WO2023118050A1

WO2023118050A1 - Use of novel markers to detect pluripotent stem cells

Info

Publication number: WO2023118050A1
Application number: PCT/EP2022/086855
Authority: WO
Inventors: Jonathan NICLIS; Josefine Rågård CHRISTIANSEN; Fabian Vinzenz ROSKE; Paschalis EFSTATHOPOULOS; Hanni WILLENBROCK
Original assignee: Novo Nordisk A/S
Priority date: 2021-12-21
Filing date: 2022-12-20
Publication date: 2023-06-29

Abstract

The present invention relates to a method of screening a cell population for undifferentiated stem cells by detecting the expression of one or more markers in the cell population, which expression is effectively downregulated as PSCs are differentiated into specialized cells of either of the three germ layers.

Description

USE OF NOVEL MARKERS TO DETECT PLURIPOTENT STEM CELLS

TECHNICAL FIELD

The present invention relates generally to the field of stem cells, such as human embryonic stem cells. Methods are provided for detecting pluripotent stem cells (PSCs) in an in vitro cell population of differentiated cells derived from PSCs.

BACKGROUND

The use of stem cells in medicine is being intensively pursued with the prospect of alleviating, potentially reversing and/or curing conditions for which only limited or no treatment is available today. The stem cell products for such treatment may be derived from human pluripotent stem cells (hPSCs) such as but not limited to embryonic stem cells or induced PSCs. Human PSCs are largely undifferentiated cells with the potential to proliferate and differentiate into a number of more specialized cells of the human body. Established methods for obtaining stem cell-derived differentiated cells for the treatment of various conditions have already been developed, including protocols for providing ventral midbrain neural cells, retinal pigment epithelium (RPE) cells, neural retina cells, pancreatic islets containing beta cells, and cardiomyocytes. Such protocols, however, are typically not completely efficient and often result in a cell population comprising the intended cells as well as other cell types that may or may not be suitable of use in a final medicinal product. Furthermore, for some treatments it may not be viable to administer the fully differentiated or matured cells. In these cases, the differentiation of the cells is not fully completed in vitro as the cells are then intended to further mature in vivo after administration into the patient. Depending on the level of maturity, the medicinal product may still contain some small fraction of cells in a mitotic stage with high capacity to proliferate. A stem cell-derived population wherein the differentiated cells have not fully matured may comprise a mixture of cells at various developmental stages. Even for cell populations derived according to a differentiation protocol for which fully matured cells are intended a subset of the cells may still be at a mitotic stage or may even be pluripotent.

When aiming to provide a patient-safe treatment it is undesirable if a stem cell- derived product for administration comprises PSCs and/or PSC-like cells with the inherent potential to proliferate and develop into almost any cell type. The major concern being the risk of uncontrollable proliferation of the cells, which could potentially develop into a teratoma or malignant tumor or a cancer-like state. Continued development of the differentiation protocols as well as optional purification processes may result in a highly pure cell population. Even still, to ensure patient safety and to comply with regulations by health authorities a quality control of stem cell- derived products is required for verifying that a product is not contaminated with residual undifferentiated cells, in particular PSCs or PSC-like cells.

Several genetic markers (and their coded proteins) are well characterized in human PSCs. As PSCs are differentiated into a specific germ layer and further into a more specialized cell type the gene expression of the cell will change. This suggests for using genetic markers to establish the type and maturity of the cell. Multiple markers identifying human PSCs are known. Depending on the cell type into which a PSC is differentiated many markers expressed at the pluripotent stage will to some extent downregulate. This can be utilized in identifying PSCs in a cell population of differentiated cells. However, the timing and extent of the expression of pluripotent markers being down regulated differs for different cell types making a generic method of detecting the PSCs difficult. Furthermore, it is known that well-established markers for pluripotency such as S0X2, NANOG, PODXL, CD9 and LIN28A may also be expressed in some cells that have differentiated and lost pluripotency.

It is therefore an objective of the present invention to overcome the aforementioned challenges, in particular it is an objective to provide a robust method to identify reliable markers to detect PSCs in a mixed cell population or a cell product or in vitro differentiation culture or tissue, with extremely high sensitivity meaning low limits of detection. It is further an objective of the present invention that the provided method may be applied across various protocols for obtaining different stem cell-derived products, i.e. applicable to multiple cell types spanning across the three germ layers and at various stages of maturity.

SUMMARY

The objectives as outlined above are achieved by the aspects of the present invention. In addition, the present invention may also solve further problems, which will be apparent from the disclosure of the exemplary embodiments.

In a first aspect of the present invention is provided a method of screening a cell population for undifferentiated stem cells comprising the step of detecting the expression of one or more markers in the cell population, wherein the marker is selected from UNC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1. The present inventors have found that the expression of these particular markers are downregulated as the stem cells lose pluripotency. This holds true for the differentiation of the PSCs into a variety of different cell types that span the three different germ layers (endoderm, mesoderm, ectoderm), which makes the method highly suitable for generic testing of stem cell derived products. The particular markers are downregulated to a level that results in a very low number of false positive.

In another aspect is provided a cell population comprising differentiated cells derived from PSCs, wherein the cell population is devoid of cells expressing one or more markers selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows combined tSNE plot of single cell sequencing results from several different cell types, every dot represents a single cell. Clusters 1-4 are endoderm/pancreatic islet cells. Clusters 5-7 and 18 are mesoderm/cardiomyocyte cells. Cluster 8 is ectoderm/ retinal pigment epithelial cells. Clusters 9-14 are undifferentiated/pluripotent stem cells. Clusters 15-17 are mesoderm/mesenchymal stem cells. Clusters 19-21 are ectoderm/ventral midbrain neural stem cells.

