CN114672455A - Method for inducing bone marrow stromal cells by utilizing pluripotent stem cells - Google Patents
Method for inducing bone marrow stromal cells by utilizing pluripotent stem cells Download PDFInfo
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Abstract
The invention discloses a method for inducing bone marrow stromal cells by utilizing pluripotent stem cells, wherein the bone marrow stromal cells are obtained by inducing the pluripotent stem cells, strictly limiting the differentiation path of the bone marrow stromal cells through specific induction culture solution and undergoing a mesoderm cell stage in a body wall. The bone marrow stromal cells induced by different pluripotent stem cell strains obtained by the invention not only have a typical homeodomain transcription factor (HOX) gene expression mode similar to that of bone mesenchymal cells, but also have better osteogenic, chondrogenic and hematopoietic support capacities compared with bone marrow mesenchymal stem cells. Therefore, the marrow stromal cells with clear sources, high proliferation speed and low heterogeneity can provide a new and high-quality cell source for clinical transformation in the field of cell therapy, and can also provide an ideal in vitro model for researching pathogenesis supporting hematopoietic stem cell transplantation, limb skeletal development and related diseases.
Description
Technical Field
The invention relates to the technical field of stem cells, in particular to a method for inducing bone marrow stromal cells by utilizing pluripotent stem cells.
Background
Stromal Cells (MSCs) are one of the most common cells in the body, and are critical to 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 developmental potential, such as responding to injury in vivo by secreting trophic factors and promoting the regeneration of extracellular matrix (ECM) molecules, and promoting the fibrotic repair process in the event of regeneration failure (Cell Stem cell.2021 Oct 7; 28(10): 1690-; the stromal cells in the bone marrow also participate in forming a bone marrow hematopoietic microenvironment to support hematopoiesis, osteogenic differentiation and the like (Nature.2014 Jan 16; 505(7483):327-34.), and meanwhile, the stromal cells have irreplaceable effects on bone injury repair, bone marrow hematopoietic microenvironment or ecological niche reconstruction and the like due to low immunogenicity, and have wide clinical application prospects.
However, the acquisition of bone marrow stromal cells not only has great traumatism, but also has great heterogeneity of bone marrow-derived stromal cells of different individuals, and is difficult to continuously passage and largely expand in vitro, thus failing to meet the requirements of clinical treatment. Therefore, how to obtain bone marrow stromal cells stably and in large quantities in vitro becomes a key problem to solve the limitation of clinical transformation.
In recent years, a number of articles have reported the use of pluripotent stem cells, including induced pluripotent stem cells and embryonic stem cells, in differentiating into various germ layer cell types, but no method has been reported for inducing differentiation of pluripotent stem cells to obtain their derived bone marrow stromal cells via the mesoderm stage of the body wall. Among them, Somatic Mesoderm cells (SM-MCs) are one of the cells belonging to the Mesoderm stage of the lateral plate during embryonic development, which are located between the ectoderm and the endosomal cavity and are progenitor cells forming connective tissue and skeletal elements (bone and cartilage) of the limbs. Previous studies have shown that somatic mesoderm cell-derived stromal cells are present in the bone marrow of long bones (Front Genet.2019 Oct 11; 10: 977; development.2020 Jun 19; 147(12): dev 175059). In addition, in the prior art (Chinese patent "immortalized rat bone marrow stromal cell line and preparation method thereof"), the catalytic subunit gene of human telomerase reverse transcriptase is introduced into rat bone marrow stromal cells through a retrovirus vector to obtain the immortalized rat bone marrow stromal cell line, which can be amplified in vitro for a long time, but cannot be applied to clinical cell therapy due to the species problem.
Disclosure of Invention
In order to overcome the problem that the prior art can not utilize pluripotent stem cells to prepare bone marrow stromal cells, the invention obtains the bone marrow stromal cells with a specific development path in vitro through a specific induced differentiation process from the perspective of cell development origin. The invention provides a method for inducing bone marrow stromal cells by utilizing pluripotent stem cells, which is a stable and efficient preparation method of the bone marrow stromal cells derived from mesoderm on the body wall induced by the pluripotent stem cells. Can provide a new and high-quality cell source for clinical transformation of cell therapy, and simultaneously can provide an ideal in vitro model for researching pathogenesis supporting hematopoietic stem cell transplantation, limb skeletal development and related diseases.
The first purpose of the invention is to provide bone marrow stromal cells.
The second purpose of the invention is to provide bone marrow stromal cells derived from mesodermal cells in a body wall.
The third purpose of the invention is to provide a culture solution for inducing pluripotent stem cells into somatic mesoderm cells.
The fourth purpose of the invention is to provide a serum-free complete culture solution for the bone marrow stromal cells.
The fifth purpose of the invention is to provide the application of any one of the culture solution and/or the bone marrow stromal cell serum-free complete culture solution in the induction of bone marrow stromal cells by pluripotent stem cells
It is a sixth object of the present invention to provide a method for inducing bone marrow stromal cells using pluripotent stem cells.
The seventh purpose of the invention is to provide the bone marrow stromal cells prepared by the method.
An eighth object of the present invention is the use of any of the bone marrow stromal cells described herein for the preparation of a medicament having the ability to promote hematopoietic support and/or bone repair.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention efficiently induces pluripotent stem cells into Mesoderm cells (SMCs) in a Mesendoderm stage by a limited induction method, replenishes and inoculates the Mesoderm cells in the body wall, and then replaces the Mesoderm cells in the body wall with a Marrow stromal cell serum-free complete culture solution (SM-MSC Medium) or a commercial MSC culture solution (StemFit) or a commercial MSC culture solution (ACF) for continuous culture, detects the phenotype of the cells, and obtains the Marrow stromal cells (SM-MSCs) from the Mesoderm cells in the body wall
As used herein, "SMCs" refers to somatic mesodermal cells.
As used herein, "SM-MSCs" refers to somatic mesodermal cell-derived bone marrow stromal cells induced by pluripotent stem cells via somatic mesodermal cells.
The invention provides a set of new, standardized and strong-feasibility bone marrow stromal cell in-vitro induced differentiation process, and the bone marrow stromal cells which can be produced in batch not only can provide a new and high-quality cell source for clinical transformation in the field of cell therapy, but also can provide an ideal in-vitro model for researching and supporting hematopoietic stem cell transplantation, limb skeletal development and pathogenesis of related diseases.
Specifically, induced pluripotent stem cells are subjected to passage amplification on a culture plate or culture dish coated with matrigel, and when the induced pluripotent stem cells grow to the amount of cells required for induction, single cell suspension is prepared for adherence, and then culture solution added with a GSK-3 inhibitor is used for directional induction for 1-3 days, so that mesendoderm cells are obtained; then, directionally inducing the mesendoderm cells for 5-7 days by using a body wall mesoderm cell induction culture solution, and inducing the mesendoderm cells into a body wall mesoderm cell population; and finally, after the mesoderm cells on the body wall in the previous step are digested again and inoculated, continuously subculturing the bone marrow stromal cells for 6-8 times by using a serum-free complete culture solution (SM-MSC Medium) or a commercially available commercial MSC culture solution (StemFit or ACF), identifying the surface markers of the bone marrow stromal cells by flow, and detecting the hematopoietic support capacity and the osteogenic chondrogenic capacity of the bone marrow stromal cells.
The invention therefore claims a bone marrow stromal cell, which is obtained by the continuous subculture in the bone marrow stromal cell culture solution after the induction of pluripotent stem cells into somatic mesoderm cells.
And somatic mesoderm cell-derived bone marrow stromal cells induced from pluripotent stem cells after undergoing the somatic mesoderm cell stage.
Preferably, the pluripotent stem cell is an induced pluripotent stem cell or an embryonic stem cell.
More preferably, the induced pluripotent stem cell induces a pluripotent stem cell.
The invention also claims a culture solution for inducing the pluripotent stem cells into the mesoderm cells in the body wall, wherein the culture solution is a basic culture solution containing the combination of the GSK-3 inhibitor and the BMP signal pathway activator.
Preferably, the basal 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 CHIR 99021.
Preferably, the BMP signal pathway activator is one or more of BMP2, BMP4 or BMP 7.
More preferably, the BMP signaling pathway activator is BMP 7.
More preferably, the culture solution contains 1-20 mu M CHIR99021 and 1-500 ng/ml BMP 7.
Even more preferably, the culture solution contains 1-5 mu M CHIR99021 and 1-100 ng/ml BMP 7.
Further preferably, the culture broth contains 3. mu.M CHIR99021 and 100ng/ml BMP 7.
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 medium further comprises 1% (V/V) ITS culture supplement, 1% (V/V) NEAA, and 1% (V/V) Glutamax.
