CN114672455B - Method for inducing bone marrow stromal cells by utilizing pluripotent stem cells - Google Patents

Abstract

The present invention discloses a method for inducing bone marrow stromal cells using pluripotent stem cells. The bone marrow stromal cells of the present invention are induced by pluripotent stem cells and their differentiation path is strictly limited by a specific induction culture medium, and are obtained after going through the body wall mesoderm cell stage. The bone marrow stromal cells induced by different pluripotent stem cell lines obtained by the present invention not only have a typical homeodomain transcription factor (HOX) gene expression pattern similar to that of bone mesenchymal cells, but also have better osteogenesis, chondrogenesis and hematopoietic support capabilities than bone marrow mesenchymal stem cells. Therefore, this type of bone marrow stromal cells with a clear source, fast proliferation rate and low heterogeneity can provide a new, high-quality cell source for clinical transformation in the field of cell therapy, and can also provide an ideal in vitro model for research on the pathogenesis of supporting hematopoietic stem cell transplantation, limb bone development and related diseases.

Description

A method for inducing bone marrow stromal cells using pluripotent stem cells

Technical Field

The present invention relates to the field of stem cell technology, and in particular to a method for inducing bone marrow stromal cells using pluripotent stem cells.

Background technique

Stromal cells (MSCs) are one of the most common cells in the body and are essential for the development, maintenance, function and regeneration of most tissues. They can differentiate along multiple connective lineages, but unlike most other stem/progenitor cells, they perform various other functions while maintaining their developmental potential, such as responding to damage in vivo by secreting trophic factors and promoting the regeneration of extracellular matrix (ECM) molecules, and promoting the fibrotic repair process when regeneration fails (Cell Stem Cell. 2021 Oct 7; 28(10): 1690-1707.); among them, stromal cells in the bone marrow are also involved in the formation of the bone marrow hematopoietic microenvironment, supporting hematopoiesis, osteogenic differentiation, etc. (Nature. 2014 Jan 16; 505(7483): 327-34.), and at the same time, due to their low immunogenicity, they play an irreplaceable role in bone damage repair and reconstruction of the bone marrow hematopoietic microenvironment or ecological niche, and have broad clinical application prospects.

However, the acquisition of bone marrow stromal cells is not only very traumatic, but also has great heterogeneity in bone marrow-derived stromal cells from different individuals. It is also difficult to continuously passage and amplify them in vitro, which cannot meet the needs of clinical treatment. Therefore, how to obtain bone marrow stromal cells stably and in large quantities in vitro has become a key issue to solve the limitation of its clinical transformation.

In recent years, a large number of articles have reported the application of pluripotent stem cells, including induced pluripotent stem cells and embryonic stem cells, in differentiating into various germ layer cell types, but no reports have been seen on the differentiation method of pluripotent stem cells through the somatic mesoderm stage to obtain the bone marrow stromal cells from which they are derived. Among them, somatic mesoderm cells (SM-MCs) are one of the cells belonging to the lateral plate mesoderm stage during embryonic development. They are located between the ectoderm and the intraembryonic body cavity and are the progenitor cells that form connective tissue and limb skeletal elements (bone and cartilage). Previous studies have shown that stromal cells derived from somatic mesoderm cells exist in the bone marrow of long bones (Front Genet. 2019 Oct 11; 10: 977; Development. 2020 Jun 19; 147 (12): dev175059). In addition, the existing technical means (Chinese patent "Immortalized rat bone marrow stromal cell line and its preparation method") is to introduce the human telomerase reverse transcriptase catalytic subunit gene into rat bone marrow stromal cells through a retroviral vector to obtain an immortalized rat bone marrow stromal cell line. Although this cell line can be expanded for a long time in vitro, it cannot be used in clinical cell therapy due to its species problem.

Summary of the invention

In order to overcome the difficulty that bone marrow stromal cells cannot be prepared using pluripotent stem cells in the prior art, the present invention obtains bone marrow stromal cells of a specific developmental path in vitro through a specific induction differentiation process from the perspective of the origin of cell development. The present invention provides a method for inducing bone marrow stromal cells using pluripotent stem cells, which is a stable and efficient preparation method of bone marrow stromal cells derived from the body wall mesoderm induced by pluripotent stem cells. The obtained body wall mesoderm-derived bone marrow stromal cells not only have the characteristics of high reproducibility, low heterogeneity, and large-scale amplification, but also have stronger bone formation ability and more superior hematopoietic support ability. It can provide a new, high-quality cell source for the clinical transformation of cell therapy, and at the same time, it can also provide an ideal in vitro model for the study of the pathogenesis of supporting hematopoietic stem cell transplantation, limb bone development and related diseases.

The first object of the present invention is to provide a bone marrow stromal cell.

The second object of the present invention is to provide bone marrow stromal cells derived from body wall mesoderm cells.

The third object of the present invention is to provide a culture medium for inducing pluripotent stem cells into parietal mesoderm cells.

The fourth object of the present invention is to provide a serum-free complete culture medium for bone marrow stromal cells.

The fifth object of the present invention is to provide the use of any of the culture medium and/or the serum-free complete culture medium for bone marrow stromal cells in inducing bone marrow stromal cells from pluripotent stem cells.

A sixth object of the present invention is to provide a method for inducing bone marrow stromal cells using pluripotent stem cells.

The seventh object of the present invention is to provide bone marrow stromal cells prepared by the method.

The eighth object of the present invention is the use of any of the above-mentioned bone marrow stromal cells in the preparation of a drug that promotes hematopoietic support and/or bone repair.

In order to achieve the above object, the present invention is implemented by the following scheme:

The present invention induces pluripotent stem cells to be efficiently transformed into somatic mesoderm cells (SMCs) at the mesendoderm stage by a limited induction method, and then digests and inoculates the somatic mesoderm cells again, replaces them with a serum-free complete culture medium for bone marrow stromal cells (SM-MSC Medium) or a commercial MSC culture medium (StemFit) or a commercial MSC culture medium (ACF) for continuous culture, and detects their phenotypes, thereby obtaining somatic mesoderm derived marrow stromal cells (SM-MSCs).

"SMCs" as used in the present invention refers to somatic mesoderm cells.

The “SM-MSCs” used in the present invention refers to marrow stromal cells derived from parietal mesoderm cells induced from pluripotent stem cells via parietal mesoderm cells.

The present invention provides a new, standardized and feasible in vitro differentiation process of bone marrow stromal cells. This mass-producible bone marrow stromal cell can not only provide a new, high-quality cell source for clinical transformation in the field of cell therapy, but also provide an ideal in vitro model for research on supporting hematopoietic stem cell transplantation, limb bone development and the pathogenesis of related diseases.

Specifically, the present invention subcultures and amplifies induced pluripotent stem cells on a culture plate or culture dish coated with matrix gel, and when the induced pluripotent stem cells grow to the required cell amount for induction, a single cell suspension is made and attached to the wall, and then a culture medium supplemented with a GSK-3 inhibitor is used for directed induction for 1 to 3 days to obtain mesendoderm cells; then, a somatic mesoderm cell induction culture medium is used for directed induction for 5 to 7 days to induce the mesendoderm cells into a somatic mesoderm cell population; finally, the somatic mesoderm cells in the previous step are re-digested and inoculated, and bone marrow stromal cell serum-free complete culture medium (SM-MSC Medium) or commercial MSC culture medium (StemFit or ACF) is used for continuous subculture for 6 to 8 times, and their surface markers are identified by flow cytometry, and their hematopoietic support ability and osteogenic and chondrogenic abilities are tested.

Therefore, the present invention claims to protect a kind of bone marrow stromal cells, which are obtained by inducing pluripotent stem cells into parietal mesoderm cells and then continuously subculturing in bone marrow stromal cell culture medium.

And a kind of bone marrow stromal cells derived from body wall mesoderm cells. The bone marrow stromal cells derived from body wall mesoderm cells are induced from pluripotent stem cells after going through the body wall mesoderm cell stage.

Preferably, the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

The present invention also claims a culture medium for inducing pluripotent stem cells into body wall mesoderm cells, wherein the culture medium is a basic culture medium containing a combination of a GSK-3 inhibitor and a BMP signaling pathway activator.

Preferably, the basal culture medium is DMEM-F12.

Preferably, the GSK-3 inhibitor is one or more of LY2090314, SB216763, CHIR99021 and CHIR99021 HCl.

More preferably, the GSK-3 inhibitor is CHIR99021.

Preferably, the BMP signaling pathway activator is one or more of BMP2, BMP4 or BMP7.

More preferably, the BMP signaling pathway activator is BMP7.

More preferably, the culture solution contains 1-20 μM CHIR99021 and 1-500 ng/ml BMP7.

Even more preferably, the culture solution contains 1-5 μM CHIR99021 and 1-100 ng/ml BMP7.

