CN114438037B

CN114438037B - Method for preparing induced mesenchymal stem cells

Info

Publication number: CN114438037B
Application number: CN202210088839.XA
Authority: CN
Inventors: 王全磊; 姜孝明; 周光前; 常崇斐; 彭冬秀; 马子玉; 马飞龙; 甘露; 施青
Original assignee: Shenzhen Letu Biomedical Co ltd
Current assignee: Shenzhen Letu Watson Precision Medical Co ltd
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2024-06-04
Anticipated expiration: 2042-01-25
Also published as: CN114438037A

Abstract

A method for preparing induced mesenchymal stem cells comprises: a stem cell culture step, comprising providing mesenchymal stem cells for culture to obtain induced mesenchymal stem cells; a differentiation step, comprising differentiating and culturing induced pluripotent stem cells into mesenchymal stem cells, namely induced mesenchymal stem cells (iMSCs). The method does not require feeder cells and fetal bovine serum, is simple to operate, avoids the risk of exogenous genome integration, and can efficiently and stably prepare human iMSCs cell lines.

Description

Method for preparing induced mesenchymal stem cells

Technical Field

The invention relates to the technical field of biology, in particular to a method for preparing induced mesenchymal stem cells.

Background

At present, mesenchymal stem cells (MESENCHYMAL STEM CELL, MSCs) are applied as cells, and two technical bottlenecks still exist, the first is that the number of cells is limited. Mesenchymal stem cells are mainly prepared by separating from body tissues such as bone marrow, fat, umbilical cord and the like, however, the content of MSCs in the tissues is low, a large amount of cell expansion processes are needed before clinical application, the in vitro expansion processes can also cause aging states of cells to different degrees, the proliferation capacity is reduced and the like, and the number of the harvested cells is difficult to meet the clinical transplanting requirement. The second is still a technical challenge in the preparation of MSCs from non-tissue sources, although research on differentiation of MSCs by using Embryonic Stem Cells (ESCs) or Induced Pluripotent Stem Cells (iPSCs) is currently being studied, the embryonic stem cells are at risk of ethical disputes, while induced pluripotent stem cells often use a virus integration system to have risk of genome integration, in addition, MSCs (ifscs) from which iPSCs are differentiated depend on treatment with animal serum and multiple biological small molecules, and have the problems of complex procedures, high heterogeneity of 2D expanded cells, and the like, which limit the further clinical application of iMSCs.

Disclosure of Invention

According to a first aspect, in an embodiment, there is provided a method of preparing an induced mesenchymal stem cell, comprising:

a stem cell culturing step, including providing mesenchymal stem cells, culturing to obtain induced pluripotent stem cells;

The differentiation step comprises the step of differentiating and culturing the induced pluripotent stem cells into mesenchymal stem cells, namely the Induced Mesenchymal Stem Cells (iMSCs).

According to a second aspect, in an embodiment, there is provided an induced mesenchymal stem cell produced by the method of the first aspect.

According to the method for preparing the induced mesenchymal stem cells, feeder cells and fetal bovine serum are not needed, the operation is simple, the risk of exogenous genome integration is avoided, and the human iMSCs cell line can be efficiently and stably prepared.

Drawings

FIG. 1 is a flow chart of one embodiment iMSCs of the preparation.

FIG. 2 is a graph of MSCs at about 25% confluency for 5 ten thousand cells/well under a microscope in example 1.

FIG. 3 is a clone embryonic form chart in example 1.

Fig. 4 is a diagram of the primary iPSCs in example 1.

Fig. 5 is a diagram of iPSCs in example 2.

Fig. 6 is a diagram of precursor iMSC in example 2.

FIG. 7 is a diagram iMSCs in example 2.

Detailed Description

The application will be described in further detail below with reference to the drawings by means of specific embodiments. Wherein like elements in different embodiments are numbered alike in association. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted, or replaced by other elements, materials, or methods in different situations. In some instances, related operations of the present application have not been shown or described in the specification in order to avoid obscuring the core portions of the present application, and may be unnecessary to persons skilled in the art from a detailed description of the related operations, which may be presented in the description and general knowledge of one skilled in the art.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning.

the differentiation step comprises the step of differentiating and culturing the induced pluripotent stem cells into mesenchymal stem cells, namely the Induced Pluripotent Stem Cells (iPSCs) differentiated source mesenchymal stem cells (iMSCs), namely the induced mesenchymal stem cells.