Figures 2-4 show gene expression distribution of cardinal lineage and cell type markers throughout multi-cell type combined dataset. Expression shown for the pluripotency gene POLI5F1 (Figure 2), retinal epithelial cell gene LHX2 (Figure 3), and ventral midbrain neural stem cell gene EN-1 (Figure 4).

Figures 5-7 show gene expression distribution of cardinal lineage and/or cell type markers throughout multi-cell type combined dataset. Expression shown for the cardiomyocyte gene NKX2.5 (Figure 5), mesenchymal stem cell gene NT5E (Figure 6), and pancreatic islets gene NKX2.2 (Figure 7).

Figures 8-10 show gene expression distribution of cardinal undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the marker LIN28A (Figure 8), NANOG (Figure 9), and POLI5F1 (Figure 10). In each figure, A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster. Figures 11-13 show gene expression distribution of cardinal undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene PODXL (Figure 11), SOX2 (Figure 12), and CD9 (Figure 13). In each figure, A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.

Figures 14-17 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene DNMT3B (Figure 14), LINC00678 (Figure 15), USP44 (Figure 16) and LINC00458 (Figure 17). In each figure, A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.

Figures 18-21 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene CNMD (Figure 18), POL3G (Figure 19), AC009446.1 (Figure 20), and SCGB3A2 (Figure 21). In each figure, A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.

Figures 22-25 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene VRTN (Figure 22), ZIC2 (Figure 23), CRABP1 (Figure 24), and CYP2S1 (Figure 25). In each figure, A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.

Figures 26-29 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene ALPL (Figure 26), AL353747.4 (Figure 27), VASH2 (Figure 28), and TNNT1 (Figure 29). In each figure, A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster. Figures 30-33 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene L1TD1 (Figure 30), GAL (Figure 31), DPPA4 (Figure 32), and TDGF1 (Figure 33). In each figure, A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.

Figures 34-37 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene FOXH1 (Figure 34), CLDN6 (Figure 35), ZFP42 (Figure 36), and CLDN7 (Figure 37). In each figure, A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.

Figures 38-40 show gene expression distribution of novel undifferentiated lineage/ pluripotent stem cell markers identified from computational investigation and shown throughout multi-cell type combined dataset. Expression shown for the gene SFRP2 (Figure 38), HSALNG0067850 (Figure 39), and AC104461.1 (Figure 40). In each figure, A are violin plots showing the expression of the marker by each group of clusters and B is a tSNE plot of all digitally merged samples showing the intensity of expression of the specific marker in each cluster.

Figures 41-44 show examples of real time qPCR results showing the mRNA expression of a selection of markers in undifferentiated hPSCs and in various differentiated cell types that represent the 3 germ layers. Expression shown for the gene SCGB3A2 (Figure 41), CYP2S1 (Figure 42), LINC00458 (Figure 43), and L1TD1 (Figure 44).

Figures 45-48 show examples of real time qPCR results showing the mRNA expression of a selection of markers in undifferentiated hPSCs and in various differentiated cell types that represent the 3 germ layers. Expression shown for the gene VRTN (Figure 45), ZFP42 (Figure 46), ALPL (Figure 47), and LIN00678(Figure 48).

Figure 49 shows results of real time qPCR showing OCT4 (POLI5F1) and VRTN genes expression in D60 hESC-RPE spiked-in with different concentrations of E1C3, hES. The data are represented as fold change compared to hES-RPEs after being normalised to ACTB expression. The VRTN fold change curve shows better linearity up to 0.001% compared to the classic pluripotency gene OCT4 and can be used for detection of pluripotent cells in the RPE drug product with a LoD of 0.001%. Figure 50 shows a histogram of mean copies/pl normalized to the input cDNA per sample. The numbers on the x axis refer to the percentage of stem cells spiked in the surrogate RPE cells. The error bars are constructed using standard error from the mean of biological repeats (n=6 for all samples). Note y axis is in logarithmic scale to allow visualization of output signal for each sample.

DESCRIPTION

Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology and pharmacology, known to those skilled in the art.

It is noted that all headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Throughout this application the terms “method” and “protocol” when referring to processes for differentiating cells may be used interchangeably. As used herein, “a” or “an” or “the” can mean one or more than one. Unless otherwise indicated in the specification, terms presented in singular form also include the plural situation. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

In general and unless otherwise stated “day 0” refers to the initiation of the protocol, this be by for example but not limited to plating the stem cells or transferring the stem cells to an incubator or contacting the stem cells in their current cell culture medium with a compound prior to transfer of the stem cells. Typically, the initiation of the protocol will be by transferring undifferentiated stem cells to a different cell culture medium and/or container such as but not limited to by plating or incubating, and/or with the first contacting of the undifferentiated stem cells with a compound that affects the undifferentiated stem cells in such a way that a differentiation process is initiated.

