cell mass of a blastocyst. The skilled person understands how to obtain such embryonic stem cells, for example as described by Chung (Chung et al (2008) Stem Cell Lines, Vol 2(2): 113- 117), which employs a technique that does not cause the destruction of the donor embryo(s). Various ESC lines are listed in the NIH Human Embryonic Stem Cell Registry.
"Exemplary": this terms means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein.
"In vivo": This term refers to an event that takes place in a subject's body
"In vitro": This term refers to an event that takes places outside of a subject's body. For example, an in vitro assay encompasses any assay conducted outside of a subject. In vitro assays encompass cell-based assays in which cells, alive or dead, are employed. In vitro assays also encompass a cell-free assay in which no intact cells are employed.
“Induced pluripotent stem cell” or “iPSC”: These terms refer to pluripotent stem cells that are derived from a cell that is not a pluripotent stem cell (i.e. , from a cell this is differentiated relative to a pluripotent stem cell). Induced pluripotent stem cell can be derived from multiple different cell types, including terminally differentiated cells. Induced pluripotent stem cell generally have an ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, induced pluripotent stem cell may express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited
to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181 , TDGF 1 , Dnmt3b, FoxD3, GDF3, Cyp26a1 , TERT, and zfp42. Examples of methods of generating and characterizing induced pluripotent stem cells may be found in, for example, U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646. To generate induced pluripotent stem cells, somatic cells may be provided with reprogramming factors (e.g. Oct4, SOX2. KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells( see, for example, Takahashi et. al, Cell. 2007 Nov. 30; 131 (5):861-72; Takahashi et. al, Nat Protoc. 2007; 2(12):3081-9; Yu et. al, Science. 2007 Dec. 21:318(5858): 1917-20. Epub 2007 Nov. 20).
“Markers” or “lineage-specific markers”: these terms refer to a characteristic specifically associated with the phenotype of cells of a lineage and can be used to assess the differentiation of cells. The terms may refer to nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. The detectable level of the marker is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
“Media”: This term refers to an aqueous solution, including buffers, suitable for maintaining human or animal cells for a sufficient period. For example, a media is suitable if it allows the treatment of cells for a period required to obtain the effect intended by the treatment. The term “media” also, and preferably, includes growth media that are suitable for the in vitro cell culture of human or animal cells. A “defined media” refers to a (growth) media suitable for the in vitro cell culture of human or animal cells and in which all of the chemical components are known. Such defined media does not or essentially not comprise any ill-defined source of nutrients and/or other ill-defined factors. Within the context of the current invention the defined media used may still contain defined amounts of products such as (purified) albumin, growth factors, and hormones, but is essential free of serum (i.e. less than 1 % w/w, preferably less than 0.5% w/w. even more preferably less than 0.1 % w/w, even more preferably less than 0.05% w/w of the medium ready for use, most preferably the medium is free of serum (i.e. 0% w/w serum; albeit it might contain defined amount of specified compounds like (recombinant) albumin. Although widely used, serum has many
limitations. It contains high levels of numerous and unknown proteins and compounds which interfere dramatically with the small quantities of the desired proteins produced by the cells. The presence of serum may also affect in vitro testing results with the cells obtained since some compounds may bind up to 99% to serum proteins. Another limitation is the serum batch-to-batch inconsistencies, resulting in serious regulatory concern about various serum protein contaminations in the product.
“Pluripotency": This term is generally understood by the skilled person and refers to an attribute of a (stem) cell that has the potential to differentiate into all cells constituting one or more tissues or organs, for example, any of the three germ layers: endoderm (e.g. interior stomach lining, gastrointestinal tract, the lungs), mesoderm (e.g. heart, muscle, bone, blood, urogenital tract), or ectoderm (e.g. epidermal tissues and nervous system).
“Pluripotent stem cell” or “PSC”: This is a stem cell capable of producing all cell types of the organism and can produce cells of the germ layers, e.g. endoderm, mesoderm, and ectoderm, of a mammal and encompasses at least pluripotent embryonic stem cells and induced pluripotent stem cells. Pluripotent stem cells can be obtained in different ways. Pluripotent embryonic stem cells may, for example, be obtained from the inner cell mass of an embryo. Induced pluripotent stem cells (iPSCs) may be derived from somatic cells. Pluripotent stem cells may also be in the form of an established cell line.
Times used for indicating organoid age: when used herein the age of cells or organoids is indicated in hours or days. When done so, the age refers to the time since initiating the first differentiation stage. For example, “3 day” or “day 3” refers to cells cultured for three days in the first differentiation stage (in a 2D culture environment) and “day 10” or “10 day” refers to organoids where the cumulative time culturing in the first and the second differentiation stage is 10 days (e.g. 7 days in the first differentiation stage and 3 days in the second differentiation stage). Alternatively the age can be annotated as “day A+B” or “A+B day”, here A indicates the number of days cultured in the first differentiation stage (in 2D culture) and B indicates the number of days cultured in the second differentiation stage. For example day 7+12 indicates organoids which are grown for 7 days in the first differentiation stage (2D culture) and 12 days in the second differentiation stage (3D culture environment), and thus are
cultured for an accumulative of 19 days in the first and second differentiation stage together.
When used herein, the term “after onset”, when for example used in the context of “from onset of the 3D culture” intends to indicate immediately after the start thereof (e.g. the 3D culture), but allows also to include shortly after onset of the intended event (e.g. 3D culture), such as 1 , 2, 3, 4, 6, 12, 24, 36 or 48 hours after initiating the referred event. Thus for example the phrase providing FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture implies that FGF9 or FGF20 is provided immediately after the 3D culture starts or within 1 , 2, 3, 4, 6, 12, 24, 36 or 48 hours of starting the 3D culture. The time period (e.g. 8 to 16 days) is calculated accordingly (e.g. starting immediately after or within 1 , 2, 3, 4, 6, 12, 24, 36 or 48 hours of starting the 3D culture, whichever applies).
Detailed description
It is contemplated that any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and/or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.'
Chronic kidney disease affects 11-13% of the global population. Worldwide, 9.7 million people need kidney replacement therapy but only 2.6 million will receive it. One regenerative medicine alternative comprises the use of iPSC-derived kidney organoids as a therapeutic engraftment to the dysfunctional kidney. However, several drawbacks must be overcome before clinical translation, among which is the presence of non-renal cell populations such as cartilage in the organoids. The inventors herein describe a modification of the culture protocol and maintained kidney organoids in medium containing FGF9 or FGF20 for one additional week compared to the control protocol (Takasato). In comparison to control, the FGF9 (or FGF20) treated kidney organoids had no cartilage at day 7+25 and diminished chondrocyte marker expression. Importantly, the renal structures assessed by immunofluorescence were unaffected by the FGF9 or FGF20 treatment. This reduction of cartilage produces a
higher quality kidney organoid that can be maintained longer in culture to improve their maturation for further in vivo work.
The present invention relates to decreasing formation of cartilage in kidney organoids. The reason for the consistent observation of chondrocytes in kidney organoid culture and transplantation is not understood. Cartilage formation involves the condensation of mesenchyme tissue, which differentiates into chondrocytes and produces the extracellular matrix protein collagen 2 (COL2A1). The pathways leading to chondrocyte differentiation mainly involve the SOX protein family, particularly SOX9.
In the study of Bantounas et al. (Stem Cell Reports 10, 766-779 (2018)), the authors used kidney progenitors to transplant mice and still observed the appearance of cartilage weeks after the graft, highlighting the presence of a potential dedifferentiation of renal structures. After injury or stress, adult kidney tissue is able to regenerate via dedifferentiation of tubes, notably through the EGFR pathway. This pathway also induces a transitory increase of SOX9 expression, particularly in proximal tubules, and can last for 2-3 days to induce healing of the injured tissue18. The transitory aspect of SOX9 expression is highly important as this nuclear factor is a key player in other processes including chondrogenesis. Indeed, during cartilage formation and maintenance, SOX9 secures the chondrocytic lineage, cell survival and regulate genes implicated in cartilage structure.