Further preferably, the culture medium contains 3 μM CHIR99021 and 100 ng/ml BMP7.

Preferably, the culture solution further contains 1-5% (V/V) ITS culture additive, 1-5% (V/V) NEAA and 1-5% (V/V) Glutamax.

More preferably, the culture solution further contains 1% (V/V) ITS culture additive, 1% (V/V) NEAA and 1% (V/V) Glutamax.

Most preferably, the culture medium is DMEM-F12 culture medium containing 1% (V/V) ITS culture supplement, 1% (V/V) NEAA, 1% (V/V) Glutamax, 3 μM CHIR99021 and 100 ng/ml BMP7.

The present invention also claims a serum-free complete culture medium for bone marrow stromal cells (SM-MSC Medium), which can induce body wall mesoderm cells into bone marrow stromal cells, and contains 1-10% (V/V) serum replacement, 1-5% (V/V) NEAA, 1-5% (V/V) ITS culture additive, 1-10 mM L-glutamic acid, 0.1-10 Mm β-mercaptoethanol, 1-100 ng/mL bFGF, 1-100 ng/mL EGF, 1-100 ng/mL VEGF, 0.1-10 mg/ml human platelet-derived growth factor, 0.1-10 mg/ml vitamin C and 0.1-5 mM uridine.

Preferably, the basal culture medium is α-DMEM culture medium.

Preferably, the serum substitute is KOSR or Ultroser ^™ G serum substitute.

More preferably, the serum replacement is KOSR.

More preferably, the human platelet-derived growth factors are PDGFAA and PDGFBB.

More preferably, the serum-free complete culture medium for bone marrow stromal cells (SM-MSC Medium) is an α-DMEM culture medium containing 1-5% (V/V) serum substitute KOSR, 1-5% (V/V) NEAA, 1-5% (V/V) ITS culture additive, 1-5mM L-glutamate, 1-5mM β-mercaptoethanol, 1-10ng/mL bFGF, 1-10ng/mL EGF, 1-10ng/mL VEGF, 1-100ng/ml human platelet-derived growth factor PDGFAA, 1-100ng/ml human platelet-derived growth factor PDGFBB, 0.1-300ng/ml vitamin C, and 0.1-500μM uridine.

Even more preferably, the serum-free complete culture medium of bone marrow stromal cells (SM-MSC Medium) is an α-DMEM culture medium containing 5% (V/V) serum substitute KOSR, 1% (V/V) NEAA, 1% (V/V) ITS culture additive, 5mM L-glutamate, 1mM β-mercaptoethanol, 2ng/mL bFGF, 2ng/mL EGF, 1ng/mL VEGF, 20ng/ml human platelet-derived growth factor PDGFAA, 20ng/ml human platelet-derived growth factor PDGFBB, 300ng/ml vitamin C, and 100μM uridine.

The present invention claims to protect the use of the culture medium and/or the serum-free complete culture medium of bone marrow stromal cells in inducing bone marrow stromal cells from pluripotent stem cells.

Preferably, the culture medium and/or the serum-free complete culture medium of bone marrow stromal cells is used in bone marrow stromal cells derived from parietal mesoderm cells induced from pluripotent stem cells.

Preferably, the combination of the culture medium and the serum-free complete culture medium for bone marrow stromal cells is used in inducing pluripotent stem cells into bone marrow stromal cells.

The present invention also claims a method for inducing bone marrow stromal cells using pluripotent stem cells, comprising the following steps:

S1. Inducing pluripotent stem cells into mesendoderm cells: After dissociating and dispersing the pluripotent stem cells, inoculate them into a culture plate or a culture dish coated with matrix gel, and culture them in a culture medium containing CHIR99021 for 1 to 3 days to obtain mesendoderm cells;

S2. Inducing the mesendoderm cells into body wall mesoderm cells: using the culture medium in which the pluripotent stem cells are induced into body wall mesoderm cells, culturing the product of the previous step for 5 to 7 days to obtain body wall mesoderm cells;

S3. Inducing the body wall mesoderm cells into body wall mesoderm cell-derived bone marrow stromal cells: After the obtained body wall mesoderm cells are digested again and inoculated into bone marrow stromal cell culture medium, they are continuously subcultured with bone marrow stromal cell culture medium for 6 to 8 times to obtain body wall mesoderm cell-derived bone marrow stromal cells.

Preferably, the bone marrow stromal cell culture medium is the bone marrow stromal cell serum-free complete culture medium (SM-MSC Medium) or a commercial MSC culture medium (eg StemFit or ACF).

More preferably, the bone marrow stromal cell culture medium is the bone marrow stromal cell serum-free complete culture medium.

Preferably, the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells.

More preferably, the pluripotent stem cells are human induced pluripotent stem cells or human embryonic stem cells.

Preferably, in step S1, the culture medium containing CHIR99021 is a DMEM-F12 culture medium containing 1-10 μM CHIR99021.

Preferably, in step S1, pluripotent stem cells are dissociated and dispersed into single cells or cell aggregates using Accutase, and the cells are seeded on a culture plate or culture dish coated with matrix gel, with the seeding number being 1×10 ³ to 1×10 ⁷ /cm ² .

More preferably, the cell seeding number is 1×10 ⁴ to 2×10 ⁴ /cm ² .

Preferably, the matrix glue is Matrigel or laminin LN.

More preferably, the matrix gel is Matrigel, which is prepared by diluting the Matrigel stock solution with pre-cooled DMEM-F12 at a volume ratio of 1:100 and then coating the culture plate or dish 1 to 12 hours in advance.

More preferably, the matrix glue is laminin LN, which is diluted with PBS at a volume ratio of 1:100 and added to the well plate for coating overnight at room temperature.

Preferably, in step S1, the proportion of double-positive cells of TBXT and MIXL1 markers of the induced mesendoderm cells can reach more than 95%, and the next step of induction can be carried out without sorting and purification.

Preferably, in step S2, the induced body wall mesoderm cells highly express their markers HAND1, FOXF1, TBX4, PRRX1 and PITX1, and the proportion of HAND1 and FOXF1 double-positive cells is more than 95%.

Preferably, in step S3, bone marrow stromal cells derived from body parietal mesoderm cells are obtained after continuous subculturing with bone marrow stromal cell culture medium for 6 to 8 times.

According to the inventor's experiments, the bone marrow stromal cells derived from the parietal mesoderm cells can be obtained by using the bone marrow stromal cell serum-free complete culture medium (SM-MSC Medium) described in the present invention or the commercial MSC culture medium (such as StemFit and ACF). However, compared with the commercial MSC culture medium (such as StemFit and ACF), the bone marrow stromal cells induced and amplified in the bone marrow stromal cell serum-free complete culture medium (SM-MSC Medium) described in the present invention have a faster proliferation rate, more proliferation generations, and better osteogenic and hematopoietic support capabilities, with significant statistical differences. Therefore, the bone marrow stromal cell serum-free complete culture medium (SM-MSC Medium) described in the present invention has obvious advantages in inducing pluripotent stem cell-derived bone marrow stromal cells.

Preferably, in step S3, when the somatic mesoderm cells in step S3 express CD44, CD90 and CD140b and do not express hematopoietic stem cell markers CD34 and CD45 after being continuously subcultured in bone marrow stromal cell culture medium for 6 to 8 times, bone marrow stromal cells derived from somatic mesoderm cells are obtained.

Bone marrow stromal cells prepared by any of the above methods also fall within the protection scope of the present invention.

The use of the bone marrow stromal cells in the preparation of drugs that promote hematopoietic support and/or bone repair also falls within the protection scope of the present invention.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention strictly limits the cell differentiation path by using a specific induction culture medium, and obtains the bone marrow stromal cells from pluripotent stem cells through the body wall mesoderm cell stage. The body wall mesoderm cells in the present invention are not only homogeneous, but also have an induction efficiency of more than 95%. At the same time, the induction differentiation system of the present invention has strong operability and standardized induction differentiation procedures, which can ensure that the bone marrow stromal cells from different cell lines and different batches have stable characteristics and good functional characteristics.

This type of bone marrow stromal cells with a clear differentiation pathway and a clear source can not only rapidly proliferate in vitro, but also have a similar typical homeodomain transcription factor (HOX) gene expression pattern to bone mesenchymal cells. In addition, in vitro and in vivo experiments have shown that bone marrow stromal cells (SM-MSCs) induced by body wall mesoderm cells have better osteogenic, chondrogenic and hematopoietic support capabilities than bone marrow mesenchymal stem cells (BMSCs) directly isolated from the bone marrow. Therefore, this type of bone marrow stromal cells with a clear source, fast proliferation rate and low heterogeneity can provide a new, high-quality cell source for clinical transformation in the field of cell therapy, and can also provide an ideal in vitro model for research on the pathogenesis of supporting hematopoietic stem cell transplantation, limb bone development and related diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a white light photograph of hiPSC cells cultured in Example 3 ( FIG. 1A ) and an immunofluorescence photograph of pluripotency markers of hiPSC cells ( FIG. 1B ).