Hereinafter, the methods according to the present invention are described in more detail by non-limiting embodiments and examples. A method is provided for screening a cell population for PSCs or PSC-like cells. By “pluripotent stem cell” (PSC) is to be understood an undifferentiated cell having differentiation potency and proliferative capacity (particularly self-renewal competence) but maintaining differentiation potency. Throughout this patent application pluripotent stem cells, undifferentiated stem cells or undifferentiated pluripotent stem cells may be used interchangeably. The stem cell includes subpopulations such as PSC, multipotent stem cell, unipotent stem cell and the like according to the differentiation potency. PSC refers to a stem cell capable of being cultured in vitro and having a potency to differentiate into any cell lineage belonging to three germ layers (ectoderm, mesoderm, endoderm). The multipotent stem cell means a stem cell having a potency to differentiate into plural types of tissues or cells, though not all kinds. The unipotent stem cell means a stem cell having a potency to differentiate into a particular tissue or cell. A PSC can be induced from fertilized egg, clone embryo, germ stem cell, stem cell in a tissue, somatic cell and the like. Examples of the PSC include embryonic stem cell (ES cell), EG cell (embryonic germ cell), induced pluripotent stem cell (iPSC) and the like. Muse cell (Multi-lineage differentiating stress enduring cell) obtained from mesenchymal stem cell (MSC), and GS cell produced from reproductive cell (e.g., testis) are also encompassed in the PSC. iPSCs are a type of PSC that can be generated directly from adult cells. By the introduction of products of specific sets of pluripotency-associated genes adult cells can be converted into PSCs. Embryonic stem cells can be produced by culturing cells from a blastomere or the inner cell mass of a blastocyst. Such cells can be obtained without the destruction of the embryo. Embryonic stem cells are available from given organizations and are also commercially available.

As used herein, cells before and after the purification step will be referred to as cell drug substance (DS) and cell drug product (DP), respectively.

In a first aspect of the present invention is provided a method of screening a cell population for undifferentiated stem cells comprising the step of detecting the expression of one or more markers in the cell population, wherein the marker is selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1. As used herein, the term “cell population” refers to a defined group of cells, which may be in vitro or in vivo. Typically, the group of cells will be isolated in vitro in a container. In a preferred embodiment, the method according to the present invention is carried out in vitro. In an embodiment, the in vitro container is a suitable substrate such as a microwell.

As used herein, the term “screening” refers to the action of examining the cell population for the presence of one or more cells having a certain genotype or phenotype, such as pluripotency. The genotype and phenotype may be established based on the expression of markers.

As used herein, the term “marker” or “markers” refers to a naturally occurring identifiable expression made by a cell, which can be correlated with certain properties of the cell. In a preferred embodiment the marker is a genetic or proteomic expression, which can be detected and correlated with the identity of the cell. The markers may be referred to by gene. This can readily be translated into the expression of the corresponding mRNA and proteins.

As used herein, the term “expression” in reference to a marker refers to the lack or presence in the cell of a molecule, which can be detected. In an embodiment, the expressed molecule is mRNA or a protein. Accordingly, in an embodiment the PSCs are detected, and optionally identified, on a transcriptomic and/or proteomic level. In an embodiment, the marker is the genetic expression of a gene, which can be correlated with pluripotency of a stem cell. The expression of the marker may be detected at any suitable level, such as at mRNA or protein level. A person skilled in the art will readily appreciate that a cell can be defined by the positive or negative expression of a marker, i.e. the properties and state of a cell may equally be correlated based on the expression of a certain marker as well as the lack thereof. When referring to specific markers the presence or lack of expression may be denoted with + (plus) or - (minus) signs, respectively.

As used herein, the term “detecting” in reference to expression means measuring a signal to establish the presence of undifferentiated stem cells in a cell population. “Detecting” according to the method does not imply that a positive signal must be obtained, which would not be the case if the cell population does not comprise any undifferentiated stem cells. Any suitable signal may be used to establish the presence of PSCs, such as by the emission of light from e.g. fluorescent molecules. Numerous techniques are readily available to detect and optionally identify markers in a cell population. In one embodiment, the cell population is screened using bulk RNA-seq (RNA sequencing) analysis. As used herein, the term “bulk” when referring to screening means analyzing the expression of a marker in a cell population not the individual cells. As used herein, “DNMT3B" refers to the gene denoted DNA Methyltransferase 3 Beta.

As used herein, “LINC00678" refers to the gene denoted Long Intergenic Non-protein Coding RNA 678.

As used herein, “USP44" refers to the gene denoted Ubiquitin Specific Peptidase 44.

As used herein, “LINC00458" refers to the gene denoted Long Intergenic Non-protein Coding RNA 458.

As used herein, “CNMD" refers to the Chondromodulin.

As used herein, “POLR3G" refers to the gene denoted RNA polymerase III subunit G.

As used herein, “AC009446.1" refers to the novel transcript also known as ENSG00000254277.

As used herein, “SCGB3A2” refers to the gene denoted Secretoglobin family 3A member 2.

As used herein, “VRTN" refers to the gene denoted Vertebrae development associated.

As used herein, “ZIC2’ refers to the gene denoted Zic family member 2.

As used herein “CRABPT’ refers to the gene denoted Cellular Retinoic Acid Binding Protein 1.

As used herein “CYP2S1” refers to the gene denoted Cytochrome P450 family 2 Subfamily S member 1.

As used herein “ALPL" refers to the gene denoted Alkaline Phosphatase, biomineralization associated.

As used herein “AL353747.4" refers to the novel transcript also known as ENSG00000280707.

As used herein “VASH2" refers to the gene denoted Vasohibin 2.

As used herein “TN NT1" refers to the gene denoted Troponin T1, slow skeletal type.

As used herein “L1TD1" refers to the gene denoted LINE1 type transposase domain containing 1.

As used herein “GAL" refers to the gene denoted Galanin and GMAP prepropeptide.

As used herein “SFRP2’ refers to the gene denoted Secreted Frizzled Related Protein 2.

As used herein “DPPA4" refers to the gene denoted Developmental Pluripotency Associated 4.