Dedifferentiation of renal structures is also linked to epithelial to mesenchymal transition (EMT). EMT is known to happen during the development of kidney organoids and is associated with expression of several markers as vimentin, alpha-smooth muscle actin (aSMA) and collagen 1a1 (COL1A1), see e.g. Rutier et al. (Adv Sci (Weinh) 9, e2200543 (2022)). During this process, epithelial cells lose their characteristics, as cell polarity and cell-cell adhesion, to a mesenchymal and ECM component — secreting cell phenotype and ability to differentiate in other cell types.
Currently the main protocols used for culturing kidney organoids are the Takasato protocol (Takasato, M. et al. Nature 526, 564-568 (2015)) and Morizane protocol (Morizane and Bonventre, Nat. Protoc. 2017 Jan;12(1):195-207). The protocols have been schematically described in Figures 1 , 2 and 3. The protocols have in common that initially stem cells (e.g. induced pluripotent stem cells (iPSCs)) are cultured as a monolayer to allow differentiation, and then transferred to an environment that allows
the formation of organoids (e.g. air-liquid interface or low adherence wells, also referred herein as the second differentiation phase or 3D culture stage), while being provided FGF9 for defined time period.
The inventors added a step in the differentiation protocol and incubated 3D kidney organoids with FGF9 or FGF20 from day 5 until day 12 (counting from onset of the 3D culture) as described in the examples below. The inventors demonstrate a clear reduction of the appearance of cartilage at the latter timepoint correlated with a reduction of EMT and cartilage markers. As demonstrated by the examples below, the inventors hereby disclose an improved protocol to generate kidney organoids at the air-liquid interface, using growth factor FGF9 or FGF20.
Thus in a first aspect, the invention relates to method for differentiating stem cells to kidney organoids, the method comprising a first differentiation stage and a second differentiation stage, wherein the first differentiation stage comprises culturing the stem cells under conditions to allow differentiation of the stem cells to a cell culture comprising cells in the intermediate mesoderm stage, wherein the second differentiation stage comprises transferring the cell culture comprising cells in the intermediate mesoderm stage to a 3D culture environment and culturing the cells under conditions that allow the formation of a kidney organoid, wherein the second differentiation step is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture, followed by culturing in growth factor free medium.
Interestingly, it is known in the cartilage field that the addition of FGF9 to a differentiation protocol will increase the formation of cartilage (see e.g. Muthikrisnan et al., Nature Comm., 6:10027 (2015); Barak et al. Dev Cell. 2012 June 12; 22(6): 1191-1207; Correa et al. Osteoarthritis Cartilage. 2015 March ; 23(3): 443-453). Indeed it is shown that addition of FGF9 during chondrogenesis will increase cartilage formation. Thus to reduce cartilage formation it would be more obvious to remove addition of FGF9 from the protocol of kidney organoid differentiation, assuming it is the culprit of cartilage appearance. However FGF9 also plays an important role in the formation of renal structures, therefore completely removing FGF9 from the culture medium is not an option. In view of the above it is surprising that increasing the exposure time of the early organoids during the second differentiation stage actually reduced formation of cartilage.
The stepwise differentiation of pluripotent stem cells to kidney begins with the induction of the primitive streak which is the -progenitor population for both endoderm and mesoderm. While the anterior primitive streak gives rise to the endoderm, the posterior primitive streak has potential to develop into the mesoderm, including the axial, paraxial, intermediate and lateral plate mesoderm. The intermediate mesoderm differentiates to the ureteric epithelium and the metanephric mesenchyme, which are two key kidney progenitor populations subsequently undergoing a reciprocal interaction to form the kidney (Takasato et al. Nat Cell Biol 16, 118-126 (2014)). When culturing kidney organoids it is attempted to recreated these developmental steps in vitro.
The most used protocols to develop kidney organoids at present are the Takasato protocol (and the Leiden adaptation thereof) and the Morizane protocol. Schematic overviews of these protocols are provided in Figures 1-3. All the protocols have in common a 2D culture phase where stem cells are differentiated to intermediate mesoderm followed by a 3D culture phase where the cells are transferred to a 3D culturing system to allow the formation of organoids. The 3D culture phase in all protocols is characterized in that FGF9 or FGF20 is provided for about 5 days starting from the onset of the 3D culture phase. The inventors now found that when the total time in the 3D culture phase that FGF9, FGF20 or an activator of FGFR1 and/or FGFR3 is provided is increased to 8 to 16 days (e.g. 8, 9, 10, 11 , 12, 13, 14, 15 or 16 days) an improved effect is observed in that the organoids eventually express less cartilage and EMT markers. In Figures 1-3 bottom parts the adapted protocols according to the invention are depicted.
When used herein days from onset of the 3D culture phase preferably means starting from the 3D culture phase, meaning that when referring to the number of days FGF9, FGF20 (or and activator of FGFR1 and/or FGFR3) is provided is also the first day of the 3D culture phase, however it is possible to start providing FGF9, FGF20 (or and activator of FGFR1 and/or FGFR3) close to the start of the 3D culture phase (e.g. after 12, 24 or 48 hours) and start the numbering of days FGF9, FGF20 (or and activator of FGFR1 and/or FGFR3) is provided accordingly.
When used herein the term first differentiation stage refers to culturing stem cells in a 2D (monolayer) culture under conditions to allow differentiation of the stem cells to a cell culture comprising cells in the intermediate mesoderm stage. The first
differentiation stage may comprise several distinct steps. Generally as a first step the stem cells are cultured in the presence of a GSK3 inhibitor or a WNT activator. Such compounds are known to the skilled person, an exemplary compound that may be used is for example CHIR99021 which is a GSK3 inhibitor. During this step induction of both the ureteric epithelium and the metanephric mesenchyme takes places in the monoculture. The ureteric epithelium is characterized by expression of the markers PAX2, GATA3 and CDH1. The metanephric mesenchyme is characterized by the expression of PAX2, Wnt4 and BMP7. Thus in an embodiment of the method of the invention the cells in the intermediate mesoderm stage comprise at least one of: ureteric epithelium, metanephric mesenchyme, progenitors of renal interstitium or endothelium.
Thus in an embodiment the first differentiation stage comprises a step (A) wherein the stem cells are cultured under conditions that allow induction of both the ureteric epithelium and the metanephric mesenchyme. Alternatively the first differentiation stage comprises a step (A) wherein the stem cells are cultured under conditions that allow induction of both cells expressing PAX2, GATA3 and CDH1 and cells expressing PAX2, Wnt4 and BMP7. In an embodiment the first differentiation stage comprises a step (A) which step comprises culturing the stem cells in the presence of a GSK3 inhibitor or a WNT activator. In an embodiment the GSK3 inhibitor is CHIR99021. In an embodiment step (A) is performed for 2 to 6 days, preferably 3 to 5 days more preferably about 4 days.
In a second step during the first differentiation stage, the metanephric mesenchyme is further differentiated to nephron progenitor cells, while further differentiating the ureteric epithelium. This step is performed by culturing the partially differentiated cells at least in the presence of FGF9, FGF20 or an activator of FGFR1 and/or FGFR3. The nephron progenitor cells are characterized by the expression of the markers SIX2, HOXD11 , WT1 and PAX2. The ureteric epithelium is at this stage also characterized by expression of the markers PAX2, GATA3 and CDH1. Thus in an embodiment the first differentiation stage comprises a step (B) which step comprises culturing the partially differentiated cells under conditions that allow differentiation of the metanephric mesenchyme into nephron progenitor cells while further differentiating the ureteric epithelium. Alternatively the first differentiation stage comprises a step (B) wherein the stem cells are cultured under conditions that allow induction of both cells
expressing PAX2, GATA3 and CDH1 and cells expressing SIX2, HOXD11 , WT1 and PAX2. In an embodiment the first differentiation stage comprises a step (B) which step comprises culturing the stem cells in the presence of FGF9, FGF20 or an activator of FGFR1 and/or FGFR3. In an embodiment step (B) is performed for 1 to 4 days, preferably 2 or 3 days. During step (B) the cells may be cultured in the presence of additional factors besides FGF or an activator of FGFR1 and/or FGFR3. For example, heparin may also be included in the culture medium.