In one embodiment, the stem cell culturing step comprises:

A somatic cell preparation step comprising culturing mesenchymal stem cells using a mesenchymal stem cell medium without feeder cells and without animal-derived components; the mesenchymal stem cell culture medium without feeder cells and animal source components can be purchased from the market;

A cell transfection step comprising pipetting the mesenchymal stem cell medium and culturing the mesenchymal stem cells using the mesenchymal stem cell medium containing the cytokine;

a dosing screening step, comprising culturing the mesenchymal stem cells after screening by using a mesenchymal stem cell culture medium containing a screening reagent;

removing the medicine and culturing, namely sucking and removing a mesenchymal stem cell culture medium containing a screening reagent, adding the medicine-removing culture medium, and culturing to obtain mesenchymal stem cells;

The clone screening step comprises sucking and removing the medicine culture medium, adding a cell culture medium without feeder cells and animal source components, and culturing to obtain mesenchymal stem cell clone;

And cloning and picking, namely picking cell clones, and subculturing to obtain the induced pluripotent stem cells.

In one embodiment, in the cell transfection step, the mesenchymal stem cell medium further comprises an mRNA in vitro transcription mixture (mRNA cocktails).

In one embodiment, the mRNA in vitro transcription mixture comprises at least one of the coding sequences of the Oct-3, oct-4, klf-4, sox2, glis1, c-Myc, puromycin (puromycin) resistance genes.

In one embodiment, in the cell transfection step, the cytokine includes, but is not limited to, B18.

In one embodiment, in the cell transfection step, the drug addition screening step and the drug removal culture step, the mesenchymal stem cell culture medium is a mesenchymal stem cell culture medium without feeder cells and animal-derived components.

In one embodiment, in the medicated screening step, the screening agent comprises puromycin. Puromycin addition was intended to screen out cells that were not successfully reprogrammed.

In one embodiment, in the step of degerming, the degerming medium includes, but is not limited to, a mesenchymal stem cell medium.

In one embodiment, in the step of degerming, the degerming medium contains cytokines.

In one embodiment, the cytokine includes, but is not limited to, B18 during the drug-free culturing step.

In one embodiment, the medium used in the clone selection step and the clone selection step is a feeder cell-free, animal-derived component-free cell medium.

In one embodiment, the feeder cells-free, animal-derived component-free cell culture medium in the clonal selection step includes, but is not limited to, mTESR ^TM medium.

In one embodiment, the medium used in the clone selection step includes, but is not limited to, teSR ^TM-E8^TM medium.

In one embodiment, the differentiating step includes primary differentiating culture, secondary differentiating culture, and tertiary differentiating culture.

In one embodiment, the primary differentiation medium used in the primary differentiation culture contains an inhibitor.

In one embodiment, the inhibitor includes, but is not limited to, a PORCN inhibitor at the time of one differentiation culture.

In one embodiment, the PORCN inhibitor includes, but is not limited to Wnt-C59 upon a single differentiation culture.

In one embodiment, the secondary differentiation medium used in the secondary differentiation culture contains cytokines.

In one embodiment, the cytokine includes, but is not limited to, structure-specific recognition protein 1 (SSRP 1) upon secondary differentiation. The structure-specific recognition protein 1 may be a recombinant structure-specific recognition protein 1 or a natural structure-specific recognition protein 1.

In one embodiment, the tertiary differentiation medium used in the tertiary differentiation culture contains cytokines.

In one embodiment, the cytokines include, but are not limited to, at least one of structure specific recognition protein 1 (SSRP 1), growth differentiation factor 5 (GDF 5) when cultured for three differentiation. The structure-specific recognition protein 1 (SSRP 1), the growth differentiation factor 5 (GDF 5) may be a recombinant protein or a natural protein.

In one embodiment, in the stem cell culturing step, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

In one embodiment, in the stem cell culturing step, the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells.

In one embodiment, the method further comprises an expansion step, wherein the mesenchymal stem cells obtained in the differentiation step are subjected to expansion culture by using an expansion culture medium, and the mesenchymal stem cells after the expansion culture are obtained. This step allows for large-scale expansion of mesenchymal stem cells.

In one embodiment, the stem cell culturing step, the differentiating step, and the expanding step are performed using a medium that does not require feeder cells and that does not have animal-derived components.

In one embodiment, the invention establishes an induced pluripotent stem cell model without serum and double antibody (penicillin streptomycin), and based on the model, the induced pluripotent stem cell model is differentiated into an induced mesenchymal stem cell, so that the invention has better clinical application advantages.