FIG2 shows the cell morphology of hiPSC cells in Example 3 after 2 days of treatment with GSK-3 inhibitor ( FIG2A ), the real-time fluorescence quantitative PCR detection results of mesendoderm markers TBXT and MIXL1 ( FIG2B ), and immunofluorescence staining images ( FIG2C ).

FIG3 is a cell morphology diagram of hiPSC-derived mesendoderm cells induced with parietal mesoderm induction medium for 6 days in Example 3 ( FIG3A ), the expression of its markers HAND1, FOXF1, TBX4, PRRX1, and PITX1 genes detected by real-time fluorescence quantitative PCR ( FIG3B ), and the expression of its markers HAND1 and FOXF1 detected by immunofluorescence staining ( FIG3C ), as well as a flow cytometry induction efficiency diagram of HAND1 and FOXF1 ( FIG3D )

FIG. 4 shows the cell morphology of the parietal mesoderm cells in Example 3 after 6 passages in bone marrow stromal cell culture medium (commercially available MSC culture medium (StemFit)), immunofluorescence staining of CD90 ( FIG. 4A ), and flow cytometry analysis of cell surface markers ( FIG. 4B ).

5 is a diagram showing the expression of homeodomain transcription factor (HOX) genes in the parietal mesoderm-derived bone marrow stromal cells induced by bone marrow stromal cell culture medium (commercially available MSC culture medium (StemFit)) using fluorescent quantitative PCR in Example 3.

Figure 6 compares the proliferation capacity of two batches (SM-MSCs 1, SM-MSCs 2) of bone marrow stromal cells derived from body wall mesoderm induced by bone marrow stromal cell culture medium (commercially available commercial MSC culture medium (StemFit)) in Example 3 and two batches (BMSCs 1, BMSCs 2) of bone marrow mesenchymal stem cells isolated from different batches in Control Example 1, which uses CCK-8 to detect cell proliferation changes and plots the results.

FIG7 shows the mesodermal cells derived from the bone marrow stromal cells prepared in different bone marrow stromal cell culture media in Example 3, and further compares the proliferation ability (FIG7 left) and proliferation generation (FIG7 right). The bone marrow stromal cell culture media include bone marrow stromal cell serum-free complete culture medium (SM-MSCs medium), commercial MSC culture medium (StemFit; Ajinomoto), and commercial MSC culture medium (ACF; STEM CELL Technologies).

FIG8 shows the bone marrow stromal cells (SM-MSCs) derived from the body wall mesoderm cells prepared by using the bone marrow stromal cell culture medium (commercially available MSC culture medium (StemFit)) in Example 3 and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1, and further compares the ability of in vitro osteogenic, chondrogenic and hematopoietic support, including in vitro osteogenic and chondrogenic staining, ELISA quantification ( FIG8A ) and fluorescence quantitative PCR detection of osteogenic (COL1A1, ALP, CON and OPN) and chondrogenic (COL2A1, A The expression levels of CAN, RUNX2 and SOX9 genes were detected (Figure 8B), the expression of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L, ANGPT1) was detected by real-time fluorescence quantitative PCR (Figure 8C), the maintenance ratio of CD34+HSC in the co-culture system was detected by flow cytometry (Figure 8D), and the clonal formation analysis of granulocyte and macrophage colony-forming units (CFU-GM) and burst erythroid colony-forming units (BFU-E) formed in LTC-IC and their statistical graphs were performed (Figure 8E).

Figure 9 shows the bone marrow stromal cells derived from the parietal mesoderm cells prepared in different bone marrow stromal cell culture media (SM-MSCs medium, StemFit, ACF) in Example 3, and further compares the osteogenic, chondrogenic and hematopoietic support abilities in vitro. Including fluorescence quantitative PCR to detect the expression of osteogenic (COL1A1, ALP, CON and OPN) and chondrogenic (COL2A1, ACAN, RUNX2 and SOX9) genes (Figure 9A), and real-time fluorescence quantitative PCR to detect the expression of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L, ANGPT1) (Figure 9B).

Figure 10 compares the in vivo osteogenesis and hematopoietic support capabilities of the body wall mesoderm-derived bone marrow stromal cells (SM-MSCs) induced in Example 3 and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1, including Masson staining of osteogenesis ability detection and its statistical graph (Figure 10A), immunofluorescence staining of osteogenic markers (OCN, OPG) (Figure 10B), hematopoietic cell clusters after HE staining and their number statistics (Figure 10C), and immunofluorescence staining of CD45+ hematopoietic progenitor cells (Figure 10D).

FIG11 is a gene expression profile analysis of two batches (SMs1 and SMs2) of different batches of body wall mesoderm induced in Example 3 and two batches (SM-MSCs 1 and SM-MSCs2) of bone marrow stromal cells derived from different batches of body wall mesoderm and two batches (BMSCs1 and BMSCs2) of bone marrow mesenchymal stem cells directly isolated from different batches of long bone marrow in Control Example 1 by RNA-Seq, gene expression profile analysis of two samples from different batches at the same differentiation stage (SMs1 vs SMs 2; SM-MSCs 1 vs SM-MSCs 2; BMSCs 1 vs BMSCs 2) ( FIG11A ), gene expression analysis of SMs 1, SMs 2; SM-MSCs 1, SM-MSCs 2; BMSCs 1, BMSCs 2 at different stages ( FIG11B ), and gene expression analysis of SM-MSCs 1, SM-MSCs 2 and BMSCs 1, BMSCs 2. 2 homeodomain transcription factor (HOX1-13) gene expression pattern (Figure 11C).

Figure 12 shows the flow cytometry surface markers (Figure 12A), morphology and in vitro osteoblastic and chondrogenic detection diagram (Figure 12B) of bone marrow stromal cells derived from embryonic stem cells (H1-ES) prepared using bone marrow stromal cell culture medium (commercially available MSC culture medium (StemFit)) in Example 7, and the bone formation (Figure 12C) and hematopoietic support ability detection diagram (Figure 12D) of bone marrow stromal cells derived from embryonic stem cells (H1-ES) prepared using bone marrow stromal cell serum-free complete culture medium (SM-MSCsmedium) compared with the bone marrow mesenchymal stem cells (BMSCs) in control example 1 obtained by culture in the corresponding culture medium.

Detailed ways

The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

The primers used in the fluorescent quantitative PCR in the following examples are shown in the following table:

The antibodies used in the immunofluorescence staining in the following examples are shown in the table below:

Antigen Host Dilution Company Cat.No. NANOG Rabbit 1:400 Cell signaling technology 4903S OCT4 Mouse 1:400 Cell signaling technology 75463S TBXT Goat 1:400 R&D Systems AF2085 MIXL1 Rabbit 1:400 Proteintech Group 22772-1-AP HAND1 Goat 1:400 R&D Systems AF3168 FOXF1 Rabbit 1:200 Abcam ab168383 OCN Mouse 1:400 R&D Systems MAB1419 OPG Rabbit 1:400 Merck Millipore ABC463 Anti-mouse CD45 Rat 1:100 eBioscience 12-0451-82

The antibodies used in the flow cytometry assays in the following examples are shown in the table below:

Method of cell immunofluorescence staining: fix the cell clones with 4% paraformaldehyde PFA, wash 3 times with PBS, then block with 2% BSA for 30 minutes, and wash twice with PBS. Add primary antibody, incubate at 4°C overnight, wash 3 times with PBS, 5 minutes each time; add secondary antibody, incubate at room temperature for 30-45 minutes; after washing 4 times with PBS, add 0.5μg/ml DAPI for staining for 10 minutes, remove DAPI, and wash three times with PBS; after staining, seal with sealing agent, and finally take pictures on confocal microscope.

Fluorescence quantitative PCR method: The cells were digested and collected with Accutase, washed 3 times with PBS, and then lysed with 1 ml RNAzol. After total RNA was extracted, it was reverse transcribed into cDNA in a 10 μl reaction system using a quantitative reverse transcription kit (Qiagen). Fluorescence quantitative PCR detection was performed using the DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Fisher Scientific) in a LightCycler 480 detection system (. The thermal cycle conditions were: 95°C denaturation for 10 s, 60°C annealing for 20 s, and 72°C extension for 30 s, for a total of 45 cycles. GAPDH was used as an internal reference, and the gene expression level was calculated using the 2-ΔΔCT method.

Method for flow cytometry of bone marrow stromal cells: digest bone marrow stromal cells with Accutase, centrifuge at 1100rpm for 4min, discard the supernatant, resuspend the cells with 50ul PBS, and use isotype homologous antibodies (BD Pharmingen) as negative control (isotype control). Add flow cytometry antibodies (CD34, CD45, CD44, CD90, CD140b) to another tube for staining for 20min, wash twice with PBS, 5min each time, centrifuge at 1100rpm for 4min, discard the supernatant, resuspend the cells with 200ul PBS, and detect the proportion of positive cells on a flow cytometer.