Most preferably, the culture is DMEM-F12 medium containing 1% (V/V) ITS culture supplement, 1% (V/V) NEAA, 1% (V/V) Glutamax, and 3. mu.M CHIR99021 and 100ng/ml BMP 7.
The invention also claims a bone marrow stromal cell serum-free complete culture solution (SM-MSC Medium), which can induce somatic mesoderm cells to be induced into bone marrow stromal cells, and comprises a basic culture solution containing 1-10% (V/V) serum substitute, 1-5% (V/V) NEAA, 1-5% (V/V) ITS culture additive, 1-10 mM L-glutamic acid, 0.1-10 Mm beta-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 Urridine.
Preferably, the basal medium is an alpha-DMEM medium.
Preferably, the serum replacement is KOSR or UltroserTM G serum substitute。
More preferably, the serum replacement is KOSR.
More preferably, the human platelet-derived growth factor is PDGFAA and PDGFBB.
Still preferably, the bone marrow stromal cell serum-free complete culture Medium (SM-MSC Medium) is an alpha-DMEM culture Medium containing 1-5% (V/V) serum substitute KOSR, 1-5% (V/V) NEAA, 1-5% (V/V) ITS culture additive, 1-5 mM L-glutamic acid, 1-5 mM beta-mercaptoethanol, 1-10 ng/mL bFGF, 1-10 ng/mL EGF, 1-10 ng/mL VEGF, 1-100 ng/mL human platelet-derived growth factor PDGFAA, 1-100 ng/mL human platelet-derived growth factor PDGFBB, 0.1-300 ng/mL vitamin C and 0.1-500 mu M Uridine Urridine.
Still more preferably, the bone marrow stromal cell serum-free complete Medium (SM-MSC Medium) is an alpha-DMEM Medium containing 5% (V/V) serum replacement KOSR, 1% (V/V) NEAA, 1% (V/V) ITS culture additives, 5mM L-glutamic acid, 1mM beta-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, 100. mu.M Uridine.
The invention claims the application of the culture solution and/or the bone marrow stromal cell serum-free complete culture solution in the induction of bone marrow stromal cells by pluripotent stem cells.
Preferably, the culture solution and/or the bone marrow stromal cell serum-free complete culture solution is used for the pluripotent stem cell-induced bone marrow stromal cells derived from mesoderm cells in a body wall.
Preferably, the combined pluripotent stem cells of the culture solution and the bone marrow stromal cell serum-free complete culture solution are used for inducing bone marrow stromal cells.
The invention also claims a method for inducing bone marrow stromal cells by utilizing the pluripotent stem cells, which comprises the following steps:
s1, inducing pluripotent stem cells into mesendoderm cells: dissociating and dispersing the pluripotent stem cells, inoculating the dissociated and dispersed pluripotent stem cells into a culture plate or a culture dish coated with matrigel, and culturing the cells for 1 to 3 days by using a culture solution containing CHIR99021 to obtain mesendoderm cells;
s2, inducing mesendoderm cells into body wall mesoderm cells: inducing the pluripotent stem cells into a culture solution of the mesoderm cells in the body wall, and culturing the product of the previous step for 5-7 days to obtain the mesoderm cells in the body wall;
s3, inducing the somatic mesoderm cells into somatic mesoderm cell-derived bone marrow stromal cells: and (3) digesting the obtained mesoderm cells on the body wall again, inoculating the cells into a bone marrow matrix cell culture solution, and carrying out continuous subculture for 6-8 times by using the bone marrow matrix cell culture solution to obtain the bone marrow matrix cells derived from the mesoderm cells on the body wall.
Preferably, the bone marrow stromal cell culture solution is the bone marrow stromal cell serum-free complete Medium (SM-MSC Medium) or a commercially available MSC culture solution (e.g., StemFit or ACF).
More preferably, the bone marrow stromal cell culture solution is the bone marrow stromal cell serum-free complete culture solution.
Preferably, the pluripotent stem cell is an induced pluripotent stem cell or an embryonic stem cell.
More preferably, the pluripotent stem cell is a human-induced pluripotent stem cell or a human embryonic stem cell.
Preferably, in step S1, the culture solution containing CHIR99021 is DMEM-F12 culture solution containing 1-10 μm CHIR 99021.
Preferably, in step S1, the pluripotent stem cells are dissociated and dispersed into single cells or cell aggregates by Accutase, and the cells are seeded on a culture plate or a culture dish coated with matrigel, wherein the seeding number is 1 × 103～1×107/cm2。
More preferably, the number of cell inoculations is 1X 104～2×104/cm2。
Preferably, the Matrigel is Matrigel or laminin LN.
More preferably, the Matrigel is Matrigel, which is prepared using pre-cooled DMEM-F12 in a volume of 1: after the Matrigel stock solution is diluted by 100 percent, the culture plate or the culture dish is coated 1 to 12 hours in advance.
More preferably, the matrigel is laminin LN, diluted 1:100 in PBS and added to the well plate for overnight coating at room temperature.
Preferably, in step S1, the proportion of cells with double positive of the markers TBXT and MIXL1 of the induced mesendoderm cells can reach more than 95%, and the next induction can be performed without sorting and purification.
Preferably, in step S2, the induced mesodermal cells in the body wall highly express their markers HAND1, FOXF1, TBX4, PRRX1 and PITX1, while the proportion of double positive cells of HAND1 and FOXF1 is more than 95%.
Preferably, in step S3, the marrow stromal cells from the mesoderm cells in the body wall are obtained after continuous subculturing with the marrow stromal cell culture solution for 6-8 times.
According to the experiments of the inventor, the marrow stromal cells from the body wall mesoderm can be obtained by using the serum-free complete culture solution (SM-MSC Medium) of the marrow stromal cells of the invention or commercial MSC culture solution (such as StemFit and ACF). However, the marrow stromal cells induced to expand in the marrow stromal cell serum-free complete Medium (SM-MSC Medium) of the invention have faster proliferation speed, more proliferation passage number, better osteogenic and hematopoietic supporting capability and significant statistical difference compared with the commercial MSC culture Medium (such as StemFit and ACF). Therefore, the serum-free complete culture solution for the bone marrow stromal cells (SM-MSC Medium) has obvious advantages in inducing the bone marrow stromal cells from the pluripotent stem cells.
Preferably, in step S3, the somatic mesoderm cells in step S3 express CD44, CD90 and CD140b after continuous subculturing in the bone marrow stromal cell culture solution for 6-8 times, and when the hematopoietic stem cell markers CD34 and CD45 are not expressed, the somatic mesoderm cell-derived bone marrow stromal cells are obtained.
The bone marrow stromal cells prepared by any one of the above methods also belong to the protection scope of the invention.
The application of the bone marrow stromal cells in preparing the medicine for promoting hematopoietic support and/or bone repair also belongs to the protection scope of the invention.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains the bone marrow stromal cells from pluripotent stem cells by inducing the mesoderm cell stage of the body wall through the pluripotent stem cells by strictly limiting the cell differentiation path by using a specific induction culture solution. The mesoderm cells in the body wall of the invention are not only homogeneous, but also have an induction efficiency of more than 95%. Meanwhile, the induced differentiation system has strong operability and standardized induced differentiation program, and can ensure stable properties and good functional characteristics of bone marrow stromal cells with different cell strain sources and different batches.
The marrow stromal cells with clear differentiation path and clear sources can be rapidly amplified in vitro, have a typical homeodomain transcription factor (HOX) gene expression mode similar to that of bone mesenchymal cells, and in vitro and in vivo experiments show that the marrow stromal cells (SM-MSCs) induced by mesoderm cells in body walls have better osteogenic, chondrogenic and hematopoietic supporting capacities compared with Bone Mesenchymal Stem Cells (BMSCs) directly separated from bone marrow. Therefore, the marrow stromal cells with clear sources, high proliferation speed and low heterogeneity can provide a new and high-quality cell source for clinical transformation in the field of cell therapy, and can also provide an ideal in vitro model for researching pathogenesis supporting hematopoietic stem cell transplantation, limb skeletal development and related diseases.
Drawings
FIG. 1 is a white light (FIG. 1A) and a photograph of pluripotent-labeled immunofluorescence (FIG. 1B) of the hipSC cells cultured in example 3.
Fig. 2 is a cell morphology map (fig. 2A) of hiPSC cells after 2 days of GSK-3 inhibitor treatment in example 3, and the results of real-time fluorescent quantitative PCR detection (fig. 2B) and immunofluorescence staining pictures (fig. 2C) of mesendoderm markers TBXT and MIXL 1.