In one embodiment, the present invention is directed to a novel iMSCs preparation method, which first uses mR NA cocktails to create a method for preparing human induced pluripotent stem cells without feeder cells and without animal-derived components. The method does not need feeder cells and fetal calf serum, has definite culture medium components and simple operation, avoids the integration risk of exogenous genome, and can efficiently and stably prepare the human iPSCs cell strain. And then, a iMSCs differentiation system is optimized to obtain iMSCs from which iPSCs differentiate, and the iPSCs have the advantages of high differentiation consistency, strong proliferation capacity, high telomerase activity, high expression of CD73, CD90 and CD105, and low expression of CD34 and CD45. Finally, the 3D micro slide system is used for completing the large-scale expansion of iMSCs, the cell uniformity is good, and a good technical foundation is laid for finally establishing human iMSCs cell resources of clinical application level.

Aiming at the technical problems of the existing MSCs preparation, in one embodiment, the invention establishes a iMSCs preparation system which is simple to operate, good in stability, has infinite amplification capability and high in quality.

In one embodiment, as shown in FIG. 1, the overall process includes the steps of:

(1) Establishing non-integrated non-viral, serum-free, medium composition-specific, feeder cell-free iPSCs cell lines.

(2) IPSCs differentiate efficiently to iMSCs.

(3) IMSCs 3D large scale amplification.

In one embodiment, the specific steps are as follows:

(1) Establishing non-integrated non-viral, serum-free, medium composition-specific, feeder cell-free iPSCs cell lines. The method comprises the following specific steps:

1) Somatic cell preparation: umbilical cord-derived Mesenchymal Stem Cells (MSCs) were resuscitated using MSCs medium (Youkang NC 0103) with clear chemical composition, without feeder cells, without animal-derived composition, and cell confluency was observed under a microscope at about 70%, cells were collected after digestion with 0.05% TrypLE ^TM Express (Thermo FISHER SCIENTIFIC), and cell viability was calculated to be greater than 90% using trypan blue staining (STEMCELL technologies) and a Counter star, and then plated into 6-well plates pre-coated with 5 μg/Kong Bo of fibronectin (Vitronnectin, STEMCELL technologies), total number of 5 to 10 ten thousand cells per well, preferably 5 ten thousand, and then transferred to a 37℃incubator for culturing for 24 hours.

2) Cell transfection: cells were observed under a microscope at a confluence of about 25%, MSCs medium was aspirated and removed, washed 1-pass with 1mLD-PBS (without Ca ²⁺ and Mg ²⁺), 1mL 100ng/mL B18 factor MSCs medium was added and incubated for 20 min in a 37℃incubator. Then mRNA co cktails reagent combinations (specifically 1 mu L ReproRNA ^TM -OKSGM, 100 mu L) after 5 minutes of standing at room temperature were added dropwise to each 6-well plateI Reduced-Ser um Medium, 2. Mu. L ReproRNA ^TM Transfection Supplement and 2. Mu. L ReproRNA ^TM Transfection Reagent) and then transferred to an incubator at 37℃for 24 hours.

3) And (3) dosing and screening: the medium in the 6-well plate was aspirated, 1.5mL of screening medium (MSCs medium+100 ng/mL B18+0.8. Mu.g/mL puromycin (Puromycin)) was added, and then transferred to an incubator at 37℃for 24 hours, the liquid change operation was repeated every day, and the operation was repeated for 2 days.

4) Removing medicine and culturing: the culture medium in the 6-well plate is sucked and removed, 1.5mL of the drug-removing culture medium (MSCs culture medium+100 ng/mL B18) is added, then the culture medium is transferred to a 37 ℃ incubator for continuous culture, the change of cell morphology is observed, at this time, cobblestone-like epithelial cells can be observed, the liquid-changing operation is carried out every day, and the operation is repeated for 2 days.

5) Clone screening: the medium in the 6-well plate was aspirated, 2mL mTeSR ^TM medium (STEMCELL techno logies) was added, transferred to a 37 ℃ incubator for continued culture, the liquid change was performed daily, and cell morphology changes were observed under a microscope with tracking until iPSCs clones appeared (after about 2 weeks).

MTeSR ^TM is a defined, feeder-free serum-free cell culture medium.

The effect of each medium on the cells is different; wherein mTESR ^TM culture medium is used for culturing induced pluripotent stem cells, and is used for clone screening in the process; whereas MSCs medium is the medium required for the step prior to clone selection.