Step (B) may directly follow step (A), or there may be an intermediate step between step (A) and step (B). For example, after step (A) a step (A1) may be included in the method wherein the cells are cultured in the presence of activin. For example, step (A1) is performed for 1 to 5 days, preferably 2 to 4 days, more preferably about 3 days. When used herein the term second differentiation stage refers to culturing differentiated cells in a 3D culture environment under conditions to allow formation of an organoid and further differentiation of the cells to a kidney organoid. The second differentiation stage may comprise several distinct steps. Generally as a first step (C) the cells are cultured in the presence of FGF9, FGF20 or an activator of FGFR1 and/or FGFR3. During this step immature organoids are formed which demonstrate renal structures and start to express renal factors such as LTL, ECAD and NPHS1.
Thus in an embodiment the second differentiation stage comprises a step (C) wherein the cells are cultured under conditions that allow the formation of immature organoids. Alternatively the second differentiation stage comprises a step (C) wherein the cells are cultured under conditions that allow induction of cells expressing LTL, ECAD and NPHS1. In an embodiment the second differentiation stage comprises a step (C) which step comprises culturing the cells in the presence of FGF9, FGF20 or an activator of FGFR1 and/or FGFR3. This is the characterizing step of the protocol and differs from existing protocols in that step (C) is performed for 8 to 16 days, preferably 9-15 days, preferably 10-14 days, more preferably 11-13 days. Existing protocols like the Morizane protocol or the Takasato protocol teach that FGF9 is provided in the 3D culture environment for 5 days only.
During step (C) the cells may further be cultured in presence of additional factors besides FGF9, FGF20 or an activator of FGFR1 and/or FGFR3. For example upon onset of the 3D cell culture a pulse treatment or short treatment of a GSK3 inhibitor or a WNT activator such as for example CHIR99021 may be provided. The pulse
treatment may for example be provided for 15 minutes to 4 hours, preferably 30 minutes to 2 hours, more preferably about 1 hour, or for a short period such as 12 hours to 4 days, preferably 1 to 3 days, more preferably about 3 days. Exemplary concentrations when using CHIR99021 are between 1 to 10 micromolar, where the concentration is preferably lower when using short treatment compared to when using pulse treatment, e.g. 5 micromolar for pulse treatment and 3 micromolar for short treatment.
Further during step (C) the culture medium may further be supplemented with heparin for the first days. For example heparin may be supplemented for the first 3 to 7 days, preferably the first 4 to 6 days more preferably for about the first 5 days after onset of the 3D culture. For example Heparin may be supplemented in a concentration of 1 microgram per millilitre culture medium.
For the purpose of the invention the term 3D culture environment should be interpreted as any culture method that allows the formation of organoids from the harvested partially differentiated cells obtained in step (B), provided the appropriate culture conditions are provided. For example the Takasato protocol describes a trans-well membrane system to culture cells on a liquid air interface, while the Morizane protocol describes the use of ultra-low attachment plates. Other culture methods that allow the formation of organoids are however known to the skilled person and should be construed as included under the term 3D culture environment. Non-limiting examples are hydrogels, scaffolds or suspension culture. Therefore in an embodiment the 3D culture environment is selected from culturing on a trans-well membrane, culturing in a low attachment well, culturing in a hydrogel, culturing on an air-liquid interface culturing, culturing on a scaffold, or suspension culture.
Thus in an embodiment of the method according to the invention the first differentiation step comprises:
(A) culturing the stem cells under conditions allowing induction of ureteric epithelium cells and metanephric mesenchyme cells, preferably wherein the ureteric epithelium cells are cells expressing PAX2, GATA3, and CDH1 and the metanephric mesenchyme cells are cells expressing PAX2, Wnt4, and BMP7, more preferably wherein the cells are cultured in the presence of a GSK3 inhibitor or a WNT activator; and
(B) differentiating the ureteric epithelium cells in nephron progenitor cells, preferably wherein the nephron progenitor cells are cells expressing SIX2, HOXD11 , WT1 , PAX2, more preferably culturing the cells in the presence of FGF9, FGF20 or an FGFR1 and/or FGFR3 activator.
In a second step during the second differentiation stage, the immature organoids are further differentiated to kidney organoids. This step is performed by culturing the immature organoids in the absence of any growth factors. The kidney organoids are expressed by an increased expression of LTL, ECAD and NPHS1 compared to the immature organoids and further express SLC12A1 . Thus in an embodiment the second differentiation stage comprises a step (D) which step comprises culturing the immature organoids under conditions that allow differentiation to kidney organoids. Alternatively the second differentiation stage comprises a step (D) wherein the immature organoids are cultured under conditions that allow induction of the organoids to express LTL, ECAD, NPHS1 and SLC12A1. In an embodiment the second differentiation stage comprises a step (D) which step comprises culturing the stem cells in the absence of growth factors. In an embodiment step (D) is performed for at least 5 days, preferably at least 6, 7, 8, 9, 10, 11 or at least 12 days.
Step (D) preferably directly follows step (C), although there may be an optional intermediate step between step (C) and step (D).
In an embodiment the second differentiation step is performed for at least 20 days, preferably at least 21 , 22, 23, 24, or at least 25 days. Thus in an embodiment combined steps (C) and (D) are performed for at least 20 days, preferably at least 21 , 22, 23, 24, or at least 25 days. Thus in an embodiment the second differentiation stage comprises transferring the cell culture comprising cells in the intermediate mesoderm stage to a 3D culture environment and culturing the cells under conditions that allow the formation of a kidney organoid, wherein the second differentiation stage is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture, followed by culturing in growth factor free medium, wherein the second differentiation stage is performed for at least 20 days, preferably at least 21 , 22, 23, 24, or at least 25 days. Thus the step of culturing in the absence of growth factors (step (D)) is performed for at least 4, for example 4, 5, 6, 7, 8 , 9, 10, 11 , 12, or 13 days or even more.
In an embodiment of the method according to the invention the second differentiation step comprises:
(C) culturing the cells under conditions that allow the formation of an immature kidney organoid in the presence of FGF9, FGF20 or the FGFR1 and/or FGFR3 activator, wherein the immature kidney organoid expresses ECAD, LTL, and NPHS1 , and
(D) culturing the immature kidney organoid in the absence of growth factors to obtain a kidney organoid, preferably wherein the kidney organoid differs from the immature kidney organoid in the increased expression of ECAD, LTL, NPHS1 , and SLC12A1.
In an embodiment step (C) further comprises culturing the cell in the presence of Heparin for the first 3-7, preferably 4-6 days after onset of the 3D culture.
Preferably after the conclusion of step (B) during the first differentiation phase (the 2D culture phase), the differentiated cells are harvested and transferred to the 3D culture environment to start step (C) of the second differentiation phase (the 3D culture phase). Suitable method for harvesting the differentiated cells are known to the skilled person, for example non limiting examples are dissociating cells with Trypsin-EDTA or Accutase.
When used herein the term FGF9, FGF20 or an activator of FGFR1 and/or FGFR3 is intended to refer to FGF9, FGF20, a functional homolog of FGF9 or FGF20 or an alternative of FGF9 or FGF20 that can be used to replace FGF9 or FGF20 in the presented method, wherein the alternative activates FGFR1 and/or FGFR3.