FIG. 3 is a diagram showing the cell morphology of the hipSC-derived mesendoderm cells in example 3 after 6 days induction with a somatic mesoderm-induced culture medium (FIG. 3A), and the expression of the markers HAND1, FOXF1, TBX4, PRRX1 and PITX1 (FIG. 3B) were detected by real-time fluorescence quantitative PCR, while the expression of the markers HAND1 and FOXF1 (FIG. 3C) and the induction efficiency of the markers HAND1 and FOXF1 by flow assay (FIG. 3D) were detected by immunofluorescence staining
FIG. 4 shows the cell morphology of the somatic mesoderm cells of example 3 after 6 passages in bone marrow stromal cell culture medium (commercial MSC culture medium (StemFit)), immunofluorescent staining pattern (FIG. 4A) and cell surface marker flow analysis pattern (FIG. 4B) of CD 90.
FIG. 5 is a graph showing the expression of homeodomain transcription factor (HOX) genes in marrow stromal cells derived from mesoderm in body wall induced by bone marrow stromal cell culture medium (commercial MSC culture medium (StemFit)) using fluorescent quantitative PCR in example 3.
FIG. 6 is a graph showing the comparison of proliferation potency of two batches (SM-MSCs 1, SM-MSCs 2) of different batches of somatic mesoderm-derived bone marrow stromal cells induced by bone marrow stromal cell culture medium (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 1, which were measured for cell proliferation change by CCK-8 and plotted.
FIG. 7 shows the proliferation potency (FIG. 7A) and proliferation passage number (FIG. 7B) of somatic mesoderm-derived bone marrow stromal cells obtained in example 3 by preparing different bone marrow stromal cell culture solutions. Wherein the bone marrow stromal CELL culture solution comprises bone marrow stromal CELL serum-free complete culture solution (SM-MSCs medium), commercially available commercial MSC culture solution (StemFit; Ajinomoto), and commercially available commercial MSC culture solution (ACF; STEM CELL Technologies).
FIG. 8 is a graph showing the in vitro osteogenesis, chondrogenesis and hematopoietic support abilities of somatic mesoderm-derived bone marrow stromal cells (SM-MSCs) prepared in example 3 using a bone marrow stromal cell culture solution (commercially available MSC culture solution (StemFit)) and mesenchymal stem cells (BMSCs) in control example 1, including in vitro osteogenesis chondrogenesis staining, enzyme-linked immunosorbent assay (FIG. 8A), quantitative fluorescence quantitative PCR assay of osteogenesis (COL1A1, ALP, CON and OPN) and chondrogenesis (COL2A1, ACAN, RUNX2 and SOX9) gene expression levels (FIG. 8B), quantitative real-time fluorescence PCR assay of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L, ANGPT1) expression (FIG. 8C), assay of CD34+ maintenance ratio in coculture system (FIG. 8D), colony formation assay of CFIC-forming granulocyte (CFU-GM-forming unit), Burst red colony forming units (BFU-E) and their statistical plots (FIG. 8E).
FIG. 9 shows the in vitro osteogenic, chondrogenic and hematopoietic support capacities of the somatic mesodermal cell-derived bone marrow stromal cells prepared in different bone marrow stromal cell cultures (SM-MSCmedium, StemFit, ACF) in example 3, respectively. Comprises the steps of detecting the expression quantity of osteogenic (COL1A1, ALP, CON and OPN) and chondrogenic (COL2A1, ACAN, RUNX2 and SOX9) genes by fluorescence quantitative PCR (figure 9A), and detecting the expression quantity of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L and ANGPT1) by real-time fluorescence quantitative PCR (figure 9B).
Fig. 10 is a graph showing the capability of bone marrow stromal cells (SM-MSCs) derived from mesoderm in body wall induced in example 3 to support osteogenesis and hematopoiesis in vivo, including a Masson staining pattern and its statistical pattern (fig. 10A) for the osteogenesis capability assay, an immunofluorescence staining pattern (fig. 10B) for osteogenic markers (OCN, OPG), an immunofluorescence staining pattern (fig. 10C) for hematopoietic cell clusters and its number statistical pattern (fig. 10C) after HE staining, and immunofluorescence staining for CD45+ hematopoietic progenitor cells (fig. 10D), as compared to bone marrow mesenchymal stem cells (BMSCs) in control example 1.
FIG. 11 is a gene expression profiling analysis of two batches (SMs 1, SMs 2) in different body wall mesoderms induced in example 3 and two batches (SM-MSCs 1 and SM-MSCs 2) in different body wall mesoderm-derived bone marrow stromal cells induced in different body wall mesoderms in example 1 and two batches (BMSCs 1 and BMSCs 2) in bone marrow mesenchymal stem cells directly isolated from different batches of long bone marrow in control example 1 by RNA-Seq, and a gene expression profiling analysis (FIG. 11A) between 2 samples of the same differentiation stage (SMs 1 vs SMs 2; SM-MSCs 1 vs SM-MSCs 2; BMSCs 1 vs BMSCs 2), for SMs 1, SMs 2; SM-MSCs 1, SM-MSCs 2; BMSCs 1 and BMSCs 2 were subjected to gene expression analysis at different stages (FIG. 11B) and gene expression pattern maps of SM-MSCs 1 and SM-MSCs 2 and homeodomain transcription factors (HOX1-13) of BMSCs 1 and BMSCs 2 (FIG. 11C).
FIG. 12 is a surface marker map (FIG. 12A), a morphology map and a map of in vitro osteogenic chondrogenic assay (FIG. 12B) of embryonic stem cell (H1-ES) -derived bone marrow stromal cells prepared from a bone marrow stromal cell culture solution (commercially available MSC culture solution (StemFit)) in example 7, and a map of bone marrow mesenchymal stem cells (H1-ES) -derived bone marrow stromal cells prepared from a bone marrow stromal cell serum-free complete culture solution (SM-MSCs medium) in comparison with bone marrow mesenchymal stem cells (BMSCs) of comparative example 1 cultured in a corresponding medium, in immunodeficient mice (FIG. 12C) and hematopoietic support ability assay (FIG. 12D).
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
The primers used in the fluorescent quantitative PCR in the following examples are shown in the following table:
the antibodies used for immunofluorescent staining in the following examples are shown in the following table:
| 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 assay of the following examples are shown in the following table:
the method for cell immunofluorescence staining comprises the following steps: after cell clones were fixed with 4% paraformaldehyde PFA, washed 3 times with PBS, followed by blocking with 2% BSA for 30 min and 2 times with PBS. Add primary antibody, go overnight at 4 ℃, wash 3 times with PBS for 5 minutes each time; adding a secondary antibody, and incubating for 30-45 minutes at room temperature; after 4 times of PBS washing, 0.5 mu g/ml DAP I is added for dyeing for 10 minutes, after removing the DAPI, PBS is used for washing for three times; after dyeing, the film is sealed by a sealing agent, and finally, a picture is taken on a confocal plane.
The fluorescent quantitative PCR method comprises the following steps: cells were collected by digestion with Accutase, washed 3 times with PBS, lysed using 1ml RNAzol, total RNA extracted, and reverse transcribed to cDNA using a quantitative reverse transcription kit (Qiagen) in a 10 μ l reaction. Gene expression was calculated using Dynamo ColorFlash SYBR Green qPCR kit (Thermo Fisher Scientific) in LightCycler 480 detection system (fluorescent quantitative PCR assay. thermal cycling conditions: denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 30s, total 45 cycles. GAPDH was used as an internal control and 2-. DELTA.CT was used.
The method for detecting the bone marrow stroma cell flow comprises the following steps: bone marrow stromal cells were digested with Accutase, centrifuged at 1100rpm for 4min, the supernatant discarded, the cells resuspended in 50ul PBS, and homologous antibody (BD Pharmingen) used as a negative control (isotype control). Flow antibodies (CD34, CD45, CD44, CD90, CD140b) are added into the other tubes respectively for staining for 20min, washed with PBS for 2 times, each time for 5min, centrifuged at 1100rpm for 4min, the supernatant is discarded, the cells are resuspended with 200ul PBS, and the proportion of positive cells is detected on a flow cytometer.
Example 1 culture solution for inducing pluripotent stem cells into Somatic Mesoderm Cells (SMCs)
DMEM-F12 medium containing 1% (V/V) ITS culture supplement, 1% (V/V) NEAA, 1% (V/V) Glutamax, and 3. mu.M CHIR99021 and 100ng/ml BMP 7.
EXAMPLE 2 serum-free complete Medium for bone marrow stromal cells
alpha-DMEM medium containing 5% (V/V) serum replacement KOSR, 1% (V/V) NEAA, 1% (V/V) ITS culture supplement, 5mM L-glutamic acid, 1mM beta-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 100. mu.M Uridine Urridine.
Comparative example 1 preparation and culture method of mesenchymal Stem cells (BMSCs)
This example is intended to illustrate the isolation, preparation and culture methods of mesenchymal stem cells (BMSCs) used in the subsequent examples of the present invention.