Example 1 A culture medium for inducing pluripotent stem cells into somatic mesoderm cells (SMCs)

DMEM-F12 culture medium containing 1% (V/V) ITS culture supplement, 1% (V/V) NEAA, 1% (V/V) Glutamax, 3 μM CHIR99021 and 100 ng/ml BMP7.

Example 2 A serum-free complete culture medium for bone marrow stromal cells

α-DMEM culture medium containing 5% (V/V) serum substitute KOSR, 1% (V/V) NEAA, 1% (V/V) ITS culture supplement, 5mM L-glutamate, 1mM β-mercaptoethanol, 2ng/mL bFGF, 2ng/mL EGF, 1ng/mL VEGF, 20ng/ml human platelet-derived growth factor PDGFAA, 20ng/ml human platelet-derived growth factor PDGFBB, 300ng/ml vitamin C and 100μM uridine.

Control Example 1 Preparation and Culture Method of Bone Marrow Mesenchymal Stem Cells (BMSCs)

The purpose of this example is to illustrate the separation, preparation and culture methods of bone marrow mesenchymal stem cells (BMSCs) used in subsequent examples of the present invention.

Density gradient centrifugation is used to separate mesenchymal stem cells from human long bone marrow; bone marrow stromal cell culture medium is used to expand and culture bone marrow mesenchymal stem cells, and the bone marrow stromal cell culture medium is: the bone marrow stromal cell serum-free complete culture medium (SM-MSCs medium) of Example 2, or commercially available MSC culture medium (StemFit; Ajinomoto) or commercially available MSC culture medium (ACF; STEM CELL Technologies).

The specific steps are as follows:

(1) Dilute 5 mL of human bone marrow fluid with an equal volume of PBS and centrifuge at 1500 rpm for 10 minutes at room temperature;

(2) After discarding the fat layer, slowly add the remaining bone marrow fluid along the wall of the centrifuge tube onto an equal volume of Ficoll separation solution (1.077 g/mL) and centrifuge at 2500 rpm for 30 minutes at room temperature;

(3) The mononuclear cells with the white film layer at the interface of the separation solution were aspirated, suspended with 40 ml of PBS, and centrifuged at 1500 rpm for 10 minutes at room temperature;

(4) Discard the supernatant and wash twice with 50 ml PBS.

(5) The cells were resuspended in bone marrow stromal cell culture medium, expanded and cultured, and seeded into a culture flask with a bottom area of 25 cm ² at a cell density of 2×10 ⁵ /mL and cultured in a 37°C, 5% CO ₂ incubator.

(6) After 3 days, the medium was changed for the first time. After discarding the suspended cells, the medium was changed every 2 to 3 days. The cell morphology and growth changes were observed under a microscope every day. After 14 days, there were basically no suspended cells in the culture flask. All cells grew in a fibrous manner and adhered to the wall. The cells were in good condition.

(7) When the cells grow to 80% to 90% confluence, they are digested with Accutase, subcultured at a ratio of 1:3, and used for subsequent experiments.

(8) The bone marrow mesenchymal stem cells isolated from different batches were labeled as BMSCs 1, BMSCs 2, BMSCs 3, etc., and used in subsequent experiments.

Example 3 Differentiation of human induced pluripotent stem cells (hiPSC) into bone marrow stromal cells (SM-MSCs) via parietal mesoderm cells

The purpose of this example is to illustrate the induction and differentiation operation based on the stable establishment and culture of human induced pluripotent stem cell line (hiPSC).

1. Cultivation of hiPSC cells and detection of the pluripotency of pluripotent stem cells

1. Experimental methods

Human induced pluripotent stem cells (hiPSC) refer to pluripotent stem cells induced by human skin fibroblasts, which are constructed by the laboratory using induced pluripotent stem cell technology (iPSCs). hiPSC cells are large-scale expanded using mTeSR culture medium from STEM CELL to maintain their undifferentiated state. At the same time, the culture medium needs to be coated with matrix gel, and the matrix gel is selected from Matrigel or laminin LN for subsequent experiments.

The specific steps are as follows:

(1) Coating culture dishes: (a) Thaw Matrigel on ice and dilute the stock solution of Matrigel with pre-cooled DMEM-F12 at a ratio of 1:100 by volume, then add it to the plate for coating overnight at room temperature; or (b) dilute laminin LN with PBS at a ratio of 1:100 by volume, then add it to the plate for coating overnight at room temperature.

(2) Resuscitation of hiPSCs: Rapidly thaw hiPSCs in a 37°C water bath, transfer to a 15 ml centrifuge tube containing 5 ml of mTeSR culture medium, and centrifuge at 1100 rpm for 4 min to collect the cells.

(3) Remove the coated Matrigel or laminin LN, resuspend the cells in 2 ml of mTeSR, evenly inoculate the cells, and place them in an incubator at 37°C, 5% _CO2 , and 95% humidity for static culture.

(4) The culture medium was replaced once a day, and the cells were observed to maintain an undifferentiated state. The culture was continued until the hiPSC cell clones grew to a density of 80-90%, and then the cells were passaged.

(5) Cell passaging: Wash the cells twice with PBS, add ReLeSR ^™ , incubate at 37°C for 1 to 5 min, discard ReLeSR ^™ , and gently pipette the cells with 1 ml of mTeSR culture medium until the cells are clumps of appropriate size.

(6) Collect the mTeSR resuspended cells and evenly inoculate the cells into a well plate pre-coated with Matrigel at a volume of 100 μl per well, and place the well plate in a 37°C, 5% CO ₂ incubator for static adherence culture.

(7) Repeat the above culture expansion steps to obtain sufficient cell quantity for the next step of differentiation induction.

(8) Perform cell immunofluorescence staining on the hiPSC cell clones amplified in the previous step to detect the pluripotent stem cell marker transcription factors NANOG and OCT4 (Cell Signaling Technology).

2. Experimental results

Figure 1 is a white light and pluripotency marker immunofluorescence photograph of hiPSC cells cultured in this embodiment; it can be seen that the hiPSC cell clones are compact, flat, and undifferentiated; and the cells express NANOG and OCT4, which are the signature transcription factors of pluripotent stem cells. Among them, laminin LN was used to coat the cell culture plate, which has a similar effect to Matrigel, and can maintain the pluripotency of pluripotent stem cells and maintain the clonal morphology. This shows that the hiPSC cells used for induction in this embodiment are indeed pluripotent stem cells.

2. Induction, differentiation and identification of somatic mesoderm cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs)

1. Experimental methods

(1) When hiPSC cells grow to a density of 80-90%, wash the cells twice with PBS, add 500 μl of Accutase and incubate at 37°C for 4 min. Observe under a microscope to dissociate the cells into single cells or small cell clumps. Remove the Accutase and gently blow the cells with PBS to disperse them evenly.

(2) Transfer the cells into a 15 ml centrifuge tube and centrifuge at 1100 rpm for 4 min to collect the cells.

(3) Resuspend the cell pellet in DMEM-F12 culture medium containing 3 μM CHIR99021, and evenly inoculate the cells into a well plate or culture dish pre-coated with Matrigel at a cell density of 1×10 ⁴ /cm ² , and place in an incubator at 37°C, 5% CO ₂ and 95% humidity for static culture.

(4) After culturing for 2 days, hiPSC cells were induced into mesendoderm cells, and mesendoderm markers TBXT and MIXL1 were detected by real-time fluorescence quantitative PCR, and mesendoderm markers TBXT (R&D Systems) and MIXL1 (Proteintech Group) were detected by immunofluorescence staining.

(5) Different batches of induced pluripotent stem cell-derived parietal mesoderm cells were labeled as SMCs 1, SMCs 2, SMCs 3, etc., and used in subsequent experiments.

(6) Thereafter, the culture medium was replaced with the culture medium of Example 1 and cultured for 5 to 7 days to induce the mesendoderm cells into parietal mesoderm cells. During this period, the medium was changed every day, and the mesoderm cell (SMCs) markers HAND1, FOXF1, TBX4, PRRX and PITX1 were detected by real-time fluorescence quantitative PCR, and the parietal mesoderm cell markers HAND1 (R&D Systems) and FOXF1 (Abcam) were detected by immunofluorescence staining.

(7) Finally, the proportion of HAND1+/FOXF1+ cells was detected by flow cytometry. The specific method was as follows: the body wall mesoderm cells were digested, fixed with 4% paraformaldehyde PFA, washed twice with PBS, washed with 1% permeabilization solution for 5 minutes, and then incubated with HAND1 and FOXF1 antibodies at room temperature for 30 minutes. Subsequently, the cells were washed twice with 1% permeabilization solution for 5 minutes each time, and the cells were resuspended in 200ul PBS and the proportion of HADN1 and FOXF1 positive cells was detected on a flow cytometer.