6) Clone picking and identification: clone shape was recorded under a microscope and the markers were photographed, single clones were transferred to a 12 well plate pre-coated with 2. Mu.g vitronectin using a 10. Mu.L pipette, 1mL TeSR ^TM-E8^TM medium (STEMCE LL technologies) was added, 10 times with a 1mL pipette tip, the markers passage 1 were then transferred to a 37℃incubator for further culture. After 3 to 5 days, the confluence of the iPSCs is about 80%, and after digestion of the iPSCs with ReLeSR digest (STEMCELL TECHN ologies), the iPSCs are not lower than 1: and carrying out passage expansion culture according to the number proportion of 10 cells. After the iPSCs are cultured to passage to be passage, carrying out quality identification, wherein the quality identification comprises the following steps: a) Cell morphology analysis; b) Alkaline phosphatase staining; c) Immunofluorescence staining includes but is not limited to OCT4, SSEA-4, TRA-1-60, and the like; d) Embryoid body trigermal differentiation; e) And (3) carrying out experimental identification on teratomas and the like, and determining a non-integrated non-viral iPSCs cell model with higher quality.

TeSR ^TM-E8^TM is a culture medium of human embryonic stem cells without feeder cells and without animal-derived components for culturing human Embryonic Stem (ES) cells and human induced pluripotent stem (hiPS) cells.

The TeSR ^TM-E8^TM culture medium is used for cloning culture after cloning screening is finished; whereas mTeSR ^TM 1 was used for clone selection. From the clinical application level, teSR ^TM-E8^TM is more stable in pH than mTESR ^TM, and the added factors are more suitable for cell growth and have the advantage of large-scale preparation.

(2) IPSCs differentiate efficiently to iMSCs. The method mainly comprises the following steps:

1) Resuscitate the non-integrated non-viral iPSCs into 6-well plates pre-coated with 5 μg vitronectin, and culture in an incubator at 37℃for 3-5 days until the iPSCs reach a confluency of about 80%.

2) TeSR ^TM-E8^TM medium was aspirated, 1mL of D-PBS (without Ca ²⁺ and Mg ²⁺) was added for 1 wash, 3mLiMSCs differentiation medium 1, comprising ：Mesenchymal Induction Medium(STEMCELL technologi es)+1μM～5μM CHIR99021(STEMCELL technologies)+0.1μM～0.5μM Wnt-C59(Selleck), preferably 1. Mu.M CHIR99021, 0.1. Mu.M Wnt-C59, was added, and after transferring to a 37℃incubator for 24 hours, the daily pipetting operation was repeated for 72 hours.

3) The iMSC differentiation medium 1 in the 6-well plate was pipetted, 1mL of D-PBS (without Ca ²⁺ and Mg ²⁺) was added for 1 time, 2mL of iMSC differentiation medium 2, the components including ：MesenCultACF Plus Medium(STEMCELL tech nologies)+5ng/mL Recombinant Structure Specific Recognition Protein 1(SSRP1, recombinant structure-specific recognition protein 1 were added, then the mixture was transferred to a 37℃incubator for culturing for 24 hours, the liquid change operation was repeated 1 time per day, the cells were observed to reach a confluence of 80% or more, the cells were digested with 0.05% TrypLE ^TM Express, the cell suspension was collected in a 15mL centrifuge tube, 300 Xg was centrifuged for 5 minutes, the supernatant was discarded, the cell pellet was resuspended in 2mL iMSCs differentiation medium 3, the components including ：MesenCultACF Plus Medium(STEMCELL technologies)+5ng/mL Recombinant Structure Specific Recognition Protein 1(SSRP1)+5ng/mL Human Recombinant GDF5. were then plated in a 6-well plate previously coated with 5. Mu.g/Kong Bo of fibronectin at 1.5-6X 10. Mu.3/cm ², and the confluence was reached about 80% after culturing in a 37℃incubator for 3 days.

(3) IMSCs 3D large-scale amplification and identification.