FGF9 is a member of the fibroblast growth factor (FGF) family. FGF family members possess broad mitogenic and cell survival activities, and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. FGF9 was isolated as a secreted factor that exhibits a growth-stimulating effect on cultured glial cells. The peptide sequence of FGF9 is represented by SEQ ID NO: 1 below. Thus in embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 1 , is provided for 8 to 16 days after onset of the 3D culture. Preferably the protein is provided in the culture medium, thus allowing contact of the cells with the protein.
It is further understood that functional homologs of FGF9 (the protein defined by SEQ ID NO: 1) can be used in the context of the invention. Therefore in an embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 1 or a functional homolog thereof, is provided for 8 to 16 days after onset of the 3D culture, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 1. Preferably the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 1 which is able to activate FGFR1 and/or FGFR3
FGF20 is also member of the fibroblast growth factor (FGF) family. The peptide sequence of FGF20 is represented by SEQ ID NO: 2 below. Thus in embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 2, is provided for 8 to 16 days after onset of the 3D culture. Preferably the protein is provided in the culture medium, thus allowing contact of the cells with the protein.
It is further understood that functional homologs of FGF20 (the protein defined by SEQ ID NO: 2) can be used in the context of the invention. Therefore in an embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 2 or a functional homolog thereof, is provided for 8 to 16 days after onset of the 3D culture, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 2. Preferably the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 2 which is able to activate FGFR1 and/or FGFR3
Fibroblast growth factor receptor 1 (FGFR1), also known as basic fibroblast growth factor receptor 1 , fms-related tyrosine kinase-2 I Pfeiffer syndrome, and CD331 , is a receptor tyrosine kinase whose ligands are specific members of the fibroblast growth factor family. Fibroblast growth factor receptor 3 is a protein that in humans is encoded by the FGFR3 gene. FGFR3 has also been designated as CD333 (cluster of
differentiation 333). The gene, which is located on chromosome 4, location q16.3, is expressed in tissues such as the cartilage, brain, intestine, and kidneys. FGFR1 and FGFR3 are cell surface membrane receptors that possess tyrosine kinase activity. A full-length representative of these four receptors consists of an extracellular region composed of three immunoglobulin-like domains which bind their proper ligands, the fibroblast growth factors (FGFs), a single hydrophobic stretch which passes through the cell's surface membrane, and a cytoplasmic tyrosine kinase domain. When bonded to FGFs, these receptors form dimers with any one of the four other FGFRs and then cross-phosphorylate key tyrosine residues on their dimer partners. These newly phosphorylated sites bind cytosolic docking proteins such as FRS2, PRKCG and GRB2 which proceed to activate cell signalling pathways that lead to cellular differentiation, growth, proliferation, prolonged survival, migration, and other functions. The FGFR1 receptor is in humans encoded by the gene annotated as
ENSG00000077782 (Ensembl) or HGNC:3688.
The FGFR3 receptor is in humans encoded by the gene annotated as
ENSG00000068078 (Ensembl) or HGNC:3690.
It is thought that the main function of FGF9 or FGF20 in the kidney organoid differentiation protocol is the activation of FGFR1 and/or FGFR3, therefore the skilled person is aware that FGF9 or FGF20 may also be replaced by other factors capable of activating these receptors. Thus in embodiment of the method of the invention the second differentiation step is characterized in that an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture. The activator of FGFR1 and/or FGFR3 may be a small molecule or a protein. Activation of FGFR1 and/or FGFR3 by a compound or protein can easily be verified by the skilled person by for example phosphorylation specific antibodies targeting the FGFR1 or FGFR3 receptor. E.g. anti phospho Tyrosine 766 or anti phospho Tyrosine 653 in (human) FGFR1 or anti phospho Tyrosine 724 in (human) FGFR3. Non limiting examples of activators of the FGFR1 and/or FGFR3 receptors are FGF20, FGF2, or Smoothened agonist (SAG). Therefore in an embodiment of the method of the invention the second differentiation step is characterized in that FGF20, FGF2, or Smoothened agonist (SAG), is provided for 8 to 16 days after onset of the 3D culture.
FGF20 is a member of the fibroblast growth factor (FGF) family and capable of activating both FGFR1 and FGFR3. The peptide sequence of FGF20 is represented
by SEQ ID NO: 2 below. Thus in embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 2, is provided for 8 to 16 days after onset of the 3D culture. Preferably the protein is provided in the culture medium, thus allowing contact of the cells with the protein.
It is further understood that functional homologs of FGF20 (the protein defined by SEQ ID NO: 2) can be used in the context of the invention. Therefore in an embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 2 or a functional homolog thereof, is provided for 8 to 16 days after onset of the 3D culture, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 2. Preferably the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 2 which able to activate FGFR1 and/or FGFR3.
FGF2 is a member of the fibroblast growth factor (FGF) family and capable of activating both FGFR1. The peptide sequence of FGF2 is represented by SEQ ID NO: 3 below. Thus in embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 3, is provided for 8 to 16 days after onset of the 3D culture. Preferably the protein is provided in the culture medium, thus allowing contact of the cells with the protein.
It is further understood that functional homologs of FGF2 (the protein defined by SEQ ID NO: 3) can be used in the context of the invention. Therefore in an embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 3 or a functional homolog thereof, is provided for 8 to 16 days after onset of the 3D culture, wherein the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, preferably 99% sequence homology with SEQ ID NO: 3. Preferably the functional homolog is a protein having or comprising a peptide sequence with 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97,
98, preferably 99% sequence homology with SEQ ID NO: 3 which able to activate FGFR1 and/or FGFR3.
Smoothened agonist (SAG) was one of the first small-molecule agonists developed for the protein Smoothened, a key part of the hedgehog signalling pathway, which is involved in brain development as well as having a number of other functions in the body. It is shown that SAG increases expression of FGF9 and thus indirectly stimulates FGFR1 and FGFR3 by increasing expression of FGF9. SAG is represented by formula 1 below and also known as 3-Chloro-/V-[trans-4-(methylamino)cyclohexyl]-/\/-[[3-(4- pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide with CAS registry number 912545-86-9
Formula 1 :
Thus in embodiment of the method of the invention the second differentiation step is characterized in that Smoothened Agonist (SAG) is provided for 8 to 16 days after onset of the 3D culture.
In a particularly attractive embodiment it is envisioned that SAG can be provided in addition to FGF9 or an activator of FGFR1 and/or FGFR3. Without wishing to be bound be theory, it is theorized that SAG increases expression of the FGF receptors and therefore renders the cells more susceptible to activation with FGF9 or other activators of FGFR1 and/or FGFR3. When provided in addition to FGF9, FGF20 or an activator of FGFR1 and/or FGFR3, SAG is preferably provided for 2 to 4 days, more preferably from the start of the second differentiation stage.
Thus in an embodiment of the method of the invention the second differentiation step is characterized in that a protein having or comprising a peptide sequence as defined in SEQ ID NO 1 or a functional homolog thereof, a protein having or comprising a peptide sequence as defined in SEQ I D NO 2 or a functional homolog thereof, a protein
having or comprising a peptide sequence as defined in SEQ ID NO 3 or a functional homolog thereof, or a small molecular compound such as Smoothened Agonist is provided for 8 to 16 days after onset of the 3D culture.