Wherein mesenchymal stem cells in human long bone marrow are separated by using a density gradient centrifugation method; the used bone marrow stromal cell culture solution is used for carrying out amplification culture on the bone marrow mesenchymal stem cells, and the bone marrow stromal cell culture solution is as follows: serum-free bone marrow stromal cells serum-free complete medium of example 2 (SM-MSCs medium), or commercially available commercial MSC medium (StemFit; Ajinomoto) or commercially available commercial MSC medium (ACF; STEM CELLTtechnologies).
The specific operation steps are as follows:
(1) diluting 5mL of human marrow fluid with PBS (equal volume), uniformly mixing, and centrifuging at 1500rpm at room temperature for 10 minutes;
(2) after the fat layer is discarded, the residual bone marrow fluid is slowly added onto the equal volume of Ficoll separating medium (1.077g/mL) along the wall of the centrifugal tube, and the mixture is centrifuged for 30 minutes at the room temperature of 2500 rpm;
(3) sucking mononuclear cells with a white membranous layer interface in the separation liquid, suspending the mononuclear cells by using 40ml of PBS, and centrifuging the mononuclear cells at room temperature of 1500rpm for 10 minutes;
(4) the supernatant was discarded and washing was repeated 2 times with 50ml PBS.
(5) Resuspending the cells in bone marrow stromal cell culture medium, performing amplification culture at 2X 105Cell density of/mL, seeded at a basal area of 25cm2The culture flask is placed at 37 ℃ and 5% CO2Culturing in an incubator.
(6) And (3) changing the liquid for the first time after 3 days, and changing the liquid every 2-3 days after suspension cells are discarded. The morphology and growth change of the cells are observed under a microscope every day, no suspension cells exist in a 14-day culture bottle, all the cells grow in a fibrous adherent manner, and the cell state is good.
(7) When the cell growth reaches 80% -90% fusion, using Accutase to digest, and performing digestion according to the proportion of 1:3 ratio, and the cells were used for subsequent experiments.
(8) Wherein the mesenchymal stem cells separated from different batches are marked as BMSCs 1, BMSCs 2, BMSCs 3 and the like and are applied to subsequent experiments.
Example 3 induced differentiation of human induced pluripotent stem cells (hipscs) into bone Marrow stromal cells (SM-MSCs) by Somatic Mesoderm cells
This example is intended to illustrate the induced differentiation procedure based on the stable establishment and culture of human induced pluripotent stem cell lines (hipscs).
Culture of first and third hiPSC cells and detection of pluripotency of pluripotent stem cells
1. Experimental methods
Human induced pluripotent stem cells (hipscs) refer to pluripotent stem cells induced by human skin fibroblasts and are constructed by using induced pluripotent stem cell technology (iPSCs) in laboratories. hiPSC CELLs were expanded in large scale using a STEM CELL mTeSR culture solution, and maintained in an undifferentiated state. Meanwhile, the culture medium is coated with Matrigel, and the Matrigel is selected from Matrigel or laminin LN for subsequent experiments.
The specific operation steps are as follows:
(1) coating a culture dish: (a) matrigel was thawed on ice and diluted with pre-cooled DMEM-F12 in volume 1: diluting a Matrigel stock solution by a ratio of 100, adding the Matrigel stock solution into a pore plate, and coating overnight at room temperature for later use; or (b) laminin LN is diluted with PBS at a volume of 1:100, and added to the well plate and coated overnight at room temperature for further use.
(2) Resuscitation hiPSC: the hiPSC cells were quickly thawed in a 37 ℃ water bath, transferred to a 15ml centrifuge tube containing 5ml mTeSR culture solution, and centrifuged at 1100rpm for 4min to collect the cells.
(3) The coated Matrigel or laminin LN was aspirated, the cells were resuspended in 2ml mTeSR, the cells were evenly seeded, and placed at 37 ℃ in 5% CO2And standing and culturing in an incubator with 95% humidity.
(4) And replacing the culture solution once a day, observing that the cells maintain an undifferentiated state, continuously culturing until the hiPSC cell clone grows to the density of 80-90%, and carrying out passage.
(5) Cell passage: the cells were washed twice with PBS and ReLeSR was addedTMIncubating at 37 ℃ for 1-5 min, discarding ReLeSRTMCells were gently pipetted into a suitably sized cell pellet with 1ml of mTeSR culture solution.
(6) The mTeSR resuspended cells were collected and seeded into Matrigel-preplated well plates uniformly in a volume of 100. mu.l per well, placed at 37 ℃ in 5% CO2The incubator is kept still and adhered to the wallAnd (5) culturing.
(7) The above culture expansion step is repeated to obtain a sufficient amount of cells for the next differentiation induction.
(8) The hiPSC Cell clones expanded in the above step were subjected to cellular immunofluorescence staining to detect pluripotent stem Cell marker transcription factors NANOG and OCT4(Cell Signaling Technology).
2. Results of the experiment
Fig. 1 is a photograph of white light and multi-potent labeled immunofluorescence of hiPSC cells cultured in this example; therefore, the hiPSC cell clone is compact, flat and undifferentiated; and the cells express the transcription factors NANOG and OCT4 marked by pluripotent stem cells. The cell culture plate coated with laminin LN has the effect similar to that of Matrigel, and can maintain the pluripotency of pluripotent stem cells and keep the clone shape. It is demonstrated that the hiPSC cells used for induction in this example were indeed pluripotent stem cells.
Second, induced differentiation and identification of human induced pluripotent stem cells (hipscs) derived Somatic Mesoderm Cells (SMCs)
1. Experimental methods
(1) And when the hiPSC cells grow to the density of 80-90%, washing the cells twice by using PBS, adding 500 mu l of Accutase, incubating for 4min at 37 ℃, observing under a mirror, dissociating the cells into single cells or small cell masses, sucking out the Accutase, and slightly blowing the cells by using PBS to uniformly disperse the cells.
(2) The cells were transferred into a 15ml centrifuge tube and centrifuged at 1100rpm for 4min to collect the cells.
(3) Resuspending the cell pellet in DMEM-F12 medium containing 3 μm CHIR99021 at 1X 104/cm2The cells were uniformly seeded into a well plate or a petri dish coated with Matrigel in advance, and then placed at 37 ℃ with 5% CO2And standing and culturing in an incubator with 95% humidity.
(4) After 2 days of culture, the hiPSC cells are induced into mesendoderm cells, and the mesendoderm markers TBXT and MIXL1 are detected by real-time fluorescence quantitative PCR (polymerase chain reaction), and the mesendoderm markers TBXT (R & DSystems) and MIXL1 (Proteintetech Group) are detected by immunofluorescence staining.
(5) Wherein the mesodermal cells of the body wall from which the pluripotent stem cells are induced in different batches are marked as SMCs1, SMCs 2, SMCs 3 and the like, and the induced pluripotent stem cells are applied to subsequent experiments
(6) Then, the medium was replaced with the medium of example 1 for 5 to 7 days, mesendoderm cells were induced into parietal mesoderm cells, during which time the medium was replaced every day, 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 thereof.
(7) Finally, detecting the cell ratio of HAND1+/FOXF1+ by flow, wherein the specific method comprises the following steps: after digestion of body wall mesodermal cells, after fixation with 4% paraformaldehyde PFA, PBS was washed 2 times, and after washing the cells with 1% rupture solution for 5 minutes, HAND1 and FOXF1 antibody were added and incubated for 30 minutes at room temperature. The cells were then washed 2 times with 1% membrane-disrupting solution for 5 minutes each, and the proportion of HADN1 and FOXF1 positive cells was determined on a flow cytometer after resuspending the cells with 200ul PBS.
2. Results of the experiment
Fig. 2 is a cell morphology graph (fig. 2A) and real-time fluorescent quantitative PCR detection results (fig. 2B) of mesendoderm markers TBXT and MIXL1 and immunofluorescent staining pictures (fig. 2C) of TBXT and MIXL1 of hiPSC cells treated with GSK-3 inhibitor (3 μm CHIR99021) for 2 days in example 3.
FIG. 3 is a cell morphology graph of the mesendoderm cells derived from hiPSC in example 3 after 6 days induction with in-vivo mesoderm induction culture medium (FIG. 3A), real-time fluorescence quantitative PCR detection of the expression of the markers HAND1, FOXF1, TBX4, PRRX1 and PITX1 (FIG. 3B), simultaneous immunofluorescence staining detection of the expression of the markers HAND1 and FOXF1 (FIG. 3C) and flow detection induction efficiency graph of HAND1 and XF FO 1 (FIG. 3D)
The results show that the pluripotent stem cells can be efficiently induced into mesendoderm cells by CHIR99021 and highly express TBXT and MIXL1, and then can be efficiently induced into mesendoderm cells in the culture solution containing the combination of GSK3 inhibitor (CHIR99021) and BMP signal pathway activator (BMP7) in example 1, and the double positive proportion of HAND1 and FOXF1 reaches more than 95%.