1) IMSCs of cells were observed under a microscope at a confluence of about 80%, collected after digestion of the cells with 0.05% TrypLE ^TM, and then cell viability was calculated to be greater than 90% using trypan blue staining and a cytometer using 3D MiniSPIN bioreactor (Beijing Hua niche organism) combined with 3D/>The cell expansion suit FK01 (Beijing Hua niche organism) is subjected to 3D culture, the rotating speed is controlled to be 30-60 rpm by adopting a magnetic field, preferably 45rpm, the speed is low, the accuracy is high, the cell growth rate can be effectively improved, and meanwhile, the damage of shearing force to cells in the stirring process is avoided. The method mainly comprises the following steps: taking 50 ten thousand cells corresponding to 1 micro slide as initial input cells corresponding to 10mL iMSCs amplification medium 1, wherein the components comprise ：Mes enCultACF Plus Medium(STEMCELL technologies)+5ng/mL Human Recombinant GDF5(STEMCELL technologies), or iMSCs amplification medium 2, and the components comprise: MSCs Medium (Youkang NC 0103) +5ng/mL Human Recombinant GDF (STEMCELL technologies), preferably iMSCs amplification Medium 2. In order to expand the quantity of a large number of cells, the method selects 250 ten thousand cells to correspond to 5 micro-slides, adds 50mL iMSCs amplification culture medium 1 or 2 into a 125mL culture bottle, detects the glucose concentration every 24 hours to determine the quantity of the supplemented liquid (the glucose concentration needs to reach the initial culture concentration), and can reach the amplification quantity of 1 to 10 times after 4 days of continuous amplification, namely more than 2500 ten thousand cells.

2) IMSCs after 3D expansion, including but not limited to, the following identification, a) cell morphology identification; b) Flow cytometry identified MSCS MARKER (CD 73, CD90, CD105, CD34, CD 45) expression; c) Analysis of telomerase activity; d) Single cell sequencing cell subpopulation identification; e) The COL2A1/SOX9 immunofluorescence staining identified iMSCs functional subgroups.

In one embodiment, the invention establishes a clinical grade iMSCs preparation method for the first time, firstly utilizes mRNA cocktails including multipotent genes Oct-3/4, klf-4, sox2, glis1, c-Myc and resistance gene puromycin (puromycin) protein coding sequences, initiates activation of a cell endogenous multipotent gene network through expression of exogenous multipotent genes, and further screens and removes unsuccessfully reprogrammed cells through puromycin drugs, thereby establishing a feeder-layer-free serum-free human induced multipotent stem cell preparation method. The method does not need feeder cells and fetal calf serum, has definite culture medium components and simple operation, avoids the integration risk of exogenous genome, and can efficiently and stably prepare the human iPSCs cell strain. Secondly, according to the prior research results, the specific factors are found to have important promotion and maintenance effects on maintaining specific cell subsets of MSCs, such as promotion of highly-proliferated MSCs by SSR P1 (structure-specific recognition protein 1), promotion of mesoderm and cartilage development by Wnt-C59 and GDF5, optimization of iMSCs differentiation system, obtaining MSCs (iMSCs) from iPSCs differentiation, higher differentiation consistency, significantly improved proliferation capacity, higher telomerase activity, high expression of CD73, CD90, CD105 and low expression of CD34 and CD45. And finally, the 3D micro-slide system is used for completing the large-scale amplification of iMSCs, so that the cell uniformity is good. Laying a good technical foundation for finally establishing human iMSCs cell resources of clinical application level.

Example 1: establishing clinical human iPSCs cell strain.

1) Somatic cell preparation: human umbilical cord-derived mesenchymal stem cells (Human Umbilical Cord MESENCHYMAL STEM CELLS, HUC-MSCs) were resuscitated and cultured using a medium (Youkang NC 0103) having a clear chemical composition, serum-free, animal-derived composition, and observed under a microscope to a confluence of about 70%, cells were collected after digestion with 0.05% TrypLE ^TM Express, and then cell viability was calculated to be greater than 90% using trypan blue staining and a cell counter, and then cells were plated into 6-well plates pre-coated with 5. Mu.g/well of Vitronectin (Vitronin), 5 ten thousand and 10 ten thousand different test conditions per well of cells, and then transferred to a 37℃incubator for culture for 24 hours.

2) Cell transfection: confluence of 5 ten thousand cells per well at about 25% (FIG. 2-MSCs) was observed under a microscope, MSCs medium (Youkang NC 0103) was pipetted, 1mL of D-PBS (without Ca ²⁺ and Mg ²⁺) was added for 1 wash, then 1mL of MSCs medium (commercial serum-free MSCs medium) with a concentration of 100ng/mL B18 factor was added, and the mixture was returned to the incubator at 37℃for 20 minutes. mRNA cocktail reagent combinations (specifically including 1. Mu. L ReproRNA ^TM -OKSGM, 100. Mu.L) after 5 minutes of standing at room temperature were then added dropwise to each 6-well plateI Reduced-Serum Me dium, 2. Mu. L ReproRNA ^TM Transfection Supplement and 2. Mu. L ReproRNA ^TM Transfection Reagent) and then transferred to an incubator at 37℃for 24 hours.