As used herein “TDGF1” refers to the gene denoted teratocarcinoma-derived growth factor 1.

As used herein “FOXHT’ refers to the gene denoted Forkhead box H1.

As used herein “CLDN6" refers to the gene denoted Claudin 6.

As used herein “ZFP42’ refers to the gene denoted ZFP42 zinc finger protein.

As used herein “CLDN7’ refers to the gene denoted Claudin 7. As used herein “AC104461.1” refers to the novel transcript also known as ENSG00000230623.

As used herein “AC0064802.1” refers to the novel transcript also known as ENSG00000254339.

In one embodiment, the expression of one or more markers selected from DNMT3B, LINC00678, USP44, CNMD, SFRP2, DPPA4, and TDGF1 is further detected. By “further detected” is meant that the expression of one or more markers is detected in a cell population in addition to the detection of the expression of other markers.

In one embodiment, the presence of undifferentiated stem cells in a cell population is established by the positive expression of either one of the markers using bulk analysis of the cell population. In a further embodiment, the bulk analysis is by RNA-seq analysis. In one embodiment a cell population or its supernatant is screened for a secreted product of markers according to the present invention.

In an embodiment, the cell population comprises differentiated cells derived from PSCs. As used herein, the term "differentiated cells" in respect to stem cells refers to PSCs, which have undergone a process wherein the cells have progressed from an undifferentiated state to a specific differentiated state, i.e. from an immature state to a less immature state or to a mature state. Changes in cell interaction and maturation occur as cells lose markers of undifferentiated cells or gain markers of differentiated cells. Loss or gain of a single marker can indicate that a cell has matured, partially differentiated or fully differentiated. “Differentiated cells” are therefore considered to be cells which have previously been classified as PSCs but allowed to differentiate into the cell type of a certain germ layer.

It follows that in an embodiment, the method comprises an initial step of differentiating PSCs into a cell population of differentiated cells derived from the PSCs. One of ordinary skill in the art will readily appreciate that as used herein the term "differentiating" refers to subjecting the PSCs to a method which progresses the cells from an undifferentiated state to a differentiated state. Typically, a step of differentiating PSCs involves culturing the cells under certain conditions and/or contacting the cells with certain factors.

In an embodiment, the PSCs are human PSCs. In a further embodiment, the PSCs are human embryonic stem cells. In another embodiment, the PSCs are induced pluripotent stem cells.

In an embodiment, the differentiated cells are selected from ventral midbrain neural stem cells, forebrain neural cells, spinal cord neural stem cells, retinal pigment epithelium (RPE) cells, pancreatic islets (containing beta cells), mesenchymal stem cells, macrophages cardiomyocytes or other differentiated cells that are not undifferentiated stem cells. A person skilled in the art will recognize suitable methods for differentiating PSCs into the aforementioned cell types. For example, a protocol for obtaining ventral midbrain neural stem cells is disclosed in patent application WO 2016/162747. Depending on the level of maturation the ventral midbrain neural stem cells may express of one or more of the markers F0XA2, LMX1B, 0TX2, EN1, PITX3, and TH. A protocol for obtaining RPE cells is disclosed by Osakada et al (J Cell Sci. 2009 Sep 1 ;122(Pt 17):3169-79. doi: 10.1242/jcs.050393. Epub 2009 Aug 11. “In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction”) or by Kuroda et al. (Stem Cell Res. 2019 Aug;39:101514. doi: 10.1016/j.scr.2019.101514. Epub 2019 Jul 25. “Robust induction of retinal pigment epithelium cells from human induced pluripotent stem cells by inhibiting FGF/MAPK signaling"). Depending on the level of maturation the RPE cells may express one or more of the markers MITF and RPE65. A protocol for obtaining pancreatic islets containing beta cells is disclosed by Robert et al. (Stem Cell Reports. 2018 Mar 13;10(3):739-750. doi:

10.1016/j.stemcr.2O18.01.040. Epub 2018 Mar 1. “Functional Beta Cell Mass from Device- Encapsulated hESC-Derived Pancreatic Endoderm Achieving Metabolic Control”) or by Bukys et al. (J Stem Cell Transplant Biol. 2016 Sep 21 ;2(1). doi: 10.19104/jorm.2017.109. “Xeno-Transplantation of macro-encapsulated islets and Pluripotent Stem Cell-Derived Pancreatic Progenitors without Immunosuppression”) or patent application WO 2017/144695. Beta cells may be defined by the expression of the markers NKX6.1+/INS+/GCG-. A protocol for obtaining cardiomyocytes is disclosed by Yap et al. (Cell Rep. 2019 Mar 19;26(12):3231- 3245. e9. doi: 10.1016/j.celrep.2019.02.083. “In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors”) or by Fernandes et al. (Stem Cell Reports. 2015 Nov 10; 5(5): 753-762. doi: 10.1016/j.stemcr.2015.09.011. “Comparison of Human Embryonic Stem Cell-Derived Cardiomyocytes, Cardiovascular Progenitors, and Bone Marrow Mononuclear Cells for Cardiac Repair”).

The present inventors analyzed cell populations of RPE cells, ventral midbrain neural stem cells, pancreatic islets containing beta cells, mesenchymal stem cells and cardiomyocytes, respectively, using single cell RNA-seq. None of the cell populations contained cells expressing the markers DNMT3B, LINC00678, USP44, LINC00458, CNMD, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, SFRP2, DPPA4, TDGF1, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1. In one embodiment, the cell population is in vitro. Most commonly, the cell population for screening will be an in vitro stem cell-derived product of differentiated cells intended for therapy. In one embodiment, the cell population is provided from a biopsy. Such biopsy may be obtained directly from a patient and analyzed in vitro to screen for PSCs.