Thirdly, induced differentiation method and identification of bone Marrow stromal cells (SM-MSCs) from Mesoderm cells in body wall
1. Experimental methods
Inducing somatic mesoderm cells obtained by inducing human induced pluripotent stem cells (hipscs) in the above step into bone marrow stromal cells bone marrow stromal cell culture solution is used, wherein the bone marrow stromal cell culture solution is: the bone marrow stromal cell serum-free complete medium (SM-MSCs medium) in example 2, or a commercially available commercial MSC medium (StemFit; Ajinomoto) or a commercially available commercial MSC medium (ACF; STEM CELLTtechnologies).
The specific operation steps are as follows:
(1) replacing the culture solution for identifying mesoderm cells in body wall in the last step with the culture solution of stroma cells, and culturing at 37 ℃ and 5% CO2And standing and culturing in an incubator with 95% humidity, and changing the culture solution every 2-3 days.
(2) After the cells grow to 80-90% of fusion, using Accutase to digest and passage, transferring the cells into a 15ml centrifuge tube, centrifuging at 1100rpm for 4min to collect the cells, discarding the supernatant, and inoculating the cells into a new culture dish according to the ratio of 1:3 to finish the passage of the cells.
(3) And (3) repeating the step 2, continuously subculturing the bone marrow stromal cell culture solution for 6-8 times, and taking a part of cells to detect the expression conditions of cell surface molecules CD34, CD45, CD44, CD90 and CD140b in a flow mode. When the cells express the bone marrow stromal cell markers CD44, CD90 and CD140b, but do not express the hematopoietic stem cell markers CD34 and CD45, the surface marker of the bone marrow stromal cells derived from the mesodermal cells of the body wall is obtained.
(4) Wherein the marrow stromal cells derived from the mesodermal cells of the body wall induced by different batches are marked as SM-MSCs 1, SM-MSCs 2, SM-MSCs 3 and the like, and are applied to subsequent experiments.
(5) Cell Counting Kit (CCK-8) CCK-8 Kit is used for detecting the proliferation comparison of the marrow stromal cells (SM-MSCs) from the mesoderm cells in the body wall prepared in the embodiment and the marrow mesenchymal stem cells (BMSCs) directly separated from the bone marrow of the long bone.
(6) After total RNA of somatic mesoderm cells obtained by inducing differentiation and bone marrow stromal cells derived from the somatic mesoderm cells is extracted by using RNAzol, the RNA is reversely transcribed into cDNA, and the gene expression condition of a homologous domain transcription factor (HOX9-13) is detected by fluorescence quantitative PCR.
2. Results of the experiment
FIG. 4 is a diagram showing the cell morphology of the somatic mesoderm cells of example 3 after passage 6 times in bone marrow stromal cell culture (commercial MSC culture (StemFit)) and the immunofluorescent staining (FIG. 4A) and cell surface marker flow analysis (FIG. 4B) of CD 90; the morphology of the induced marrow stromal cells derived from the mesoderm cells of the body wall is similar to that of the marrow stromal cells, the marrow stromal cells are represented by typical fiber shapes, and immunofluorescence staining indicates that the marrow stromal cells highly express CD 90; and flow detection results show that marrow stromal cells derived from mesoderm cells in body walls express marrow stromal cell surface markers CD44, CD90 and CD140b, and do not express hematopoietic stem cell surface markers CD34 and CD 45.
Fig. 5 shows that the fluorescent quantitative PCR is used to detect the homeodomain transcription factor (HOX) gene expression of the marrow stromal cells from the body wall mesoderm derived marrow stromal cells induced by the marrow stromal cell culture solution (commercial MSC culture solution (StemFit)) in example 3, and the results show that the induced bone marrow stromal cells from the body wall mesoderm and the bone mesenchymal cells have similar typical homeodomain transcription factor (HOX) gene expression patterns.
FIG. 6 is a graph showing the comparison of proliferation potency of two batches (SM-MSCs 1, SM-MSCs 2) of different batches of somatic mesoderm-derived bone marrow stromal cells induced by bone marrow stromal cell culture medium (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 1, which were measured for cell proliferation change by CCK-8 and plotted. The result shows that the induced somatic mesoderm cell derived bone marrow stromal cells have stronger proliferation capacity.
FIG. 7 shows a comparison of proliferation potency (FIG. 7A) and proliferation passage number (FIG. 7B) of somatic mesoderm-derived bone marrow stromal cells obtained in different bone marrow stromal cell cultures in example 3. Wherein the bone marrow stromal CELL culture solution comprises bone marrow stromal CELL serum-free complete culture solution (SM-MSCs medium), commercially available commercial MSC culture solution (StemFit; Ajinomoto), and commercially available commercial MSC culture solution (ACF; STEM CELL Technologies). The results show that in addition to commercially available commercial MSC culture solutions (StemFit and ACF), bone marrow stromal cells can be induced, and the bone marrow stromal cells serum-free complete culture solution (SM-MSCs medium) in example 2 can also induce bone marrow stromal cells to be obtained, and the proliferation speed is faster, the proliferation generation number is more, and there is a significant statistical difference. Example 4 in vitro osteogenic chondrogenic and hematopoietic support Capacity test of bone Marrow stromal cells (SM-MSCs) derived from Mesoderm cells in body wall
First, experiment method
In this example, the somatic mesoderm-derived bone marrow stromal cells prepared in example 3 and the bone marrow mesenchymal stem cells isolated in control example 1 were used. And were cultured using commercially available commercial MSC culture medium (StemFit; Ajinomoto) or bone marrow stromal cell serum-free complete medium (SM-MSCs medium) of example 2 as the bone marrow stromal cell culture medium.
1. In-vitro osteogenic and chondrogenic capacity of bone marrow stromal cells derived from mesodermal cells in body wall
(1) In vitro osteogenic differentiation: when the growth density of the bone marrow stromal cells derived from the mesodermal cells in the body wall prepared in example 3 and the bone marrow mesenchymal stem cells (BMSCs) isolated in control example 1 reached 80 to 90%, the culture medium was replaced with an osteogenic differentiation inducing culture medium (DMEM (L) supplemented with 10% (V/V) FBS, 10mM β -glycophorosphate, 50 μ g/ml vitamin C, and 100nM dexamethasone. The culture medium was changed every 3 days, and alizarin red staining was performed 21 days after 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 collected by digestion at a ratio of 2.0 to 3.0X 105The cell amount in 15ml centrifuge tube was divided into 15ml centrifuge tubes, centrifuged at 300 Xg for 5min, the supernatant was discarded, and 1ml cartilage differentiation induction culture medium (DMEM (H)) was added10ng/ml recombinant human transforming growth factor (TGF-beta 3), 100nM dexamethasone, 50. mu.g/ml vitamin C, 1mM sodium pyruvate, 40. mu.g/ml proline and ITS culture additive), and placing in an incubator for incubation and culture. Changing the culture solution every 3 days, and carrying out Ascinum blue staining after 21 days of induction culture.
(3) Total osteocyte RNA induced by bone differentiation of bone marrow stromal cells (SM-MSCs) of example 3 and total chondrocyte RNA induced by cartilage differentiation of bone marrow mesenchymal stem cells (BMSCs) of control example 1, both of which were induced and amplified by commercially available MSC culture medium (StemFit; Ajinomoto) were extracted using RNAzol, and after reverse transcription into cDNA, the expression of genes of osteogenic genes (COL1A1, ALP, OCN and OPN) and chondrogenic genes (COL2A1, ACAN, RUNX2 and SOX9) were detected by fluorescence quantitative PCR.
(4) Total RNA of bone marrow stromal cells derived from mesoderm cells in the body wall prepared in different bone marrow stromal cell culture solutions (SM-mscmedium, StemFit, ACF) in example 3 was extracted using RNAzol, and after reverse transcription into cDNA, expression of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L, ANGPT1) was detected by fluorescence quantitative PCR; and extracting the total RNA of bone marrow cells derived from mesoderm cells of the body wall prepared in the culture solution of different bone marrow stromal cells (SM-MSCs medium, StemFit, ACF) in example 3, performing reverse transcription to obtain cDNA, and performing fluorescent quantitative PCR to detect the gene expression of osteogenic genes (COL1A1, ALP, OCN and OPN) and chondrogenic genes (COL2A1, ACAN, RUNX2 and SOX 9).
2. In-vitro hematopoiesis supporting capability detection of bone Marrow stromal cells (SM-MSCs) derived from Mesoderm cells in body wall
(1) Total RNAs of somatic mesoderm-derived bone marrow stromal cells (SM-MSCs) prepared in example 3 and bone marrow mesenchymal stem cells (BMSCs) in control example 1 were extracted with RNAzol, and then reverse-transcribed into cDNAs, and expression levels of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L, and ANGPT1) were compared by quantitative fluorescence PCR.