The main advantage of the protocol of the invention is that, when compared to protocols known in the art, the protocols described herein result in reduced expression of EMT and cartilage markers. When used herein reduced expression of EMT and cartilage markers should be interpreted as either a reduced number of cells in the organoid show expression of EMT and cartilage markers, or the expression levels of the of EMT and cartilage markers is significantly lower in kidney organoids grown according to the invention when compared to kidney organoids grown according to the reference protocols (e.g. the Takasato protocol or the Morizano protocol). Thus in an embodiment the kidney organoids grown according to the method of the invention have a lower expression of EMT and/or cartilage markers compared to kidney organoids cultured under conditions wherein the second differentiation step comprises providing FGF9, FGF20 or the FGFR1 and/or FGFR3 activator for fewer than 8 days after onset of the 3D culture. Typical EMT and cartilage markers are COL2A1 , SOX9, Aggrecan, COL1A1 COL10, Vimentin and/or ACTA2, although other markers are known in the field. Thus in an embodiment the EMT and/or cartilage markers are selected from COL2A1 , SOX9, Aggrecan, COL1A1 , COL10, Vimentin and/or ACTA2. The skilled person is aware how to determine markers in an organoid, e.g. immunohistochemistry staining of markers can be used to determine number of cells within the organoid expressing a certain marker, while qPCR may be used to determine overall expression levels in the organoid or isolated cells to compare expression levels of the markers. The EMT or cartilage markers are preferably determined on day 20 or later after onset of the 3D culture stage, e.g. day 21 , 22, 23, 24, 25 or later after onset of the 3D culture stage.
In a second aspect the invention relates to an organoid obtained or obtainable by the method according to the first aspect of the invention. Organoids produced according to the method of the invention differ from organoids grown according to the reference protocols in that they display reduced expression of EMT and/or cartilage markers. Therefore in an embodiment the organoids according to the second aspect of the invention has reduced expression of EMT and/or cartilage markers. In a preferred
embodiment the organoid has reduced expression of a EMT and/or cartilage marker selected from COL2A1 , SOX9, Aggrecan, COL1A1 , COL10, Vimentin and/or ACTA2. In a third aspect the invention relates to an organoid according to the second aspect of the invention for use as a medicament. Alternatively the invention relates to a method of treatment, the method comprising providing an organoid as defined herein to a subject in need thereof.
More specifically, the invention relates to an organoid according to the second aspect of the invention for use in the treatment of a kidney disease. In a preferred embodiment the kidney disease is chronic kidney disease. Alternatively the invention relates to a method of treating a kidney disease in a subject, the method comprising providing an organoid as defined herein to the subject.
In a further aspect the invention relates to the in vitro or ex vivo use of an organoid according to the second aspect of the invention for one or more of:
- target identification for a drug;
- target validating for a drug;
- performing safety studies for a drug;
- performing pharmacodynamics studies for a drug;
- drug development;
- drug discovery;
- performing stratification studies for a drug;
- predicting an individual patient’s response to a drug;
- modelling a disease;
- modelling development; or
- studying kidney biology.
In a fifth aspect the invention relates to a method for differentiating intermediate mesoderm cells under conditions that allow for the formation of an immature kidney organoid expressing ECAD, LTL, and NPHS1 , wherein the cells are cultured in a 3D culture environment, and wherein the method is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture.
In a sixth aspect the invention relates to an immature kidney organoid obtained or obtainable by the method according to the fifth aspect of the invention.
Sequences described in the application:
SEQ ID NO: 1 (FGF9) maplgevgny fgvqdavpfg nvpvlpvdsp vllsdhlgqs eagglprgpa vtdldhlkgi Irrrqlycrt gfhleifpng tiqgtrkdhs rfgilefisi avglvsirgv dsglylgmne kgelygsekl tqecvfreqf eenwyntyss nlykhvdtgr ryyvalnkdg tpregtrtkr hqkfthflpr pvdpdkvpel ykdilsqs
SEQ ID NO: 2 (FGF20)
MAPLAEVGGF LGGLEGLGQQ VGSHFLLPPA GERPPLLGER RSAAERSARG GPGAAQLAHL HGILRRRQLY CRTGFHLQIL PDGSVQGTRQ DHSLFGILE F I SVAVGLVS I RGVDSGLYLG MNDKGELYGS EKLTSECI FR EQFEENWYNT YSSNIYKHGD TGRRY FVALN KDGTPRDGAR SKRHQKFTHF LPRPVDPERV PELYKDLLMY T
SEQ ID NO: 3 (FGF2) mvgvgggdve dvtprpggcq isgrgargcn gipgaaawea alprrrprrh psvnprsraa gsprtrgrrt eerpsgsrlg drgrgralpg grlggrgrgr apervggrgr grgtaapraa paargsrpgp agtmaagsit tlpalpedgg sgafppghfk dpkrlyckng gfflrihpdg rvdgvreksd phiklqlqae ergvvsikgv canrylamke dgrllaskcv tdecffferl esnnyntyrs rkytswyval krtgqyklgs ktgpgqkail flpmsaks
Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention.
Reference Example 1 - Reference protocol
Kidney organoids were generated at the air liquid interface using previously described protocol (Takasato, M. et al. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564-568 (2015)). Briefly, 105 cells were seeded in a 6-well plate and cultured in Stemdiff APEL2 medium (StemCell Technologies), supplemented with 1 % Protein-Free Hybridoma Medium II (ThermoFischer Scientific) and 1% antibiotic-antimycotic, and treated with CHIR99021 (8pM GSK-3 inhibitor, R&D system) and FGF9 (fibroblast growth factor 9, 200ng/ml, R&D system)/heparin (1 pg/ml, Sigma-Aldrich) cocktail. After 7 days of differentiation
(denoted day 7+0), cells were treated with CHIR99021 for 1 h and trypsinized. 500000 cells were aggregated by centrifugation at 425 RCF and cultured at the air-liquid interface on transwell tissue culture plates 1 pm pore polyester membrane inserts (CelIQart, Sabeu). Organoids are kept in complete Stemdiff APEL2 medium with a FGF9/heparin cocktail for 5 additional days (denoted day 7+5), after which the cocktail was removed. Kidney organoids were them cultures until day 7+25 and 7+32 in complete Stemdiff APEL2 medium. The protocol is schematically depicted in Figure 1 , bottom part.
4 days of CHIR99021 resulted in the induction of both the ureteric epithelium and the metanephric mesenchyme in monolayer culture and is followed by 3 days of FGF9/heparin. Maximal nephron number per organoid required a pulse of CHIR99021 for one hour. The continued presence of FGF9 after this CHIR99021 pulse was essential for nephrogenesis.
Example 2 - materials and method use in the experiments
Culture and maintenance of human induced pluripotent stem cells (hiPSCs)
The hiPSC line LUMC0072iCTRL01 was provided by the hiPSC core facility at the U.M.C. Leiden (the Netherlands). Cells are cultured in 6-well plates coated with vitronectin (Fisher Scientific) using E8 medium (Thermo Fisher Scientific) supplemented with 1% penicillin-streptomycin (Gibco). The medium was refreshed daily and the cells were passaged twice weekly using TrypLE dissociation reagent (Invitrogen). After passaging, cells were seeded in the presence of RevitaCell (Fisher Scientific) overnight. Pluripotency and karyotype of the iPSCs were verified. iPSC-derived kidney organoid generation and treatment
Kidney organoids were generated at the air-liquid interface using a the Takasato protocol. Briefly, 100,000 cells were seeded in a vitronectin-coated 6-well plate. After 24 h, regular E8 medium was replaced by STEMdiff APEL2 medium (STEMCELL Technologies) supplemented with 1% protein-free hybridoma medium II (PFHMII) (Fisher Scientific), 1% antibiotic-antimycotic (AA), and 8 pM CHIR99021 for 4 days to induce differentiation. This step was followed by an incubation with 200 ng/mL fibroblast growth factor 9 (FGF9, R&D Systems), and 1 pg/ml heparin (Sigma-Aldrich) for 3 days. After these 7 days of differentiation in 2D (denoted as day 7+0), cells were
treated with CHIR99021 for 1 h, trypsinised and counted. 500,000 cells were centrifuged at 425 RCF to form aggregates and placed on polyester membrane cell culture inserts with 1 pm pores (CelIQART) to create a 3D culture at the air-liquid interface. The organoids were cultured in complete STEMdiff APEL2 medium supplemented with 200 ng/ml FGF9 and 1 pg/ml heparin for 5 additional days (denoted as day 7+5), after which the cocktail of growth factors was removed (Fig. 1A). Kidney organoids were then cultured until day 7+25 or day 7+32 in STEMdiff APEL2 medium supplemented with 1% PHFMII and 1% AA at 37 °C and 5% CO2.