In an embodiment, the method as disclosed herein is carried out in vitro. In an embodiment, the cell population is derived in vitro. In an embodiment, the method is carried out on an in vitro stem cell-derived cell culture, which has not been directly taken from a human or animal body. Accordingly, in an embodiment the cell population is not provided from a biopsy.

In an embodiment, the method comprises the step of identifying PSCs or PSC-like cells in the cell population. As used herein, the term “PSC-like cells” means cells that have lost pluripotency but are still sharing some characteristics with PSCs such as some gene expression, capacity to proliferate or any other feature similar to PSCs. By the term “identifying” is meant establishing or indicating a strong link between detecting the expression of certain markers in a cell population and a specific cell of that cell population. In an embodiment, PSCs or PSC-like cells are detected, and optionally identified, by single cell sequencing. In one embodiment, the cell population is screened using fluorescence-activated cell sorting (FACS).

In another aspect, it is provided that a cell population comprising differentiated cells derived from PSCs, wherein the cell population is devoid of cells expressing one or more of the marker selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYPS21, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, DPPA4, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.

In an embodiment, if the cell population screened contains a PSC, it has a limit of detection (LoD) value of the expression of one or more of the markers LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1 below 0.1 , 0.01 , 0.001 , 0.0001 or 0.00001 % of hPSC mixed in the differentiated cells compared to a spike-in reference cell population.

In another embodiment, the cell population has a limit of detection value of the expression of one or more of the markers below 0.1, 0.01 , or 0.001 % of hPSCs mixed in the differentiated cells compared to a spike-in reference cell population.

As used herein, the term “devoid” is defined by the negative detection of one or more of the expression markers selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1. In a preferred embodiment, the cell population has been screened according to the method of the first aspect of the present invention.

Particular embodiments

The aspects of the present invention are now further described by the following nonlimiting embodiments:

1. A method of screening a cell population for undifferentiated stem cells comprising the step of detecting the expression of one or more markers in a cell population, wherein the marker is selected from LINC00458, POLR3G, AC009446. 1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.

2. The method according to any one of the preceding embodiments, wherein the expression of one or more markers selected from DNMT3B, LINC00678, USP44, CNMD, SFRP2, DPPA4, and TDGF1 is further detected.

3. The method according to any one of the preceding embodiments, wherein the limit of detection is 0.1%, 0.01%, 0.001%, 0.0001%, or 0.00001% of undifferentiated cells in the cell population.

4. The method according to any one of the preceding embodiments, wherein the cell population comprises differentiated cells derived from Pluripotent Stem Cells (PSCs).

5. The method according to any one of the preceding embodiments, comprising the initial step of differentiating PSCs into a cell population of differentiated cells derived from the PSCs.

6. The method according to any one of embodiments 4 and 5, wherein the differentiated cells are selected from ventral midbrain neural stem cells, forebrain neural cells, spinal cord neural stem cells, retinal pigment epithelium (RPE) cells, pancreatic islets, mesenchymal stem cells, macrophages and cardiomyocytes.

7. The method according to embodiments 4 to 6, wherein the PSCs are human PSCs. 8. The method according to embodiment 7, wherein the PSCs are human embryonic stem cells or induced pluripotent stem cells.

9. The method according to any one of the preceding embodiments, wherein the cell population is in vitro.

10. The method according to the preceding embodiment, wherein the cell population is derived in vitro.

11. The method according to any one of the preceding embodiments, wherein the method is in vitro.

12. The method according to any one of the preceding embodiments, wherein the cell population is provided from a biopsy.

13. The method according to any one of the preceding embodiments, wherein the cell population is not provided from a biopsy.

14. The method according to any one of the preceding embodiments, comprising the step of identifying PSCs or PSC-like cells in the cell population.

15. The method according to any one of the preceding embodiments, wherein PSCs or PSC-like cells are detected, and optionally identified, on a transcriptomic and/or proteomic level.

16. The method according to any one of the preceding embodiments, comprising a step of amplifying cDNA prior to the step of detecting the marker.

17. The method according to embodiment 16, wherein the cDNA (complementary DNA, DNA synthesized from a single-stranded RNA) is amplified using RT-PCR, qPCR, or ddPCR, or a combination thereof.

18. The method according to any one of the previous embodiments, wherein the cell population is screened using fluorescence-activated cell sorting (FACS). 19. The method according to any one of the previous embodiments, wherein the cell population is screened using bulk analysis.

20. The method according to embodiment 19, wherein the cell population is screened using western blotting, ELISA, RNA-seq, RT-PCR, nested PCR, ddPCR, or a combination thereof.

21. The method according to embodiment 20, wherein the cell population is screened using RT-PCR.

22. The method according to any one of the previous embodiments, wherein the cell population is screened by single cell analysis.

23. The method according to embodiment 22, wherein the cell population is screened using immunostaining, flow cytometry, or a combination thereof.

24. A method of screening a cell population comprising dopaminergic progenitor cells for undifferentiated stem cells comprising the step of detecting the expression of the marker ZFP42.

25. A cell population comprising differentiated cells derived from PSCs, wherein the population is devoid of cells expressing one or more markers selected from LINC00458, POLR3G, AC009446.1, SCGB3A2, VRTN, ZIC2, CRABP1 , CYPS21, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, DPPA4, FOXH1, CLDN6, CLDN7, ZFP42, AC104461.1 , AC0064802.1, and AC0064802.7.