(2) In vitro maintenance assay of Hematopoietic Stem Cells (HSCs)
Bone marrow stromal cells (SM-MSCs) derived from mesodermal cells in the body wall prepared in example 3 and mesenchymal stem cells (BMSCs) in control example 1 were pre-seeded in a 24-well plate at a density of 90%, cultured overnight, and then 0.5mg/ml mitomycin C was added to the culture solution to treat BMSCs and SM-MSCs the next day, after 3 hours, the culture solution containing mitomycin C was discarded, washed 3 times with PBS, and replaced with fresh culture solution, to obtain mitomycin C-treated BMSCs and SM-MSCs. Subsequently, the flow-sorted human cord blood CD34+ HSCs were used in StemBanTmCD34+ amplification medium (Stem Cell Technologies) was resuspended and plated at 5000 cells per well in treated BMSCs and SM-MSCs. The culture was continued for 9 days, during which half-changes were made every two days. Simultaneously with the same StemBanTmCD34+ HSCs cultured alone in CD34+ expansion medium served as controls. After 9 days of co-culture, cells suspended in the culture broth were collected and maintained by flow assay of CD34+ cells under different co-culture conditions.
(3) In vitro Long-Term co-Culture (LTC-IC) assay of Hematopoietic Stem Cells (HSCs) and marrow stromal cells derived from somatic mesoderm cells
Bone marrow stromal cells (SM-MSCs) derived from mesodermal cells in the body wall prepared in example 3 and bone marrow mesenchymal stem cells (BMSCs) in control example 1 were pre-seeded at a density of 90% in a 24-well plate, cultured overnight, and the BMSCs obtained in control example 1 and the SM-MSCs prepared in example 3 were treated the next day with mitomycin C at a concentration of 0.5mg/ml, and after 3 hours, the culture containing mitomycin C was discarded, washed with PBS 3 times, and replaced with fresh culture to obtain mitomycin C-treated BMSCs and SM-MSCs. CD34+ HSC (cells) were seeded at 1000 cells per well into BMSCs and SM-MSCs and StemBanTmCD34+ amplification medium (Stem Cell Technologies) was cultured with half-changes every two days. CD34+ HSCs cultured separately in the same medium were also used as controls. 10000 cells isolated from the supernatant of the co-culture system after 35 days of co-cultureSeeded on methylcellulose (Stem Cell Technologies) plates at 37 ℃ and 5% CO2After 14 days of medium incubation, the colonies formed were photographed, counted and analyzed.
Second, experimental results
FIG. 8 is a graph showing the in vitro osteogenesis, chondrogenesis and hematopoietic support abilities of somatic mesoderm-derived bone marrow stromal cells (SM-MSCs) prepared in example 3 using a bone marrow stromal cell culture solution (commercially available MSC culture solution (StemFit)) and mesenchymal stem cells (BMSCs) in control example 1, including in vitro osteogenesis chondrogenesis staining, enzyme-linked immunosorbent assay (FIG. 8A), quantitative fluorescence quantitative PCR assay of osteogenesis (COL1A1, ALP, CON and OPN) and chondrogenesis (COL2A1, ACAN, RUNX2 and SOX9) gene expression levels (FIG. 8B), quantitative real-time fluorescence PCR assay of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L, ANGPT1) expression (FIG. 8C), assay of CD34+ maintenance ratio in coculture system (FIG. 8D), colony formation assay of CFIC-forming granulocyte (CFU-GM-forming unit), Burst red colony forming units (BFU-E) and their statistical plots (FIG. 8E).
The results show that alizarin red S staining of the bone marrow stromal cells (SM-MSCs) from mesoderm in body wall is darker and appears deep red compared with bone marrow mesenchymal stem cells (BMSCs), and meanwhile, the absorbance measured at 562nm by a microplate reader also shows that the alizarin red S staining of the SM-MSCs is darker (fig. 8A), which indicates that calcium salt generated during the bone formation of the SM-MSCs is induced to be more abundant in deposition and stronger in bone formation capacity; for cartilage differentiation, toluidine blue staining showed that SM-MSCs induced chondrocytes were purplish blue, the generated cartilage matrix was abundant, the chondrocyte diameter was larger (fig. 8A), and fluorescent quantitative PCR further detected the corresponding markers of osteogenesis (COL1a1, ALP, CON, OPN) and chondrogenesis (COL2a1, ACAN, RUNX2, SOX9), and the results also showed that SM-MSCs had stronger chondrogenesis ability in vitro (fig. 8B). The above evidence indicates that bone marrow stromal cells (SM-MSCs) derived from mesodermal cells in body walls have superior osteogenic and chondrogenic capacity in vitro compared to BMSCs. In addition, in vitro fluorescence quantitative PCR detected SM-MSCs to express more hematopoietic support genes than BMSCs (VCAM1, CXCL12, MCP1, KITLG, FLT3L, ANGPT1) (fig. 8C) and had statistical differences. The in vitro hematopoietic support capacity assay showed that the proportion of CD34+ HSC co-cultured with SM-MSCs remained positive was significantly higher than that of BMSCs and controls (FIG. 8D). CFU analysis also showed that SM-MSCs produced the highest number of granulocyte and macrophage colony forming units (CFU-GM), burst erythroid colony forming units (BFU-E) (FIG. 8E).
The evidence shows that SM-MSCs have stronger osteogenic and chondrogenic differentiation capacity in vitro and express higher hematopoietic support genes compared with BMSCs, and can better maintain the expression of CD34+ HSCs and better promote the in vitro clone formation and proliferation capacity of hematopoietic stem cells.
FIG. 9 shows the in vitro osteogenic, chondrogenic and hematopoietic support capacities of the somatic mesodermal cell-derived bone marrow stromal cells prepared in different bone marrow stromal cell cultures (SM-MSCmedium, StemFit, ACF) in example 3, respectively. Comprises the steps of detecting the gene expression quantity of osteogenesis (COL1A1, ALP, CON and OPN) and chondrogenesis (COL2A1, ACAN, RUNX2 and SOX9) by fluorescence quantitative PCR (figure 9A), and detecting the expression of hematopoietic support genes (VCAM1, CXCL12, MCP1, KITLG, FLT3L and ANGPT1) by real-time fluorescence quantitative PCR (figure 9B).
The results show that the bone marrow stromal cells obtained by inducing the bone marrow stromal cells in the serum-free complete culture medium (SM-MSCs medium) in example 2 have better osteogenic, chondrogenic (FIG. 9A) and hematopoietic support (FIG. 9B) capabilities, and have significant statistical differences.
The above-mentioned evidence in fig. 7 and fig. 9 indicates that the serum-free complete culture Medium (SM-MSC Medium) for bone marrow stromal cells in the present invention not only can stably and efficiently induce and amplify bone marrow stromal cells, but also the obtained bone marrow stromal cells have better osteogenic, chondrogenic and hematopoietic supporting abilities. Therefore, the serum-free complete culture solution for the bone marrow stromal cells (SM-MSC Medium) has certain advantages in inducing the bone marrow stromal cells from the pluripotent stem cells.
Example 5 in vivo osteogenic and hematopoietic support Capacity assay of bone Marrow stromal cells (SM-MSCs) derived from Mesoderm cells in body wall
First, experiment method
In this example, the somatic mesoderm-derived bone marrow stromal cells prepared in example 3 and the bone marrow mesenchymal stem cells isolated in control example 1 were used. And were cultured using a commercially available commercial MSC culture solution (StemFit; Ajinomoto) as a bone marrow stromal cell culture solution.
(1) Sample preparation: bone marrow stromal cells (SM-MSCs) derived from somatic mesoderm cells prepared in example 3 were selected as controls for bone marrow mesenchymal stem cells (BMSCs) in control example 1. Two groups of cells were made into 2X 10 cells simultaneously7cells/ml cell suspension, each 50. mu.l of cell suspension was pipetted and mixed well into 30mg of hydroxyapatite support material previously soaked overnight in 50. mu.l of chondrogenic induction solution, the vessel lid was loosened slightly, and the mixture was placed at 37 ℃ with 5% CO2After incubation in an incubator for about 2 hours, the cells were embedded together under the skin of immunodeficient mice.
(2) In vivo transplantation: the animals were anesthetized with 1% pentobarbital at a dose of 50mg/kg per immunodeficient mouse, 5 per group. After anesthesia succeeds, the iodine wine is used for disinfecting the skin surface, a wound with the length of about 1cm is cut at the position of the uppermost back and the right side by scissors, the wound is separated bluntly by a separating forceps, the left side of the wound is deepened, BMSCs group cells and materials are sent to the left side of the neck in multiple times, and then the wound is sutured and disinfected. Then, a wound of about 1cm was cut on the right side of the abdomen, and the SM-MSCs group cells were sent to the right side of the abdomen together with the material, sutured, and the wound was sterilized. After it is awake, put into the mouse cage. The status of the mice was then observed daily and kept routinely for 8 weeks.