2. Experimental results

Figure 2 shows the cell morphology of hiPSC cells in Example 3 of this embodiment after 2 days of treatment with GSK-3 inhibitor (3 μm CHIR99021) (Figure 2A) and the real-time fluorescence quantitative PCR detection results of mesendoderm markers TBXT and MIXL1 (Figure 2B) and TBXT and MIXL1 immunofluorescence staining images (Figure 2C).

FIG3 is a cell morphology diagram of hiPSC-derived mesendoderm cells in Example 3 after induction with parietal mesoderm induction medium for 6 days ( FIG3A ), real-time fluorescence quantitative PCR detection of the expression of its markers HAND1, FOXF1, TBX4, PRRX1, and PITX1 genes ( FIG3B ), and immunofluorescence staining detection of the expression of its markers HAND1 and FOXF1 ( FIG3C ), as well as a flow cytometry induction efficiency diagram of HAND1 and FOXF1 ( FIG3D )

The results showed that pluripotent stem cells can be efficiently induced into mesendoderm cells by CHIR99021, highly expressing TBXT and MIXL1, and then in the culture medium containing a combination of a GSK3 inhibitor (CHIR99021) and a BMP signaling pathway activator (BMP7) in Example 1, they can be efficiently induced into body wall mesoderm cells, with a HAND1 and FOXF1 double-positive ratio of more than 95%.

3. Induction and differentiation method of somatic mesoderm derived marrow stromal cells (SM-MSCs) and identification

1. Experimental methods

The parietal mesoderm cells obtained by inducing human induced pluripotent stem cells (hiPSC) in the previous step are induced into bone marrow stromal cells using bone marrow stromal cell culture medium, wherein the bone marrow stromal cell culture medium is: the bone marrow stromal cell serum-free complete culture medium (SM-MSCs medium) in Example 2, or commercially available MSC culture medium (StemFit; Ajinomoto) or commercially available MSC culture medium (ACF; STEM CELL Technologies).

The specific steps are as follows:

(1) The culture medium of the body wall mesoderm cells identified in the previous step was replaced with the stromal cell culture medium, and the cells were cultured statically in an incubator at 37°C, 5% _CO2 , and 95% humidity, with the medium changed every 2 to 3 days.

(2) After the cells grow to 80-90% confluence, they are digested and passaged using Accutase. The cells are transferred into a 15 ml centrifuge tube and centrifuged at 1100 rpm for 4 min to collect the cells. After discarding the supernatant, the cells are inoculated into a new culture dish at a ratio of 1:3 to complete cell passage.

(3) Repeat step 2, and after continuous subculturing in the bone marrow stromal cell culture medium for 6 to 8 times, take some cells to detect the expression of cell surface molecules CD34, CD45, CD44, CD90 and CD140b by flow cytometry. When the cells express bone marrow stromal cell markers CD44, CD90 and CD140b, but do not express hematopoietic stem cell markers CD34 and CD45, the bone marrow stromal cells derived from the parietal mesoderm cells are obtained.

(4) Different batches of induced parietal mesoderm cells derived from bone marrow stromal cells were labeled as SM-MSCs 1, SM-MSCs 2, SM-MSCs 3, etc., and used in subsequent experiments.

(5) Cell Counting Kit (CCK-8) was used to detect the proliferation of the somatic mesoderm cell-derived bone marrow stromal cells (SM-MSCs) prepared in this example and the bone marrow mesenchymal stem cells (BMSCs) directly isolated from human long bone marrow.

(6) Total RNA from the induced differentiated parietal mesoderm cells and their derived bone marrow stromal cells was extracted using RNAzol and then reverse transcribed into cDNA. The expression of the homeodomain transcription factor (HOX9-13) gene was detected by fluorescence quantitative PCR.

2. Experimental results

Figure 4 shows the cell morphology of the parietal mesoderm cells in Example 3 after 6 passages in bone marrow stromal cell culture medium (commercially available MSC culture medium (StemFit)), immunofluorescence staining of CD90 (Figure 4A), and flow cytometry analysis of cell surface markers (Figure 4B); the morphology of the induced parietal mesoderm cell-derived bone marrow stromal cells is similar to that of the bone marrow stromal cells, showing a typical fibrous shape, and immunofluorescence staining shows that they highly express CD90; and the flow cytometry detection results show that the parietal mesoderm cell-derived bone marrow stromal cells express bone marrow stromal cell surface markers CD44, CD90, and CD140b, and do not express hematopoietic stem cell surface markers CD34 and CD45.

Figure 5 shows the expression of homeodomain transcription factor (HOX) genes in the marrow stromal cells derived from the parietal mesoderm induced by the marrow stromal cell culture medium (commercially available MSC culture medium (StemFit)) using fluorescent quantitative PCR in Example 3. The results show that the induced marrow stromal cells derived from the parietal mesoderm and the skeletal mesenchymal cells have similar typical homeodomain transcription factor (HOX) gene expression patterns.

Figure 6 compares the proliferation ability of two batches (SM-MSCs 1, SM-MSCs 2) of marrow stromal cells derived from parietal mesoderm in different batches induced by marrow stromal cell culture medium (commercially available commercial MSC culture medium (StemFit)) in Example 3 and two batches (BMSCs 1, BMSCs 2) of marrow mesenchymal stem cells isolated from different batches in Control Example 1, which is detected by CCK-8 and plotted. The results show that the marrow stromal cells derived from parietal mesoderm cells induced have stronger proliferation ability.

FIG7 shows the mesodermal cell-derived bone marrow stromal cells prepared in different bone marrow stromal cell culture media in Example 3, and further compares the proliferation ability (FIG7 left) and proliferation generation (FIG7 right). The bone marrow stromal cell culture media include bone marrow stromal cell serum-free complete culture medium (SM-MSCs medium), commercial MSC culture medium (StemFit; Ajinomoto), and commercial MSC culture medium (ACF; STEM CELL Technologies).

The results showed that in addition to commercial MSC culture media (StemFit and ACF) that can induce bone marrow stromal cells, the bone marrow stromal cell serum-free complete culture medium (SM-MSCs medium) in Example 2 can also induce bone marrow stromal cells, and their proliferation rate is faster and the number of proliferation generations is more, with significant statistical differences.

Example 4 Detection of the osteogenic, chondrogenic and hematopoietic supporting abilities of somatic mesoderm derived marrow stromal cells (SM-MSCs) in vitro

1. Experimental Methods

In this example, the somatic mesoderm cell-derived bone marrow stromal cells prepared in Example 3 and the bone marrow mesenchymal stem cells isolated in Control Example 1 were used. They were cultured using a commercial MSC culture medium (StemFit; Ajinomoto) or the bone marrow stromal cell serum-free complete culture medium (SM-MSCs medium) in Example 2 as the bone marrow stromal cell culture medium.

1. The ability of bone marrow stromal cells derived from body wall mesoderm cells to form osteoblasts and chondrocytes in vitro

(1) In vitro osteogenic differentiation: When the growth density of the bone marrow stromal cells derived from the parietal mesoderm cells prepared in Example 3 and the bone marrow mesenchymal stem cells (BMSCs) isolated in Control Example 1 reached 80-90%, the culture medium was replaced with osteogenic differentiation medium (DMEM (L) supplemented with 10% (V/V) FBS, 10 mM β-glycerophosphate, 50 μg/ml vitamin C and 100 nM dexamethasone). The medium was changed every 3 days, and Alizarin red staining was performed after 21 days of induction culture.

(2) In vitro chondrogenic differentiation: The bone marrow stromal cells derived from the somatic mesoderm cells prepared in Example 3 and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1 were digested and collected, and the cells were divided into 15 ml centrifuge tubes at a volume of 2.0-3.0×10 ⁵ /15 ml centrifuge tubes, centrifuged at 300×g for 5 min, the supernatant was discarded, and 1 ml of chondrogenic differentiation induction medium (DMEM (H) supplemented with 10 ng/ml recombinant human transforming growth factor (TGF-β3), 100 nM dexamethasone, 50 μg/ml vitamin C, 1 mM sodium pyruvate, 40 μg/ml proline and ITS culture additive) was added, and the cells were placed in an incubator for incubation. The medium was changed every 3 days, and alcian blue staining was performed after 21 days of induction culture.

(3) RNAzol was used to extract the total RNA of osteoblasts induced by the bone differentiation method of the bone marrow stromal cells (SM-MSCs) in Example 3 and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1, which were induced and proliferated using commercial MSC culture medium (StemFit; Ajinomoto) and the total RNA of chondrocytes induced by the cartilage differentiation method. After reverse transcription into cDNA, fluorescence quantitative PCR was used to detect the gene expression of osteogenic genes (COL1A1, ALP, OCN and OPN) and chondrogenic genes (COL2A1, ACAN, RUNX2 and SOX9).