To assess the impact of a prolonged FGF9 treatment on the organoids, 200 ng/ml FGF9 (without the simultaneous addition of heparin) was added from day 7+5 to day 7+12. Beginning on day 7+12, the organoids were maintained in complete STEMdiff APEL2 medium supplemented with 1 % PHFMII and 1 % AA until day 7+25 or day 7+32.
Alcian blue staining of whole kidney organoids
To assess the onset of cartilage, the kidney organoids were fixed on days 7+18, 7+25 or 7+32 on the cell culture inserts in 70% ethanol overnight (ON) at 4°C. They were then incubated at room temperature (RT) for 1 h in 95% ethanol followed by acetone overnight. Alcian blue (0.03% (w/v)) diluted in 20% acetic acid was then added to the organoids for 4 h at RT followed by a rinse in 1% potassium hydroxide (KOH) (w/v) until the tissue was transparent. The organoids were stored in glycerol at 4°C before imaging on a Nikon SMZ25 automated stereomicroscope equipped with a 1 * objective and a PHOTONIC LED-Set Ringlight stereomicroscope (SMZ25, Nikon). Z stack images were taken every 15 pm, images were analyzed using NIS-Elements software (version 5.30.06, Nikon).
Gelatin/sucrose embedding of organoids for cryosections
On days 7+18, 7+25, or 7+32, kidney organoids were incubated for 20 min in 2% (v/v) paraformaldehyde diluted in phosphate-buffered saline (PBS) at 4°C. After a rinse in PBS, organoids were incubated in 15% (w/v) sucrose solution (in 0.1 M phosphate buffer pH 7.4) at 4°C for 24 h under agitation. The organoids are then transferred for 48 h into a 30% (w/v) sucrose solution at 4°C and set in a cryomold with the freezing buffer (7.5% (w/v) gelatin and 15% (w/v) sucrose in 0.1 M phosphate buffer). After letting the freezing medium harden on ice, the organoids were snap-frozen in liquid
nitrogen. Cryosections were cut 16 m thick on a CM3050S cryostat (Leica), mounted on Superfrost slides (Fisher Scientific), and used for immunostaining or Alcian blue staining. To remove the gelatin/sucrose embedding before any staining, cryosections were placed in PBS for 15 min at 37°C.
Alcian blue and Toluidine Blue staining on sections
After removal of embedding and a quick wash in distilled water, the sections were incubated for 3 min in 3% (v/v) acetic acid diluted in water followed by incubation in a 1% (w/v) Alcian blue diluted in 3% acetic acid for 30 min at RT protected from light. After washing, samples were stained for 3 min with a nuclear fast red solution (Sigma- Aldrich). Slides were dehydrated and mounted in Ultrakitt mounting medium (VWR). For Toluidine Blue, ultrathin sections are dried and incubated for 30 s with one drop of staining consisting of 1 % Toluidine Blue and 1% Sodium tetraborate diluted in milliCi water. After a rinse, slides are mounted. Imaging was performed on an inverted Nikon Ti-S/L100 microscope, equipped with a Nikon DS-Ri2 camera using a CFI Plan Apochromat K 20xobjective (NA: 0.75, WD: 1.0). Images were analyzed using NIS- Elements software (version 5.30.06, Nikon).
Immunofluorescence
After embedding removal and in order to assess the renal structures, the slides were incubated for 20 min at RT in a blocking buffer containing 10% bovine serum albumin (BSA), 0.1 M glycine and 0.2% Tween-20 in PBS. Primary antibodies (Table 1), diluted in PBS with 1% BSA, 0.1 M glycine and 0.2% Tween-20, were incubated overnight at 4°C. After rinsing in PBS with 0.2% Tween-20, the slides were incubated with secondary antibodies diluted in PBS with 1% BSA, 0.1 M glycine and 0.2% Tween-20 for 1 h at RT. Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI, 0.1 pg/ml) in PBS with 0.2% Tween-20 for 5 min. Slides were mounted using Dako fluorescence mounting medium (Agilent Technologies). Imaging was performed on an automated inverted Nikon Ti-E microscope, equipped with a Lumencor Spectra light source, an Ador Zyla 5.5 sCMOS camera, and an MCL NANO Z200-N Tl z-stage. The objective used was CFI PLAN APO LBDA 10* 0.45/4 mm. Images were analyzed using NIS-Elements software (version 5.30.06, Nikon).
Western blot
Proteins were extracted from the kidney organoids using TRIzol (Invitrogen) as described by the manufacturer. After performing a bicinchoninic acid protein assay (Pierce), 15 pg of protein was loaded per well of an 8% or 10% acrylamide gel. Migration was performed at 120 V in a migration buffer (pH 8.3) consisting of 25 mM Tris-Base, 192 mM glycine, and 0.1 % sodium dodecylsulfate (SDS) (Bio-Rad). Transfer to nitrocellulose membrane was performed at 4 C for 90 min in a transfer buffer consisting of 50 mM Tris, 40 mM glycine, and 1.5 mM SDS at a constant amperage of 350 mA. The membranes were then incubated in a blocking solution consisting of Tris-buffered saline (TBS) added with 5% BSA and 0.1% Tween-20 for 1 h at RT. Primary antibodies diluted in blocking solution were incubated overnight at 4°C. Primary antibodies used were the following: anti-SOX-9 (1/1000, Cell Signaling Technology, 82630), anti-GAPDH (1/10000, Cell Signaling Technology, 2118), anti- vimentin (1/500, Thermo Fisher Scientific, MA5-16409), anti-a-SMA (1/1000, Cell Signaling, 19245S) and anti-COL2 (1/1000; Abeam, ab34712). Membranes were rinsed twice in TBS with 0.1 % Tween and incubated for 1 h with secondary antibodies coupled to a horseradish peroxidase (HRP) (1/3000; BioRad) at RT. After two additional washes, the membranes were developed using a chemiluminescence substrate (Clarity Western ECL Substrate, Bio-Rad) and detected for 10 s to 5 min on CL-Xposure film (Thermo Fisher Scientific) or using a Chemidoc (Bio-Rad). Films were scanned and the protein bands were quantified by measuring density via Imaged software (National Institutes of Health, Imaged 1.53e). GAPDH was used as the reference protein for the normalization of the proteins of interest.
RNA extraction
Kidney organoids were manually crushed and lysed in TRIzol (Invitrogen) and mixed with chloroform (200 pl/ml Trizol). After homogenization, the samples were incubated for 15 min at RT followed by a centrifugation at 18000 RCF for 15 min at 4°C. The clear supernatant was collected, and isopropanol (500 pl/ml Trizol) was added to precipitate the RNA. The samples were homogenized by vortexing and incubated for 15-20 min at RT and centrifuged again at 4°C and 18000 RCF for 15 min. The RNA pellets were then washed several times with ethanol. After centrifugation at 4°C and
6800 RCF for 5 min, the ethanol was removed, and the pellet was air-dried before being eluted in RNAse-free water (Qiagen).
Reverse transcription and qPCR
Reverse transcription was performed using 500 ng of RNA mixed with water, reverse transcription enzyme and iScript buffer (BioRad) in a final volume of 20 pl. The samples were heated for 5 min at 25°C for priming followed by 20 min at 46°C for reverse transcription and 1 min at 95°C for enzyme inactivation. The obtained cDNA was diluted five times in RNAse-free water, and 2 pl were prepared for qPCR with a reaction mix comprising 250 nM of each primer (Table 2), 4 pl of water and 10 pl SYBR (Bio-Rad) Green Master Mix. The real-time PCR CFX96 (Bio-Rad) was programmed to perform 40 cycles of 2-steps amplification consisting of 10 s of denaturation at 95°C, and 60 s of combined annealing and extension at 60°C. Experiments were performed in technical duplicate and normalized to GAPDH as a housekeeping gene.