(3) Samples were harvested after 8 weeks: (a) collecting a specimen, fixing, decalcifying, carrying out Masson staining and immunofluorescence staining on the section, and comparing the in vivo bone formation capability of SM-MSCs and BMSCs; (b) after fixation, decalcification and HE staining of the sections, 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 capacity of SM-MSCs to BMSCs.
Second, experimental results
Fig. 10 is a graph showing the capability of bone marrow stromal cells (SM-MSCs) derived from mesoderm in body wall induced in example 3 compared with bone marrow mesenchymal stem cells (BMSCs) in control example 1 in supporting osteogenesis and hematopoiesis in vivo, including Masson staining pattern and its statistical chart for osteogenesis capability test (fig. 10A), immunofluorescence staining pattern for osteogenesis markers (OCN, OPG) (fig. 10B), hematopoietic cell cluster pattern and its number statistical chart after HE staining (fig. 10C), and immunofluorescence staining pattern for CD45+ hematopoietic progenitor cells (fig. 10D).
The results show that the marrow stromal cells (SM-MSCs) from mesoderm in the body wall prepared in example 3 have stronger osteogenesis capacity in the immunodeficient mice compared with the marrow mesenchymal stem cells (BMSCs) in the control example 1, and specifically show that Masson staining is darker and wider in staining range, and meanwhile, immunofluorescence staining detects more Osteoprotegerin (OPG) and Osteocalcin (OCN) positive cells. In addition, the induced SM-MSCs in example 3 could support more hematopoietic cluster formation and more CD45+ hematopoietic progenitor cells at the implantation site under the skin of immunodeficient mice.
The evidence indicates that the bone marrow mesenchymal stem cells (BMSCs) directly separated from the bone marrow of the adult long bones have stronger bone formation capability and more excellent hematopoietic support capability in vivo compared with the bone marrow stromal cells (SM-MSCs) derived from the mesoderm of the body wall induced by the invention.
Example 6 sequencing analysis of stromal cells of bone Marrow stromal cells derived from Mesoderm in body wall, obtained from stromal cells, SM-MSCs)
First, experiment method
In this example, the somatic mesoderm-derived bone marrow stromal cells prepared in example 3 and the bone marrow mesenchymal stem cells isolated in control example 1 were used. And were cultured using a commercially available commercial MSC culture solution (StemFit; Ajinomoto) as a bone marrow stromal cell culture solution.
(1) Sample preparation: collecting bone marrow stromal cells (SM-MSCs) from mesodermal cells in the body wall prepared in example 3, extracting total RNA of the Bone Marrow Stromal Cells (BMSCs) from the control example 1 according to the operation steps of an RNA extraction kit manufactured by QIAGEN, and performing the next operation after the integrity evaluation of the total RNA is completed;
(2) building a library: using reagents provided by Illumina, mRNA was isolated from total RNA and reverse-transcribed into cDNA according to the corresponding procedures, and then the modified cDNA fragments were PCR amplified, purified and enriched to thereby create cDNA libraries.
(3) Sequencing and analyzing: using the established cDNA library, RNA sequencing was performed by a GA high throughput sequencer (Illumina, San Diego, USA) and gene expression profiles were analyzed.
Second, experimental results
FIG. 11 is a gene expression profiling analysis (FIG. 11A) of two batches (SMs 1 and SMs 2) in different-batch parietal mesoderms induced by example 3 and bone marrow stromal cells derived therefrom (SM-MSCs 1 and SM-MSCs 2) by RNA-Seq and two batches (BMSCs 1 and BMSCs 2) in bone marrow mesenchymal stem cells directly isolated from different-batch long bone marrow in control example 1, and 2 samples (SMs 1 vs SMs 2; SM-MSCs 1 vs SM-MSCs 2; BMSCs 1 vs BMSCs 2) of different batches in the same differentiation stage, for SMs 1 and SMs 2; SM-MSCs 1, SM-MSCs 2; BMSCs 1 and BMSCs 2 were subjected to gene expression analysis at different stages (FIG. 11B) and gene expression pattern maps of SM-MSCs 1 and SM-MSCs 2 and homeodomain transcription factors (HOX1-13) of BMSCs 1 and BMSCs 2 (FIG. 11C).
The results show that the gene expression profiles between the 2 samples of the same differentiation stage from batch to batch have high similarity (SMCs1 vs SMCs 2; SM-MSCs 1 vs SM-MSCs 2; BMSCs 1 vs BMSCs 2), indicating high reproducibility of the induction method of the invention (FIG. 11A). RNA-Seq gene expression profiling revealed that SMCs highly expressed somatic mesoderm markers such as PITX1, HAND1, TBX4, while bone marrow stromal cell expression patterns induced by somatic mesoderm backward were similar to BMSCs, and that somatic mesoderm markers PITX1, HAND1, TBX4 were no longer expressed, but expressed, for example, as the bone marrow stromal cell surface marker of CD44 (FIG. 11B), and SM-MSCs expressed the homeodomain transcription factor (HOX9-13) gene similar to that expressed by skeletal mesenchymal cells (FIG. 11C).
Example 7 induced differentiation of Embryonic Stem Cells (Human Embryonic Stem Cells; H1-ES) into bone marrow stromal Cells through the Embryonic layer in the body wall
This example is intended to illustrate the operation of induced differentiation on the basis of stable establishment and culture of a human embryonic stem cell line (H1-ES).
First, experiment method
1. Culture of Embryonic Stem Cells (Human Embryonic Stem Cells; H1-ES)
Embryonic stem cells (H1-ES) were obtained commercially. H1-ES CELLs were also expanded in a large scale using a STEM CELL mTeSR culture medium to maintain the undifferentiated state. Meanwhile, Matrigel is required to be coated on the culture medium, and Matrigel or laminin LN and the like can be selected.
The specific operation steps are as follows:
(1) coating a culture dish: matrigel was thawed on ice and diluted with pre-cooled DMEMF12 in volume 1: the Matrigel stock solution was diluted at a ratio of 100 and added to a well plate and allowed to incubate overnight for use.
(2) Resuscitating embryonic stem cells (H1-ES): H1-ES cells were quickly thawed in a 37 ℃ water bath, transferred to a 15ml centrifuge tube containing 5ml mTeSR medium, and centrifuged at 1100rpm for 4min to collect the cells.
(3) Blotted to remove the coated Matrigel or laminin LN, resuspended cells in 2ml mTeSR, homogenized, placed at 37 deg.C, 5% CO2And (5) standing and culturing in an incubator.
(4) And replacing the culture solution once a day, observing that the cells maintain an undifferentiated state, continuously culturing until the H1-ES cell clone grows to the density of 80-90%, and carrying out passage.
(5) Cell passage: the cells were washed twice with PBS and ReLeSR was addedTMIncubating at 37 ℃ for 1-5 min, discarding ReLeSRTMCells were gently pipetted into a suitably sized cell pellet with 1ml of mTeSR culture solution.
(6) The mTeSR resuspended cells were collected and the cells were seeded evenly in 100. mu.l volume per well into a Matrigel-preplated well plate, placed at 37 ℃ in 5% CO2And (5) standing in an incubator for adherent culture.
(7) The above culture expansion step is repeated to obtain a sufficient amount of cells for the next differentiation induction.
2 embryonic Stem cell (H1-ES) origin induced differentiation and identification of Somatic Mesoderm Cells (SMCs)
(1) And when the cells grow to the density of 80-90%, washing the cells twice by using PBS, adding 500 mu l of Accutase, incubating for 4min at 37 ℃, observing under a mirror, dissociating the cells into single cells or small cell masses, sucking out the Accutase, and slightly blowing the cells by using PBS to uniformly disperse the cells.
(2) The cells were transferred into a 15ml centrifuge tube and centrifuged at 1100rpm for 4min to collect the cells.
(3) Resuspend the cell pellet with DMEM-F12 medium containing 3 μm CHIR99021 at 1X 104/cm2The cells were uniformly seeded into a Matrigel-coated well plate or petri dish at 37 ℃ in 5% CO2And (5) standing and culturing in an incubator.
(4) After 2 days of culture, mesendoderm cells were induced.
(5) Then, the culture medium of example 1 was replaced for 5 to 7 days of induction culture, and mesendoderm cells were induced into parietal mesoderm cells, during which the culture medium was replaced every day.
2. Embryonic stem cell (H1-ES) derived induced differentiation and identification of Mesoderm cells in body wall into bone Marrow stromal cells (SM-MSCs)
Further inducing the mesoderm cells in the body wall obtained by inducing the embryonic stem cells (H1-ES) in the above step into bone marrow stromal cells by using bone marrow stromal cell culture solution, wherein the bone marrow stromal cell culture solution is: the bone marrow stromal cell serum-free complete medium (SM-MSCs medium) in example 2, or a commercially available commercial MSC medium (StemFit; Ajinomoto) or a commercially available commercial MSC medium (ACF; STEM CELLTtechnologies).