(4) RNAzol was used to extract total RNA from the marrow stromal cells derived from the body wall mesoderm cells prepared in different marrow stromal cell culture media (SM-MSCs medium, StemFit, ACF) in the above Example 3, and after reverse transcription into cDNA, fluorescence quantitative PCR was used to detect the expression of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L, ANGPT1); and total RNA of osteoblasts induced by the bone differentiation method and total RNA of chondrocytes induced by the cartilage differentiation method were extracted from the marrow stromal cells derived from the body wall mesoderm cells prepared in different marrow stromal cell culture media (SM-MSCs medium, StemFit, ACF) in the above Example 3, and after reverse transcription into cDNA, fluorescence quantitative PCR was used to detect the gene expression of osteogenic genes (COL1A1, ALP, OCN and OPN) and chondrogenic genes (COL2A1, ACAN, RUNX2 and SOX9).

2. Detection of in vitro hematopoietic support ability of somatic mesoderm derived marrow stromal cells (SM-MSCs)

(1) Total RNA was extracted from the somatic mesoderm cell-derived bone marrow stromal cells (SM-MSCs) prepared in Example 3 and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1 using RNAzol, and then reverse transcribed into cDNA. The expression of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L and ANGPT1) was compared by fluorescence quantitative PCR.

(2) In vitro maintenance assay of hematopoietic stem cells (HSCs)

The somatic mesoderm cell-derived bone marrow stromal cells (SM-MSCs) prepared in Example 3 and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1 were pre-seeded on a 24-well plate at a density of 90%, cultured overnight, and 0.5 mg/ml mitomycin C was added to the culture medium on the second day to treat BMSCs and SM-MSCs. After 3 hours, the culture medium containing mitomycin C was discarded, washed 3 times with PBS, and replaced with fresh culture medium to obtain BMSCs and SM-MSCs treated with mitomycin C. Subsequently, the CD34+HSCs in human umbilical cord blood sorted by flow cytometry were resuspended with StemSpan ^Tm CD34+ expansion culture medium (Stem Cell Technologies) and inoculated into the treated BMSCs and SM-MSCs at a volume of 5000 cells per well. The culture was carried out for 9 days, and the medium was half-changed every two days. At the same time, CD34+HSCs cultured alone in the same StemSpan ^Tm CD34+ expansion culture medium were used as controls. After 9 days of co-culture, the cells suspended in the culture medium were collected and flow cytometry was used to detect the maintenance of CD34+ cells under different co-culture conditions.

(3) Long-Term Culture Initiating Cell Assays (LTC-IC) in vitro assay of hematopoietic stem cells (HSCs) and bone marrow stromal cells derived from parietal mesoderm

The somatic mesoderm cell-derived bone marrow stromal cells (SM-MSCs) prepared in Example 3 and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1 were pre-seeded on a 24-well plate at a density of 90% and cultured overnight. The next day, 0.5 mg/ml mitomycin C was added to the culture medium to treat the BMSCs obtained in Control Example 1 and the SM-MSCs prepared in Example 3. After 3 hours, the culture medium containing mitomycin C was discarded, washed 3 times with PBS, and replaced with fresh culture medium to obtain BMSCs and SM-MSCs treated with mitomycin C. CD34+HSC (cells) were inoculated into BMSCs and SM-MSCs at 1000 cells per well and cultured with StemSpan ^Tm CD34+ expansion culture medium (Stem Cell Technologies), with half of the medium replaced every two days. At the same time, CD34+HSC cultured alone in the same culture medium was used as a control. After 35 days of co-cultivation, 10,000 cells isolated from the supernatant of the co-cultivation system were seeded in methylcellulose (Stem Cell Technologies) plates and incubated at 37°C and 5% _CO2 for 14 days. The colonies formed were photographed, counted, and analyzed.

2. Experimental Results

FIG8 shows the bone marrow stromal cells (SM-MSCs) derived from the body wall mesoderm cells prepared by using the bone marrow stromal cell culture medium (commercially available MSC culture medium (StemFit)) in Example 3 and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1, and further compares the ability of in vitro osteogenic, chondrogenic and hematopoietic support, including in vitro osteogenic and chondrogenic staining, ELISA quantification ( FIG8A ) and fluorescence quantitative PCR detection of osteogenic (COL1A1, ALP, CON and OPN) and chondrogenic (COL2A1, A The expression levels of CAN, RUNX2 and SOX9 genes were detected (Figure 8B), the expression of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L, ANGPT1) was detected by real-time fluorescence quantitative PCR (Figure 8C), the maintenance ratio of CD34+HSC in the co-culture system was detected by flow cytometry (Figure 8D), and the clonal formation analysis of granulocyte and macrophage colony-forming units (CFU-GM) and burst erythroid colony-forming units (BFU-E) formed in LTC-IC and their statistical graphs were performed (Figure 8E).

The results showed that compared with bone marrow mesenchymal stem cells (BMSCs), the body wall mesoderm-derived bone marrow stromal cells (SM-MSCs) showed a darker dark red color when stained with Alizarin Red S. At the same time, the absorbance measured by the microplate reader at 562 nm also showed that the Alizarin Red S staining of SM-MSCs was darker (Figure 8A), indicating that the calcium salt deposition generated when SM-MSCs were induced to osteogenesis was richer and the osteogenic ability was stronger. For chondrogenic differentiation, toluidine blue staining showed that the chondrocytes induced by SM-MSCs were purple-blue, the generated cartilage matrix was rich, and the diameter of the chondrocytes was larger (Figure 8A). Fluorescence quantitative PCR further detected the corresponding osteogenic (COL1A1, ALP, CON, OPN) and chondrogenic (COL2A1, ACAN, RUNX2, SOX9) markers. The results also showed that SM-MSCs had a stronger chondrogenic differentiation ability in vitro (Figure 8B). The above evidence shows that the bone marrow stromal cells (SM-MSCs) derived from the body wall mesoderm cells have superior osteogenic and chondrogenic abilities in vitro compared with BMSCs. In addition, in vitro fluorescence quantitative PCR detected that SM-MSCs expressed more hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L, ANGPT1) than BMSCs (Figure 8C), and there was a statistical difference. At the same time, the in vitro hematopoietic support ability test showed that the proportion of CD34+HSCs co-cultured with SM-MSCs to maintain positive was significantly higher than that of BMSCs and the control group (Figure 8D). CFU analysis also showed that SM-MSCs produced the largest number of granulocyte and macrophage colony-forming units (CFU-GM) and burst erythroid colony-forming units (BFU-E) (Figure 8E).

The above evidence shows that SM-MSCs have stronger osteogenic and chondrogenic differentiation ability in vitro than BMSCs, and at the same time express higher hematopoietic support genes, can better maintain the expression of CD34+HSCs and have better ability to promote hematopoietic stem cell cloning and proliferation in vitro.

Figure 9 shows the bone marrow stromal cells derived from the parietal mesoderm cells prepared in different bone marrow stromal cell culture media (SM-MSCs medium, StemFit, ACF) in Example 3, and further compares the osteogenic, chondrogenic and hematopoietic support abilities in vitro. Including fluorescence quantitative PCR to detect the expression of osteogenic (COL1A1, ALP, CON and OPN) and chondrogenic (COL2A1, ACAN, RUNX2 and SOX9) genes (Figure 9A), and real-time fluorescence quantitative PCR to detect the expression of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L, ANGPT1) (Figure 9B).

The results showed that the bone marrow stromal cells induced by the serum-free complete culture medium of bone marrow stromal cells (SM-MSCs medium) in Example 2 had better osteogenic, chondrogenic ( FIG. 9A ) and hematopoietic support ( FIG. 9B ) abilities, with significant statistical differences.

The evidences in Figures 7 and 9 above show that the serum-free complete culture medium for bone marrow stromal cells (SM-MSCMedium) described in the present invention can not only stably and efficiently induce and expand bone marrow stromal cells, but also the obtained bone marrow stromal cells have better osteogenic, chondrogenic and hematopoietic support capabilities. Therefore, the serum-free complete culture medium for bone marrow stromal cells (SM-MSCMedium) described in the present invention has certain advantages in inducing pluripotent stem cell-derived bone marrow stromal cells.

Example 5 Detection of the in vivo osteogenic and hematopoietic support ability of somatic mesoderm derived marrow stromal cells (SM-MSCs)

1. Experimental Methods

This example uses the somatic mesoderm cell-derived bone marrow stromal cells prepared in Example 3 and the bone marrow mesenchymal stem cells separated in Control Example 1. Both cells are cultured using commercial MSC culture medium (StemFit; Ajinomoto) as the bone marrow stromal cell culture medium.