26. The cell population according to the preceding embodiment, wherein the cell population has been screened according to the method of any one of the embodiments 1 to 23.

27. The cell population according to any one of embodiments 25 and 26, wherein the PSCs are human PSCs. 28. The cell population according to embodiment 27, wherein the PSCs are human embryonic stem cells or induced pluripotent stem cells.

Examples

The following are non-limiting examples of protocols for carrying out the invention.

Example 1 : RNA sequencing methodology and experimental design to identify markers unique to undifferentiated PSCs

Several different hPSC samples and differentiated cell types were processed with single cell RNA sequencing in order to compare the transcriptomic signature for the purpose of identifying, in an unbiased manner, genes unique to or upregulated in undifferentiated hPSCs.

In order to identify genes whose expression is unique to the state of pluripotency and not those unique to a genetically distinct individual hPSC line nor those unique to a particular culture condition (i.e. type of media or type of extracellular matrix) many hPSCs samples were analysed. Firstly, three independent genetically distinct hPSC cell lines, one of which was a human induced pluripotent stem cell line and two of which were human embryonic stem cell lines were analysed. Further, three different culture conditions were selected that covered several different commercial medias (including mTeSR, iPSC-Brew and NutriStem) and different matrices including human laminins.

Differential gene expression comparisons were made between these diverse undifferentiated hPSC samples and differentiated cell samples to identify genes upregulated in undifferentiated hPSCs. Importantly, a variety of differentiated cells were also sequenced, covering all three major developmental lineages (or germ layers namely ecotoderm, mesoderm and endoderm; see Example 2), to ensure gene expression comparison results were robust and universally applicable for all cell types. A recent report by Sekine et al. (Robust detection of undifferentiated iPSC among differentiated cells, Sci Rep. 2020 Jun 24;10(1):10293), claimed to have identified three such universal pluripotency genes, but these were identified by comparing only one (hepatic) differentiated cell type to hPSCs.

To perform single cell RNA sequencing (scRNA-seq), cell clusters of undifferentiated PSCs as well as those of differentiated cells were dissociated into single cell suspensions with accutase, tryple select or other such reagents and 3000-10000 cells were processed using the 10X Genomics Chromium Platform and sequenced on a NextSeq550. Data was processed using 10X cellranger and the Seurat analysis package in R programming language. Samples were analysed, filtered for low quality or multiplet cells and analyzed separately for each individual experiment before combining the cells of the selected differentiated cell lineages of choice as well as the hPSCs into one dataset that were then analysed using the standard Seurat workflow as outlined for Seurat version 3, i.e. normalizing using SCTransform and finally using the first 29 principal components for the unified tSNE plot (Fig.1).

Example 2: Methods of generating differentiated cells of all three germ layers from hPSCs and positions in combined dataset

Differentiated cells derived from hPSCs were obtained according to several published protocols that generate cells that cover all three major germ layers, these are represented in clusters 1-8 and 15-21 (Fig. 1). Differentiation was performed to two ectoderm lineages, one to ventral midbrain neural stem cells following the Nolbrant et al 2017 (Nat Protoc. 2017 Sep;12(9):1962-1979.doi: 10.1038/nprot.2017.078. Epub 2017 Aug 31.’’Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation”) methodology and this sample is contained in clusters 19-21 (Fig.1). A second ectodermal lineage of the forebrain was differentiated following the Plaza Reyes et al 2020 protocol that produces retinal epithelium cells and corresponded to cluster 8 (Fig.1 ).

Differentiation was also performed to two mesoderm lineages, one following the Halloin et al 2019 (Methods Mol Biol. 2019;1994:55-70.doi: 10.1007/978-1-4939-9477-9_5. “Production of Cardiomyocytes from Human Pluripotent Stem Cells by Bioreactor Technologies”) methodology to produce cardiomyocytes and this sample is contained in clusters 5-7 and 18 (Fig.1 ). A second distinct mesodermal lineage was also differentiated to, specifically mesenchymal stem cells and corresponds to clusters 15-17 (Fig.1 ).

Differentiation was also performed to the endodermal lineage, following the Jensen et al 2021 protocol that generates pancreatic endoderm (clusters 1,4) and beta-islet like cells (clusters 2-3).

To confirm that the sequenced samples were differentiated correctly the expression of cardinal genes (typically transcription factors) that are well known to identify the various differentiated cell types were assessed in the combined dataset. Importantly, all cardinal markers were enriched in their appropriate clusters, with the pluripotency transcription factor POLI5F1 being principally expressed in hPSC sample clusters 9-14 (Fig.2). The cardinal retinal lineage transcription factor LHX2 was principally expressed in the RPE cell cluster 8 (Fig.3) and cardinal midbrain transcription factor EN-1 was principally expressed in ventral midbrain neural SC clusters 19-21 (Fig.4). The cardinal cardiac lineage transcription NKX2.5 was principally expressed in cardiomyocyte cell clusters 5-7 and 18 (Fig.5), while the cardinal mesenchymal plasma membrane marker NT5E was expressed in the mesenchymal stem cell population. The pancreatic endoderm marker NKX2.2 (Doyle et al., 2007) was expressed in pancreatic islet clusters 1-4 (Fig.7).