Statistics
All data are expressed as the mean ± standard deviation (SD). Experiments were performed at least 3 independent times (N=3). Normality was checked using the Shapiro-Wilk normality test. Significant differences (p < 0.05) between groups were assessed using a Student T-test (2 groups) or a two-way ANOVA followed by a Tukey- Kramer post hoc ANOVA test (> 3 groups). Nonparametric data were analyzed using a Mann- Whitney or Kruskal-Wallis test.
Example 3 - Results
Non-renal cell populations appeared between day 7+18 and 7+25 of kidney organoid culture
To induce the formation of kidney organoids from iPSCs, we used a protocol comprising 2D and 3D differentiation steps as illustrated in Figure 4A. We maintained the organoids in culture for up to 7+25 days. While organoids at day 7+18 presented a good overall shape (Fig. 1 B), we observed that their shape became less regular by day 7+25 (Fig. 40). Upon assessing the consequence of the prolonged culture on the development of renal structures, we observed that at day 7+18, the kidney organoids
comprised glomeruli (NPHS1), tubules (ECAD), proximal tubules (LTL) (Fig. 4D) as well as loops of Henle (SLC12A1) and the expected stromal population (MEIS) (Fig. 4E). By day 7+25, the kidney organoids still showed positive immunostaining for tubules, glomeruli, and loops of Henle, but also showed the appearance of an off- target population absent of renal markers (asterisks in Fig. 4F and 4G).
The non-renal cell population was identified as cartilage progressively developing over time in culture
As described previously in the literature (Nam, S.A. etal. Exp Mol Med 51, 1-13 (2019); Bantounas, I. et al. Stem Cell Reports 10, 766-779 (2018)), we showed that the predominant off-target cell population observed at day 7+25 is cartilage. While Alcian blue staining of the whole kidney organoid and cryosections did not show cartilage in organoids at day 7+18 (Fig. 5A and 5B), by day 7+25 the staining revealed abundant cartilage in the non-renal cell population (Fig. 5C and 5D). This observation was correlated with increased expression of the cartilage-related transcripts, S0X9 (p < 0.05), COL2A1 (p < 0,001), ACAN (p < 0.01), COL1A1 (p < 0.001), and COL10A 1 (p < 0.01) in day 7+25 kidney organoids measured by qPCR (Fig. 5E-I). Some of these markers, such as COL2A1 and SOX-9, were also assessed at the protein level using western blotting (Fig. 5J). COL2A1 protein level presented a 3.5 times increase (p < 0.01) at day 7+25 compared to day 7+18 (Fig. 5J and 5L), whereas SOX-9 levels remained unchanged (Fig. 5J and 5K).
At day 7+25, cartilage did not form in FGF9-treated kidney organoids
With the aim to prevent the cartilage formation detected at day 7+25, FGF9 without heparin was added from day 7+5 until day 7+12 (Fig. 6A). Organoids that underwent this treatment are referred to as FGF9-treated organoids (day 7+25+FGF9), while the organoids cultured in regular conditions are referred to as control organoids (day 7+25). Using Alcian blue to stain the whole organoids and cryosections, we observed that the cartilage present in control organoids (Fig. 6B and 6C) was absent in the FGF9-treated organoids at day 7+25 (Fig. 6D and 6E). At the molecular level, S0X9, ACAN, COL2A1, COL1A 1, and COL10 mRNA expression were also decreased (Fig. 7A-E, p < 0.01). A three times decrease of SOX9 protein levels was also observed in FGF9-treated organoids compared to control organoids at day 7+25 (Fig. 7F and G, p
< 0.01), associated with a four times decrease of COL2 protein levels (Fig. 7F and H, p < 0.01).
FGF9-treated organoids correctly developed renal structures
To ensure the FGF9 treatment had no negative impact on the development of renal structures in kidney organoids, cryosections were stained for renal markers (Fig. 8A- D): NPHS1 (glomeruli), ECAD (distal tubules), LTL (proximal tubules) and SLC12A1 (loops of Henle). Unlike the control organoids, we found that FGF9-treated organoids at day 7+25 possessed typical renal structures (Fig. 8C and 8D). Indeed, specifically SLC12A1 was hardly present by day 7+25 but the FGF9 treatment restored it to levels similar to day 7+18. This observation correlated with the expression of the renal structures’ markers measured by qPCR, showing no detrimental impact of the FGF9 treatment in organoids at day 7+25 (Fig. 10). This result suggests that the phenotype of FGF9-treated organoids at day 7+25 is close to the one observed in control organoids at day 7+18.
FGF9-treated kidney organoids developed vessel-like structures
While control organoids do not contain vessels at day 7+25 (Fig. 8E), FGF9-treated organoids developed vessel-like structures, as observed by toluidine blue staining (Fig. 8F). This observation was correlated with an increased level of the vascularization marker CD31 (PECAM1) in immunofluorescence (Fig. 8G and 8H) and PECAM1 expression measured by qPCR (Fig. 11) in FGF9-treated organoids compared to control organoids.
FGF9-treated organoids at day 7+25 showed low levels of EMT
We assessed the levels of EMT markers vimentin and a-SMA at days 7+5, +10, +14, + 18, +25 and +25+FGF9. We observed that EMT markers increased from day 7+5 until day 7+25 in control organoids. FGF9-treated organoids at day 7+25 had levels of vimentin and a-SMA similar to day 7+5, showing that EMT was halted with the treatment (Fig. 9A-C).
Cartilage was found in FGF9-treated organoids at day 7+32
To assess if the FGF9 treatment allows kidney organoids to be kept longer in culture with no off-target appearance, we maintained control and FGF9 organoids in culture for 32 days after aggregation (7+32). In the control organoids, we observed that cartilage formation continued to increase from day 7+25 to day 7+32 (Fig. 11A and 11 B). In the FGF9-treated organoids at day 7+32, we did not observe the appearance of large islands of cartilage in Alcian blue-stained whole organoids (Fig. 11 C) , but we did notice small islands of cartilage at the organoid edges (Fig. 11 D), albeit less than control organoids at day 7+25. Moreover, the expression of S0X9 (p < 0.001), COL2A 1 (p < 0.001), ACAN (p < 0.001), and COL10A 1 (p < 0.001) (Fig. 11 E-I) was decreased in FGF9-treated organoids compared to control organoids. These findings were confirmed by COL2A1 protein levels in western blots (Fig. 11 F).