(1) Sample preparation: The somatic mesoderm cell-derived bone marrow stromal cells (SM-MSCs) prepared in Example 3 were selected, and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1 were used as controls. The two groups of cells were simultaneously prepared into a 2×10 ⁷ cells/ml cell suspension, and 50 μl of the cell suspension was taken from each group and fully mixed into 30 mg of hydroxyapatite support material that had been soaked in 50 μl of chondrogenic induction solution overnight. The tube cover was loosened slightly, and the tube was placed in a 37°C, 5% CO ₂ incubator for incubation for about 2 hours, and then embedded together into the subcutaneous tissue of immunodeficient mice.

(2) In vivo transplantation: 1% pentobarbital was used for intraperitoneal anesthesia at a dosage of 50 mg/kg per immunodeficient mouse, with 5 mice in each group. After successful anesthesia, the skin surface was disinfected with iodine, and a wound about 1 cm long was cut with scissors on the upper right side of the back. The wound was bluntly separated with separation forceps and deepened to the left side of the wound. The BMSCs group cells and materials were sent to the left side of the neck in batches, and then the wound was sutured and disinfected. Then, a wound about 1 cm long was cut on the right side of the abdomen, and the SM-MSCs group cells and materials were sent to the right side of the abdomen, sutured, and disinfected. After they woke up, they were placed in a mouse cage. The mice were then observed daily and fed routinely for 8 weeks.

(3) Samples were collected after 8 weeks: (a) The collected specimens were fixed, decalcified, and the sections were subjected to Masson staining and immunofluorescence staining to compare the in vivo bone formation ability of SM-MSCs and BMSCs; (b) The specimens were fixed, decalcified, and sections were stained with HE, then photographed and the number of hematopoietic cell clusters formed was counted, and immunofluorescence staining of CD45+ hematopoietic progenitor cells was performed to compare the in vivo hematopoietic support ability of SM-MSCs and BMSCs.

2. Experimental Results

Figure 10 compares the in vivo osteogenesis and hematopoietic support capabilities of the body wall mesoderm-derived bone marrow stromal cells (SM-MSCs) induced in Example 3 and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1, including Masson staining of osteogenesis ability detection and its statistical graph (Figure 10A), immunofluorescence staining of osteogenic markers (OCN, OPG) (Figure 10B), hematopoietic cell clusters after HE staining and their number statistics (Figure 10C), and immunofluorescence staining of CD45+ hematopoietic progenitor cells (Figure 10D).

The results showed that the somatic mesoderm-derived bone marrow stromal cells (SM-MSCs) prepared in Example 3 had stronger osteogenic ability in immunodeficient mice than the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1, which was specifically manifested by deeper and wider Masson staining, and more osteoprotegerin (OPG) and osteocalcin (OCN) positive cells detected by immunofluorescence staining. In addition, the SM-MSCs induced in Example 3 can also support the formation of more hematopoietic cell clusters and the presence of more CD45+ hematopoietic progenitor cells in the subcutaneous transplantation site of immunodeficient mice.

The above evidences indicate that the induced somatic mesoderm-derived bone marrow stromal cells (SM-MSCs) of the present invention have stronger bone formation ability and more superior hematopoietic support ability in vivo compared with bone marrow mesenchymal stem cells (BMSCs) directly isolated from adult long bone marrow.

Example 6 Sequencing Analysis of Somatic Mesoderm Derived Marrowstromal Cells (SM-MSCs)

1. Experimental Methods

This example uses the somatic mesoderm cell-derived bone marrow stromal cells prepared in Example 3 and the bone marrow mesenchymal stem cells separated in Control Example 1. Both cells are cultured using commercial MSC culture medium (StemFit; Ajinomoto) as the bone marrow stromal cell culture medium.

(1) Sample preparation: The somatic mesoderm cell-derived bone marrow stromal cells (SM-MSCs) prepared in Example 3 were collected, and the bone marrow mesenchymal stem cells (BMSCs) in Control Example 1 were used as a control to extract total RNA according to the operating procedures of the RNA extraction kit produced by QIAGEN, and the next step was performed after the integrity evaluation of the total RNA was completed;

(2) Library construction: Using the reagents provided by Illumina and following the corresponding operating procedures, mRNA is isolated and purified from total RNA and reverse transcribed into cDNA. The modified cDNA fragments are then PCR amplified, purified, and enriched to establish a cDNA library.

(3) Sequencing and analysis: The established cDNA library was used to perform RNA sequencing and analyze the gene expression profile using a GA high-throughput sequencer (Illumina, San Diego, USA).

2. Experimental Results

FIG11 is a gene expression profile analysis of two batches (SMs1, SMs2) of different batches of body wall mesoderm induced in Example 3 and two batches of bone marrow stromal cells (SM-MSCs 1 and SM-MSCs 2) derived therefrom and two batches of bone marrow mesenchymal stem cells (BMSCs 1 and BMSCs2) directly isolated from different batches of long bone marrow in Control Example 1 by RNA-Seq, gene expression profile analysis between two samples from different batches at the same differentiation stage (SMs 1 vs SMs 2; SM-MSCs 1 vs SM-MSCs 2; BMSCs 1 vs BMSCs 2) ( FIG11A ), gene expression analysis of SMs1, SMs 2; SM-MSCs 1, SM-MSCs 2; BMSCs 1, BMSCs 2 at different stages ( FIG11B ), and gene expression analysis of SM-MSCs 1, SM-MSCs2 and BMSCs 1, BMSCs 2. 2 homeodomain transcription factor (HOX1-13) gene expression pattern (Figure 11C).

The results showed that the gene expression profiles between two samples of different batches at the same differentiation stage were highly similar (SMCs1 vs SMCs 2; SM-MSCs 1 vs SM-MSCs 2; BMSCs 1 vs BMSCs 2), showing the high reproducibility of the induction method of the present invention (Figure 11A). RNA-Seq gene expression profile analysis showed that SMCs highly expressed body wall mesoderm markers, such as PITX1, HAND1, and TBX4, while the expression pattern of bone marrow stromal cells induced from the body wall mesoderm was similar to that of BMSCs, no longer expressing body wall mesoderm markers PITX1, HAND1, and TBX4, but expressing bone marrow stromal cell surface markers such as CD44 (Figure 11B). In addition, SM-MSCs expressed similar homeodomain transcription factor (HOX9-13) genes to bone mesenchymal cells (Figure 11C).

Example 7 Induction of differentiation of embryonic stem cells (H1-ES) into bone marrow stromal cells via parietal mesoderm

The purpose of this example is to illustrate the induction of differentiation based on the stable establishment and culture of human embryonic stem cell line (H1-ES).

1. Experimental Methods

1. Culture of Human Embryonic Stem Cells (H1-ES)

Embryonic stem cells (H1-ES) are purchased commercially. H1-ES cells are also expanded on a large scale using mTeSR culture medium from STEM CELL to maintain their undifferentiated state. At the same time, the culture medium needs to be coated with matrix gel, which can be Matrigel or laminin LN.

The specific steps are as follows:

(1) Coating culture dishes: Thaw Matrigel on ice and dilute the stock solution of Matrigel with pre-cooled DMEMF12 at a volume ratio of 1:100. Add the solution to the well plate and coat overnight at room temperature.

(2) Thawing embryonic stem cells (H1-ES): H1-ES cells were quickly thawed in a 37°C water bath, transferred to a 15 ml centrifuge tube containing 5 ml of mTeSR culture medium, and centrifuged at 1100 rpm for 4 min to collect the cells.

(3) Remove the coated Matrigel or laminin LN, resuspend the cells in 2 ml of mTeSR, inoculate the cells evenly, and place them in a 37°C, 5% _CO2 incubator for static culture.

(4) The culture medium was replaced once a day, and the cells were observed to maintain an undifferentiated state. The culture was continued until the H1-ES cell clones grew to a density of 80-90%, and then the cells were subcultured.

(5) Cell passaging: Wash the cells twice with PBS, add ReLeSR ^™ , incubate at 37°C for 1 to 5 min, discard ReLeSR ^™ , and gently pipette the cells with 1 ml of mTeSR culture medium until the cells are clumps of appropriate size.

(6) Collect the mTeSR resuspended cells and evenly inoculate the cells into a well plate pre-coated with Matrigel at a volume of 100 μl per well, and place the well plate in a 37°C, 5% CO ₂ incubator for static adherence culture.

(7) Repeat the above culture expansion steps to obtain sufficient cell quantity for the next step of differentiation induction.

2 Induction, differentiation and identification of somatic mesoderm cells (SMCs) derived from embryonic stem cells (H1-ES)

(1) When the cells grow to 80-90% of their density, wash the cells twice with PBS, add 500 μl of Accutase and incubate at 37°C for 4 min. Observe under a microscope to dissociate the cells into single cells or small cell clumps. Aspirate the Accutase and gently blow the cells with PBS to disperse them evenly.

(2) Transfer the cells into a 15 ml centrifuge tube and centrifuge at 1100 rpm for 4 min to collect the cells.