Example 3: Computational comparison between hPSC and differentiated cell samples: Computational comparisons can be made between any cell clusters. To identify novel genes highly enriched or exclusively expressed in undifferentiated cells, the clusters of hPSCs (9-14; Fig.1 ) were compared to all differentiated cell clusters (clusters 1-8, 15-21; Fig.1 ). Highly enriched or exclusively expressed genes in undifferentiated cells were defined using the Wilcoxon Rank Sum test for differential gene expression, while only considering genes with a foldchange of minimum 0.25 between the 2 groups and where a gene is expressed in at least 25% of the hPSC cells. As a result of differential gene expression comparison between these two sets of clusters, a list of 3674 genes was produced, with 2064 upregulated in all hPSC clusters compared to all differentiated cell clusters. A list of these genes with average Iog10 fold change greater than 1.0 between undifferentiated and differentiated cell cluster are shown in Table 1. Genes are listed in order of their average fold change expression level difference between hPSC clusters and all differentiated cell clusters.

Table 1 :

We found many cardinal pluripotency genes including POLI5F1 , LIN28A, SOX2, PODXL, CD9 and NANOG (Yuin-Han Loh et al., 2006) as shown in Table 1 (results in bold numbered 1, 12, 32, 33, 41 and 66 respectively) confirming the validity of our method for identifying markers associated with undifferentiated pluripotent stem cells. The expression of cardinal pluripotency markers was checked in our combined dataset and in all cases genes were expressed throughout pluripotency cell clusters (Fig.8-10 and 11-13). However, many of these cardinal pluripotency markers were found also to be expressed in differentiated cell clusters and were not confined to hPSC cells, e.g. LIN28A was expressed in 2 different differentiated cell types and 22,8% of all differentiated cells (Fig.8). Many other cardinal pluripotency markers were found not to be expressed by all undifferentiated cells, e.g. NANOG which was not expressed in 18,8% of undifferentiated cells (Fig 9). Surprisingly, only one of the cardinal pluripotency markers, POLI5F1 , appeared to uniquely identify all pluripotent cells (Fig.10) and thus would avoid the risk of false positive or negative results when used to screen a population of cells for the presence of undifferentiated cells (for which our mixed cell dataset is a theoretical example of). This indicates there is a need for more markers to identify undifferentiated cells with high fidelity so that cell therapy products can be screened for safety i.e. the absence of all pluripotent cells or differentiation protocol output efficacy can be measured. Relying on one marker for detecting tumorigenic cells in a cell therapy product most obviously places great reliance on one metric for the most substantial safety concern for these regenerative medicine products and a panel of markers is most preferred.

We then analysed the expression profile in our combined dataset of all of the noncardinal pluripotency genes identified in Table 1 to assess whether any could be superior to POLI5F1 and act as universal markers for screening a cell population for undifferentiated pluripotent stem cells.

Example 4: Identification of universal markers of undifferentiated pluripotent stem cells.

Of the genes listed in above table 1, those that were expressed in a high percentage of undifferentiated cells (typically above 90%) and all undifferentiated clusters, as well as those with low expression levels in differentiated cells clusters and in few differentiated cells were deemed universal genes for identifying undifferentiated pluripotent cells. These universal genes are summarised in Table 2 and their expression profiles in the combined dataset are shown in Figs.14-40. Intriguingly, many identified genes have completely unknown functions e.g. AC104461.1 and AC0064802.1.

Table 2:

Example 5: Use of Quantitative real-time PCR (qRT-PCR) to compare expression levels in pure hPSC and differentiated cell populations

To validate our results using another technique that assesses RNA expression and one that would also be more suitable than scRNA-sequencing for QC purposes in a cell manufacturing/GMP setting, we selected some of the genes in Table 2 (SCGB3A2, CYP2S1, LINC00458, L1TD1, VRTN, ZFP42, ALPL, LINC00678) and performed qPCR on samples of pure undifferentiated hPSCs and differentiated cells covering all three lineages/germ layers (Figs.41-44).

In the traditional RT-PCR (or qPCR for simplification), the amplification of a sequence is followed by emerging fluorescence during the PCR reaction (Higuchi et al., Biotechnology (N Y). 1992 Apr;10(4):413-7. doi:10.1038/nbt0492-413). qPCR is usually conducted to compare relative amounts of a target sequence between samples. This technique monitors the amplification of the target in real-time via a target-specific fluorescent signal emitted during amplification. In qPCR, the threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels. The Ct (threshold cycle) or otherwise Cp (crossing point) is the intersection between an amplification curve and a threshold line (Bustin et al., Clin Chem. 2009 Apr;55(4):611-22. doi: 10.1373/clinchem.2008.112797) and it is used as a measure of the amount of RNA for the gene of interest that exists in a given sample. In order to compare the expression levels in different samples analyzed simultaneously, equal amount of total RNA is loaded per reaction.

All genes tested showed higher levels of expression in hPSCs compared to differentiated cell types, which indicates they would be useful to identify undifferentiated cells within a mixed cell population (Figs.41-48).

The level of expression of the gene in hPSCs vs the levels of expression in the cell type of interest is the fold change difference which indicates how good is this marker for detecting a potential hPSC contaminant in the respective drug product. A fold change over 1000 times is expected to give a good sensitivity for detection of PSCs using an RNA based assay e.g. VRTN for RPEs and ZFP42 for vmDA neurons.

Example 6: Use of a novel universal marker to identify residual hPSCs in a mixed population, at a lower level of detection than the most robust traditional marker (POU5F1)

Finally, in order to determine how robust these newly identified universal marker candidates are at detecting pluripotent cells in a mixed population, and whether they might be superior to the best cardinal pluripotency marker (POU5F1, Fig.10), we compared the limit of detection with quantitative RT-PCR for one of our universal candidates, VRTN, against POU5F1 (OCT4).