The specific operation steps are as follows:
(1) replacing the culture solution of the mesodermal cells in the body wall induced by the previous step with the culture solution of bone marrow stromal cells at 37 ℃ and 5% CO2And (5) carrying out standing culture in an incubator, and changing the culture solution every 2-3 days.
(2) After the cells grow to be 80-90% fused, using Accutase to digest and passage, transferring the cells into a 15ml centrifuge tube, centrifuging at 1100rpm for 4min to collect the cells, discarding the supernatant, and inoculating the cells into a new culture dish according to the ratio of 1:3 to finish passage of the cells for 6-8 times.
(3) And (3) repeating the step 2, and taking a part of cells to detect the expression conditions of cell surface molecules CD34, CD45, CD44, CD90 and CD140b in a flow mode after the cells are continuously subcultured for 6-8 times. When the cells express the bone marrow stromal cell markers CD44, CD90 and CD140b, but do not express the hematopoietic stem cell markers CD34 and CD45, the surface marker of the bone marrow stromal cells derived from the mesodermal cells of the body wall is obtained.
3. In vivo formation and hematopoietic support capacity detection of bone Marrow stromal cells (SM-MSCs) obtained from Somatic Mesoderm cells induced by embryonic stem cells (H1-ES)
Bone marrow stromal cells (SM-MSCs) obtained from somatic mesoderm cells induced by embryonic stem cells (H1-ES) prepared in example 7 and bone marrow mesenchymal stem cells (BMSCs) isolated in comparative example 1 were used in this step. And all were cultured with the bone marrow stromal cell serum-free complete medium (SM-MSCs medium) of example 2 as a bone marrow stromal cell culture medium.
(1) Sample preparation: bone marrow stromal cells (SM-MSCs) obtained from somatic mesoderm cells induced by human embryonic stem cells (H1-ES) in example 7 were selected and bone marrow mesenchymal stem cells (BMSCs) isolated in comparative example 1 were used as a control. Two groups of cells were made into 2X 10 cells simultaneously7cells/ml cell suspension, each 50. mu.l of cell suspension was pipetted and mixed well into 30mg of hydroxyapatite support material previously soaked overnight in 50. mu.l of chondrogenic induction solution, the vessel lid was loosened slightly, and the mixture was placed at 37 ℃ with 5% CO2After incubation in an incubator for about 2 hours, the cells were embedded together under the skin of immunodeficient mice.
(2) Transplanting in vivo: the mice were anesthetized intraperitoneally with 1% pentobarbital at a dose of 50mg/kg per immunodeficient mouse, 5 mice each. After anesthesia succeeds, the iodine wine is used for disinfecting the skin surface, a wound with the length of about 1cm is cut at the position of the uppermost back and the right side by scissors, the wound is separated bluntly by a separating forceps, the left side of the wound is deepened, BMSCs group cells and materials are sent to the left side of the neck in multiple times, and then the wound is sutured and disinfected. Then, a wound of about 1cm was cut on the right side of the abdomen, and the SM-MSCs group cells were sent to the right side of the abdomen together with the material, sutured, and the wound was sterilized. After it is awake, put into the mouse cage. The status of the mice was then observed daily and kept routinely for 8 weeks.
(3) Samples were harvested after 8 weeks: (a) collecting a specimen, fixing, decalcifying, carrying out Masson staining and immunofluorescence staining on the section, and comparing the in vivo bone formation capability of SM-MSCs and BMSCs; (b) fixing, decalcifying, taking pictures after HE staining the slices, counting the number of formed hematopoietic cell clusters, performing immunofluorescence staining on CD45+ hematopoietic progenitor cells, and comparing the in vivo hematopoietic support capacity of SM-MSCs and BMSCs.
Second, experimental results
FIG. 12 is a surface marker map (FIG. 12A), a morphology map and a map of in vitro osteogenic chondrogenic assay (FIG. 12B) of embryonic stem cell (H1-ES) -derived bone marrow stromal cells prepared from a bone marrow stromal cell culture solution (commercially available MSC culture solution (StemFit)) in example 7, and a map of bone marrow mesenchymal stem cells (H1-ES) -derived bone marrow stromal cells prepared from a bone marrow stromal cell serum-free complete culture solution (SM-MSCs medium) in comparison with bone marrow mesenchymal stem cells (BMSCs) of comparative example 1 cultured in a corresponding medium, in immunodeficient mice (FIG. 12C) and hematopoietic support ability assay (FIG. 12D).
The results show that the bone marrow stromal cells (SM-MSCs) obtained by somatic mesoderm cells induced by the human embryonic stem cells (H1-ES) of the embodiment have similar morphology with the bone marrow stromal cells, show adherent growth and typical fiber shape, express the bone marrow stromal cell markers CD44, CD90 and CD140b, and do not express the surface markers CD34 and CD45 of hematopoietic stem cells; alizarin red S staining shows that the induced osteoblasts are colored in deep red, toluidine blue staining shows that the induced chondrocytes are purple blue, and the generated cartilage matrix is rich.
In addition, in vivo transplantation experiments in immunodeficient mice showed that the bone marrow stromal cells (SM-MSCs) induced by somatic mesoderm cells by embryonic stem cells (H1-ES) prepared from bone marrow stromal cell serum-free complete medium (SM-MSCs medium) in example 7 also have good functional properties, and have stronger bone formation capability (FIG. 12C) and superior hematopoietic support capability (FIG. 12D) in immunodeficient mice compared with bone marrow mesenchymal stem cells (BMSCs) directly isolated from adult long bone marrow cultured in the same bone marrow stromal cell medium.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A bone marrow stromal cell, wherein said bone marrow stromal cell is obtained by inducing pluripotent stem cells into somatic mesoderm cells and then subjecting the cells to continuous subculture in a stromal cell culture medium.
2. A somatic mesoderm-derived bone marrow stromal cells derived from a pluripotent stem cell induced after undergoing a somatic mesoderm cell stage.
3. A culture solution for inducing pluripotent stem cells into somatic mesoderm cells, wherein the culture solution is a basal culture solution comprising a combination of a GSK-3 inhibitor and a BMP signaling pathway activator.
4. The culture solution of claim 3, wherein the GSK-3 inhibitor is one or more of LY2090314, SB216763, CHIR99021 and CHIR99021 HCl; the BMP signal pathway activator is one or more of BMP2, BMP4 or BMP 7.
5. The serum-free complete culture solution for the bone marrow stromal cells is characterized by comprising 1-10% (V/V) serum substitute, 1-5% (V/V) NEAA, 1-5% (V/V) ITS culture additive, 1-10 mM L-glutamic acid, 0.1-10 Mm beta-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 Urridine.
6. Use of the culture medium of any one of claims 3 to 4 and/or the serum-free complete culture medium of bone marrow stromal cells of claim 5 for the induction of bone marrow stromal cells by pluripotent stem cells.
7. A method for inducing bone marrow stromal cells by utilizing pluripotent stem cells, which comprises the following steps:
s1, inducing pluripotent stem cells into mesendoderm cells: dissociating and dispersing the pluripotent stem cells, inoculating the dissociated and dispersed pluripotent stem cells into a culture plate or a culture dish coated with matrigel, and culturing for 1-3 days by using a culture solution containing CHIR99021 to obtain mesendoderm cells;
s2, inducing mesendoderm cells into somatic mesoderm cells: culturing the product of the previous step for 5-7 days by using a culture solution for inducing the pluripotent stem cells into body wall mesoderm cells according to claim 3 or 4 to obtain body wall mesoderm cells;
s3, inducing the somatic mesoderm cells into somatic mesoderm cell-derived bone marrow stromal cells: and (3) digesting the obtained mesoderm cells on the body wall again, inoculating the cells into a bone marrow matrix cell culture solution, and carrying out continuous subculture for 6-8 times by using the bone marrow matrix cell culture solution to obtain the bone marrow matrix cells derived from the mesoderm cells on the body wall.
8. The method according to claim 7, wherein the bone marrow stromal cell culture solution is the bone marrow stromal cell serum-free complete culture solution of claim 5 or a commercially available commercial MSC culture solution.
9. Bone marrow stromal cells produced by the method of claim 7 or 8.
10. Use of bone marrow stromal cells according to any one of claims 1, 2 or 8, for the preparation of a medicament having the function of promoting hematopoietic support and/or bone repair.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115247152A (en) * | 2022-09-21 | 2022-10-28 | 呈诺再生医学科技(北京)有限公司 | Method for preparing hematopoietic stem cells or hematopoietic stem and progenitor cells and method for culturing long-term regenerative hematopoietic stem cells |
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