Table 1 : Primary and secondary antibodies used for immunofluorescence
Table 2: Primers used in qPCR
Primers Sequences forward (F) and reverse (R) 5' to 3’
F: CTGGGCTACACTGAGCACC
GAPDH
R: AAGTGGTCGTTGAGGGCAATG
F: GAGCCGAAAGCGGAGCTGGAA
S0X9
R: ACAGCTGCCCGCTCCAAGTG
F: AACCAGATTGAGAGCATCCG
C0L2A1
R: ACCTTCATGGCGTCCAAG
F: TCGAGGACAGCGAGGCC
AC AN
R: TCGAGGGTGTAGCGTGTAGAGA
F: CGGTGGTTTCTTGGTCGGT
C0L1A1
R: GTGCGATGACGTGATCTGTGA
F: AAGAATGGCACCCCTGTAATGT
COL10A1
R: ACTCCCTGAAGCCTGATCCA
F: TCCACCCGTTGAGTGTGTG
CUBN
R: AGGAACCTAGAGTTGAGGAGC
F: TCACCGTGAATGTTCTGTTCC
NPHS1
R: AGTGTGGCTAAGGGATTACCC
F: AGTGCCCAGTAATACCAATCGC
SLC12A1
R: GCCTAAAGCTGATTCTGAGTCTT
F: GGGCATGGATGGAGTAGGC
MEIS
R: GGGTACTGATGCGAGTGCAG
F: AACAGTGTTGACATGAAGAGCC
PECAM1
R: TGTAAAACAGCACGTCATCCTT
Discussion
With the increasing burden of renal disease and the lack of donor kidneys, the possibility of creating kidney organoids derived from human iPSCs for transplantation is a promising therapeutic approach. However, despite their ability to recapitulate renal structures and function, several drawbacks prevent their translation to the clinic. For example, the emergence of off-target differentiation products such as cartilage remains a major issue as it profoundly disrupts organoid structure. In this study, we aimed to adjust the differentiation protocol developed by Takasato et al. in order to prevent the appearance of cartilage in vitro. We found that extending the FGF9 incubation from day 7+0 to day 7+12 after aggregation at the air-liquid interface significantly reduced the off-target cartilage until day 7+25. This presence of cartilage was assessed with Alcian blue staining and the measurement of cartilage markers, such as SOX9 and COL2A1 , in qPCR and western blot. Importantly, this modified protocol did not negatively affect renal structures as evaluated by immunofluorescence and PCR. Indeed, the organoids still presented nephron and tubular structures, with an increased number of vessel-like structures.
Example 4 - FGF20
Next the inventors tested if other activators of FGFR1 and/or FGFR3 such as FGF20 can replace FGF9 in the above protocol.
At day 7+25, cartilage did not form in FGF20-treated kidney organoids
With the aim to prevent the cartilage formation detected at day 7+25, FGF20 without or with heparin was added from day 7+5 until day 7+12. Organoids that underwent this treatment are referred to as FGF20-treated organoids (day 7+25+FGF20 or day 7+25+FGF20+heparin), while the organoids cultured in regular conditions are referred to as control organoids (day 7+25). Using Alcian blue to stain the whole organoids and cryosections, we observed that the cartilage present in control organoids (Fig. 12 left panel) was absent in the FGF20-treated organoids at day 7+25 (Fig. 12 middle and right panels).
FGF20-treated organoids correctly developed renal structures
To ensure the FGF20 treatment had no negative impact on the development of renal structures in kidney organoids, cryosections were stained for renal markers (Fig. 13): NPHS1 (glomeruli), ECAD (distal tubules), and LTL (proximal tubules). Unlike the control organoids, we found that FGF20-treated organoids at day 7+25 possessed typical renal structures (Fig. 13). This result suggests that the phenotype of FGF9- treated organoids at day 7+25 can also be achieved with a different activator of FGFR1 and/or FGFR3 such as FGF20. Treating organoids with FGF20 until day 5+12 resulted in less cartilage formation as organoids cultured using common protocols (treating with FGF9 until day 7+5).
Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.
All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.
Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the
art (including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.
Claims
1. A method for differentiating stem cells to kidney organoids, the method comprising a first differentiation stage and a second differentiation stage, wherein the first differentiation stage comprises culturing the stem cells under conditions to allow differentiation of the stem cells to a cell culture comprising cells in the intermediate mesoderm stage, wherein the second differentiation stage comprises transferring the cell culture comprising cells in the intermediate mesoderm stage to a 3D culture environment and culturing the cells under conditions that allow the formation of a kidney organoid, wherein the second differentiation stage is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture, followed by culturing in growth factor free medium.
2. Method according to claim 1 wherein the second differentiation stage is performed for at least 20 days, preferably at least 21 , 22, 23, 24, or at least 25 days.
3. Method according to claim 1 or 2, wherein the FGFR1 and/or FGFR3 activator is selected from FGF20, FGF2, or Smoothened agonist (SAG).
4. Method according to any one of the preceding claims wherein the second differentiation step is characterized in that FGF9, FGF20 or an activator of FGFR1 and/or FGFR3 is provided for 9 to 15 days after onset of the 3D culture, preferably 10 to 14 days, more preferably 11 to 13 days.
5. Method according to any one of the preceding claims wherein in the second differentiation stage SAG is provided in addition to FGF9 or FGF20, preferably wherein SAG is provided for the first 2 to 4 days of the second differentiation stage.
6. Method according to any one of the preceding claims wherein the cells in the intermediate mesoderm stage comprise at least one of: ureteric epithelium, metanephric mesenchyme, progenitors of renal interstitium or endothelium.
7. Method according to any one of the preceding claims wherein the 3D culture environment is selected from culturing on a trans-well membrane, culturing in a low attachment well, culturing in a hydrogel, culturing on an air-liquid interface culturing, culturing on a scaffold, or suspension culture.
8. Method according to any one of the preceding claims wherein the kidney organoids have a lower expression of EMT and/or cartilage markers compared to kidney organoids cultured under conditions wherein the second differentiation step comprises providing FGF9, FGF20 or the FGFR1 and/or FGFR3 activator for fewer than 8 days after onset of the 3D culture.
9. Method according to claim 8 wherein the EMT or cartilage markers are selected from COL2A1 , SOX9, Aggrecan, COL1A1 COL10, Vimentin and/or ACTA2.
10. Method according to any one of the preceding claims, wherein the first differentiation step comprises:
(A) culturing the stem cells under conditions allowing induction of ureteric epithelium cells and metanephric mesenchyme cells, preferably wherein the ureteric epithelium cells are cells expressing PAX2, GATA3, and CDH1 and the metanephric mesenchyme cells are cells expressing PAX2, Wnt4, and BMP7, more preferably wherein the cells are cultured in the presence of a GSK3 inhibitor or a WNT activator; and
(B) differentiating the ureteric epithelium cells in nephron progenitor cells, preferably wherein the nephron progenitor cells are cells expressing SIX2, HOXD11 , WT1 , PAX2, more preferably culturing the cells in the presence of FGF9, FGF20 or an FGFR1 and/or FGFR3 activator.
11. Method according to any one of the preceding claims, wherein the second differentiation step comprises:
(C) culturing the cells under conditions that allow the formation of an immature kidney organoid in the presence of FGF9, FGF20 or the FGFR1 and/or FGFR3 activator, wherein the immature kidney organoid expresses ECAD, LTL, and NPHS1 , and
(D) culturing the immature kidney organoid in the absence of growth factors to obtain a kidney organoid, preferably wherein the kidney organoid differs from the immature kidney organoid in the increased expression of ECAD, LTL, NPHS1 , and SLC12A1.
12. Method according to claim 11 , wherein step (C) further comprises culturing the cell in the presence of Heparin for the first 3-7, preferably 4-6 days after onset of the 3D culture.
13. Organoid obtained or obtainable by the method according to any one of claims 1 to 12.
14. Organoid according to claim 13 for use as a medicament.
15. Organoid according to claim 13 for use in the treatment of a kidney disease, preferably wherein the kidney disease is chronic kidney disease.
16. The in vitro or ex vivo use of an organoid according to claim 13 for one or more of:
- target identification for a drug;
- target validating for a drug;
- performing safety studies for a drug;
- performing pharmacodynamics studies for a drug;
- drug development;
- drug discovery;
- performing stratification studies for a drug;
- predicting an individual patient’s response to a drug;
- modelling a disease;
- modelling development; or
- studying kidney biology.
17. A method for differentiating intermediate mesoderm cells under conditions that allow for the formation of an immature kidney organoid expressing ECAD, LTL, and NPHS1 , wherein the cells are cultured in a 3D culture environment, and wherein the method is characterized in that FGF9, FGF20, or an activator of FGFR1 and/or FGFR3, is provided for 8 to 16 days after onset of the 3D culture, followed by culturing in growth factor free medium.
18. Immature kidney organoid obtained or obtainable by the method according to claim 17.
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