Briefly, RNA was extracted using RNeasy mini kit (Qiagen, 74104) following manufacturer instructions including DNAse I treatment. 500ng RNA was converted to cDNA using the SuperScript™ IV VI LO™ Master Mix (Invitrogen, 11756050). cDNA was diluted 1:10 and 1 ul was used per reaction. TaqMan fast advanced master mix (applied biosystems, 4444556) was used for the qPCR run in viia7 Real time PCR system.

The fold change relative to RPE expression was calculated using the ddCt (delat- delta Ct) method and GAPDH expression (housekeeping gene) as an endogenous control. Briefly, for each sample the dCt was initially calculated by subtracting the Ct value for the gene of interest from the Ct value of GAPDH. Subsequently, since we wanted to normalize everything to the expression level of the RPE cells, the dCt of the RPE sample for each gene was subtracted from the dCt of each of the other samples in order to calculate the ddCt. Finally, the fold change was calculated using the formula 2^A(-ddCt).

This was performed with a series of spike-in samples of hPSCs into d60 differentiated RPE cells. Using this technique, we observed that there was a higher fold change between hPSCs and d60 RPEs for VRTN than POU5F1 at all spike-in RPE samples down to a limit of detection of 0.001% spiked-in hPSCs. Furthermore, VRTN expression was able to out-perform the best cardinal marker, POU5F1, and detect up to 0,001% hPSCs spiked-in the d60 RPE sample rather than 0.01% for POU5F1. The superiority of this novel marker for pluripotent cell detection compared to the best cardinal marker was further demonstrated by a higher R^A2-value of linearity (0.9913 vs 0.9546; Fig.49).

Example 7: Use of the novel universal marker ZFP42 to identify residual hPSCs in a mixed population of vmDA neurons, with high sensitivity

We used one of the markers proven to have a high fold change difference in expression between stem cells and one of the differentiated cell populations, the vmDA neurons, in order to test the lowest LoD that we can achieve by using the sensitive method of ddPCR.

Briefly, a series of spike-in samples of hPSCs into d14 vmDA neurons was prepared for 0 to 2% and RNA was extracted using QIAcube RNeasy mini kit (Qiagen, 74116) following manufacturer instructions High-Capacity cDNA Reverse Transcription Kit (Thermo fisher, 4368814). cDNA was diluted 1:100 and 5 ul were used per reaction. ddPCR is performed according to Bio-Rad instructions. The reactions are set up according to ddPCR Supermix for Probes (No dUTP) (Biorad, 1863023) and the droplets are generated using the Automated Droplet Generator.

Droplet digital PCR offers absolute quantification of nucleic acid targets by counting discrete water-in-oil droplets encapsulating nucleic acid molecules. The massive sample partitioning into -20000 droplets effectively enriches rare templates and thus provides a sensitive detection of rare sequences in a high background. The PCR amplification is carried out within each droplet, and the target concentration is calculated based on the number of positive and negative droplets. Positive droplets contain at least one copy of the target molecule, and thus exhibit a higher fluorescent signal compared to the negative droplets. The fraction of positive droplets is then fitted into a Poisson distribution-based algorithm by the QX manager standard Edition software version 1.2 or newer. The readout is the target concentration in units of copies/pl, which can be used for downstream calculations to determine copies/cell in the overall population. This value, together with the evaluation of stem cells, can be used to give an estimate of the presence and potentially also the fraction of pluripotent cells in the sample.

We observed by using the method of ddPCR for ZFP42 gene, that we could detect with confidence up to 0.01% of PSCs in the vmDA product while we obtain a linearity (R²=0,96) up to 0.1% (LoQ) (Figure 50).

Claims

1. An in vitro method of screening a cell population for undifferentiated stem cells comprising the step of detecting the expression of one or more markers in the cell population, wherein the marker is selected from LINC00458, POLR3G, AC009446. 1, SCGB3A2, VRTN, ZIC2, CRABP1, CYP2S1, ALPL, AL353747.4, VASH2, TNNT1, L1TD1, GAL, F0XH1, CLDN6, ZFP42, CLDN7, AC104461.1, and AC0064802.1.

2. The method according to claim 1 , wherein the expression of one or more markers selected from DNMT3B, LINC00678, USP44, CNMD, SFRP2, DPPA4, and TDGF1 is further detected.

3. The method according to any one of the preceding claims, wherein limit of detection is 0.1%, 0.01%, 0.001%, 0.0001%, or 0.00001% of undifferentiated cells in a cell population.

4. The method according to any one of the preceding claims, wherein the cell population comprises differentiated cells derived from Pluripotent Stem Cells (PSCs).

5. The method according to the preceding claim, wherein the PSCs are human embryonic stem cells or induced pluripotent stem cells.

6. The method according to any one of the preceding claims, wherein the cell population comprises differentiated cells selected from ventral midbrain neural stem cells, forebrain neural cells, spinal cord neural stem cells, retinal pigment epithelium (RPE) cells, pancreatic islets, mesenchymal stem cells, macrophages and cardiomyocytes or other differentiated cells that are not undifferentiated stem cells.

7. The method according to any one of the preceding claims, wherein the cell population is provided from a biopsy.

8. The method according to any one of the preceding claims, wherein the cell population is screened using bulk analysis.

9. The method according to claim 8, wherein the cell population is screened using western blotting, ELISA, RNA-seq, RT-PCR, nested PCR, digital droplet PCR, or a combination thereof.

10. The method according to claim 9, wherein the cell population is screened using RT-

PCR.

11. The method according to claims 1 to 7, wherein the cell population is screened using single cell analysis.

12. The method according to claim 11, wherein the cell population is screened using immunostaining, flow cytometry, or a combination thereof.