(3) Resuspend the cell pellet in DMEM-F12 culture medium containing 3 μM CHIR99021, and evenly inoculate the cells into a well plate or culture dish pre-coated with Matrigel at a cell density of 1×10 ⁴ /cm ² , and place in a 37°C, 5% CO ₂ incubator for static culture.

(4) After 2 days of culture, mesendoderm cells were induced.

(5) Thereafter, the culture medium was changed to the culture medium of Example 1 and induced for 5 to 7 days to induce the mesendoderm cells into parietal mesoderm cells, during which the medium was changed every day.

2. Induction and identification of embryonic stem cell (H1-ES)-derived parietal mesoderm cells into bone marrow stromal cells (SM-MSCs)

The somatic mesoderm cells obtained by induction of embryonic stem cells (H1-ES) in the previous step are further induced into bone marrow stromal cells using bone marrow stromal cell culture medium, wherein the bone marrow stromal cell culture medium is: the bone marrow stromal cell serum-free complete culture medium (SM-MSCs medium) in Example 2, or commercially available MSC culture medium (StemFit; Ajinomoto) or commercially available MSC culture medium (ACF; STEM CELL Technologies).

The specific steps are as follows:

(1) Replace the culture medium of the parietal mesoderm cells induced in the previous step with the culture medium of bone marrow stromal cells, and culture them statically in an incubator at 37°C and 5% _CO2 , changing the medium every 2 to 3 days.

(2) After the cells grow to 80-90% confluence, they are digested and passaged using Accutase. The cells are transferred into a 15 ml centrifuge tube and centrifuged at 1100 rpm for 4 min to collect the cells. After discarding the supernatant, the cells are inoculated into a new culture dish at a ratio of 1:3, completing 6-8 cell passages.

(3) Repeat step 2, and after 6 to 8 consecutive subcultures, take some cells to detect the expression of cell surface molecules CD34, CD45, CD44, CD90 and CD140b by flow cytometry. When the cells express bone marrow stromal cell markers CD44, CD90 and CD140b, but do not express hematopoietic stem cell markers CD34 and CD45, the surface markers of bone marrow stromal cells derived from body parietal mesoderm cells are obtained.

3. In vivo development and hematopoietic support capacity of bone marrow stromal cells (SM-MSCs) induced by embryonic stem cells (H1-ES) via parietal mesoderm cells

In this step, the bone marrow stromal cells (SM-MSCs) obtained by inducing the embryonic stem cells (H1-ES) prepared in Example 7 through the parietal mesoderm cells and the bone marrow mesenchymal stem cells (BMSCs) separated in Control Example 1 were used. And both were cultured using the bone marrow stromal cell serum-free complete culture medium (SM-MSCs medium) in Example 2 as the bone marrow stromal cell culture medium.

(1) Sample preparation: bone marrow stromal cells (SM-MSCs) obtained by inducing human embryonic stem cells (H1-ES) via parietal mesoderm cells in Example 7 were selected, and bone marrow mesenchymal stem cells (BMSCs) isolated in Control Example 1 were used as controls. Both groups of cells were simultaneously prepared into a 2×10 ⁷ cells/ml cell suspension, and 50 μl of the cell suspension was taken from each group and fully mixed into 30 mg of hydroxyapatite support material that had been soaked in 50 μl of chondrogenic induction solution overnight. The tube cap was loosened slightly, and the tube was placed in an incubator at 37°C and 5% CO ₂ for incubation for about 2 hours, and then embedded together into the subcutaneous tissue of immunodeficient mice.

(2) In vivo transplantation: 1% pentobarbital was used for intraperitoneal anesthesia at a dosage of 50 mg/kg per immunodeficient mouse, with 5 mice in each group. After successful anesthesia, the skin surface was disinfected with iodine, and a wound about 1 cm long was cut with scissors on the upper right side of the back. The wound was bluntly separated with separation forceps and deepened to the left side of the wound. The BMSCs group cells and materials were sent to the left side of the neck in batches, and then the wound was sutured and disinfected. Then, a wound about 1 cm long was cut on the right side of the abdomen, and the SM-MSCs group cells and materials were sent to the right side of the abdomen, sutured, and disinfected. After they woke up, they were placed in a mouse cage. The mice were then observed daily and fed routinely for 8 weeks.

(3) Samples were collected after 8 weeks: (a) The collected specimens were fixed, decalcified, and the sections were subjected to Masson staining and immunofluorescence staining to compare the in vivo bone formation ability of SM-MSCs and BMSCs; (b) The sections were fixed, decalcified, and stained with HE, then photographed and the number of hematopoietic cell clusters formed was counted, and immunofluorescence staining of CD45+ hematopoietic progenitor cells was performed to compare the in vivo hematopoietic support ability of SM-MSCs and BMSCs.

2. Experimental Results

Figure 12 shows the flow cytometry surface markers (Figure 12A), morphology and in vitro osteoblastic and chondrogenic detection diagram (Figure 12B) of bone marrow stromal cells derived from embryonic stem cells (H1-ES) prepared using bone marrow stromal cell culture medium (commercially available MSC culture medium (StemFit)) in Example 7, and the bone formation (Figure 12C) and hematopoietic support ability detection diagram (Figure 12D) of bone marrow stromal cells derived from embryonic stem cells (H1-ES) prepared using bone marrow stromal cell serum-free complete culture medium (SM-MSCsmedium) compared with the bone marrow mesenchymal stem cells (BMSCs) in control example 1 obtained by culture in the corresponding culture medium.

The results showed that the bone marrow stromal cells (SM-MSCs) induced by human embryonic stem cells (H1-ES) in this example via body wall mesoderm cells had a morphology similar to that of bone marrow stromal cells, exhibited adherent growth and typical fibrous shape, expressed bone marrow stromal cell markers CD44, CD90, and CD140b, and did not express surface markers of hematopoietic stem cells CD34 and CD45; Alizarin Red S staining showed that the induced osteoblasts were colored dark red, and toluidine blue staining showed that the induced chondrocytes were purple-blue, and the generated cartilage matrix was rich.

In addition, the in vivo transplantation experiment in immunodeficient mice showed that the bone marrow stromal cells (SM-MSCs) induced by embryonic stem cells (H1-ES) prepared using the bone marrow stromal cell serum-free complete culture medium (SM-MSCs medium) in Example 7 also had good functional characteristics. Compared with the bone marrow mesenchymal stem cells (BMSCs) directly isolated from the adult long bone marrow cultured in the same bone marrow stromal cell culture medium, they had stronger bone formation ability (Figure 12C) and better hematopoietic support ability (Figure 12D) in immunodeficient mice.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims (4)

1. A use of a culture medium for inducing pluripotent stem cells into parietal mesoderm cells, characterized in that the culture medium is a DMEM-F12 culture medium containing 1% by volume ITS culture additive, 1% by volume NEAA, 1% by volume Glutamax, 3 μM CHIR99021 and 100 ng/ml BMP7. 2. A serum-free complete culture medium for bone marrow stromal cells, characterized in that the culture medium is an α-DMEM medium containing: 5% volume serum replacement, 1% volume NEAA, 1% volume ITS culture additive, 5mM L-glutamate, 1Mm β-mercaptoethanol, 2ng/mL bFGF, 2ng/mL EGF, 1ng/mL VEGF, 20ng/ml human platelet-derived growth factor, 300ng/ml vitamin C, and 100μM uridine. 3. Use of the serum-free complete culture medium of bone marrow stromal cells according to claim 2 in inducing pluripotent stem cells into bone marrow stromal cells. 4. A method for obtaining bone marrow stromal cells by inducing pluripotent stem cells through the parietal mesoderm cell stage, characterized in that it comprises the following steps: S1. Inducing pluripotent stem cells into mesendoderm cells: After dissociating and dispersing the pluripotent stem cells, inoculate them into a culture plate or dish coated with matrix gel, and culture them in DMEM-F12 culture medium containing 3 μM CHIR99021 for 1 to 3 days to obtain mesendoderm cells; S2. Inducing the mesendoderm cells into the body wall mesoderm cells: using the culture medium for inducing pluripotent stem cells into the body wall mesoderm cells, culturing the product of the previous step for 5 to 7 days to obtain the body wall mesoderm cells, wherein the culture medium for inducing the pluripotent stem cells into the body wall mesoderm cells is the culture medium described in claim 1; S3. Inducing the body wall mesoderm cells into body wall mesoderm cell-derived bone marrow stromal cells: after the obtained body wall mesoderm cells are digested again and inoculated into bone marrow stromal cell culture medium, they are continuously subcultured with bone marrow stromal cell culture medium for 6 to 8 times to obtain body wall mesoderm cell-derived bone marrow stromal cells, wherein the bone marrow stromal cell culture medium is the bone marrow stromal cell serum-free complete culture medium as described in claim 2.