3) And (3) dosing and screening: the medium in the 6-well plate was aspirated, 1.5mL of screening medium (MSCs medium+100 ng/mL B18+0.8. Mu.g/mL puromycin (Puromycin)) was added, and then transferred to a 37℃incubator for 24 hours, and the pipetting operation was repeated every day.

4) Removing medicine and culturing: the medium in the 6-well plate was aspirated, 1.5mL of the drug-free medium (MSCs medium+100 ng/mL B18) was added, and then the mixture was transferred to a 37℃incubator to culture and continued to culture, and the change in cell morphology was observed, at which time the occurrence of cobblestone-like epithelial-like cell clone was observed (FIG. 3-clone embryonic form).

5) Clone screening: the culture medium in the 6-well plate was aspirated, 2mL mTESR medium was added, and the mixture was transferred to a 37℃incubator to continue the culture, and the morphological changes of the cells were observed under a microscope in a tracking manner until iPSCs clone (FIG. 4-primary iPSCs) appeared.

6) Clone picking and identification: clone shape was recorded under a microscope and the markers were photographed, single clones were transferred to a 12 well plate pre-coated with 2. Mu.g vitronectin using a 10. Mu.L pipette, 1mL TeSR ^TM-E8^TM medium was added, 10 times with a 1mL pipette tip, the markers passage 1 were then transferred to a 37℃incubator for further culture. After 3 to 5 days, iPSCs were confluent at about 80%, and after digestion of iPSCs with ReLeSR digest (STEMCELL technologies), passaging expansion culture was performed at a cell number ratio of not less than 1:10. After the iPSCs are cultured to passage to be passage, carrying out quality identification, wherein the quality identification comprises the following steps: a) Cell morphology identification; b) Alkaline phosphatase staining; c) Immunofluorescence staining includes but is not limited to OCT4, SSEA-4, TRA-1-60, and the like; d) Embryoid body trigermal differentiation; e) And (3) carrying out experimental identification on teratomas and the like, and determining a non-integrated non-viral iPSCs cell model with higher quality.

Example 2: iPSCs differentiate efficiently to iMSCs.

1) The non-integrated non-viral iPSCs were resuscitated into 6 well plates pre-coated with 5. Mu.g vitronectin, and cultured in an incubator at 37℃for 3-5 days until iPSCs reached a confluency of about 80% (FIG. 5-iPSCs).

2) TeSR ^TM-E8^TM medium was aspirated, 1mL of D-PBS (without Ca ²⁺ and Mg ²⁺) was added and washed 1 time, 3mLiMSCs of differentiation medium 1 was added, and the components of differentiation medium 1 included: MESENCHYMAL INDUCTION MEDIUM +1. Mu.M CHIR 99021+0.1. Mu.M Wnt-C59; then, after transferring to an incubator at 37℃for 24 hours, the daily liquid change operation was repeated for 72 hours.

3) The iMSC differentiation medium 1 in the 6-well plate was pipetted, 1mL of D-PBS (without Ca ²⁺ and Mg ²⁺) was added for 1 time, 2mL iMSCs differentiation medium 2 was added, the components of differentiation medium 2 were also worked up under ：MesenCultACF Plus Medium+5ng/mL Recombinant Structure Specific Recognition Protein 1(SSRP1)(10ng/mL SSRP1 conditions, then the mixture was transferred to a 37℃incubator for culturing for 24 hours, the liquid change operation was repeated 1 time per day, the cells were observed to reach a confluence of 80% or more (FIG. 6-precursor IMSC) by microscopy, the cells were digested with 0.05% TrypLE ^TM Express, the cell suspension was collected in a 15mL centrifuge tube, 300 Xg was centrifuged for 5 minutes, the supernatant was discarded, the cell pellet was resuspended in 2mL iMSCs differentiation medium 3, the components of differentiation medium 3 were ：MesenCultACF Plus Medium(STEMCELL technolo gies)+5ng/mL Recombinant Structure Specific Recognition Protein 1(SSRP1)+5ng/mL Huma n Recombinant GDF5. and then plated in a 6-well plate pre-coated with 5. Mu.g/Kong Bo of desmin 1.5-6X 10. Lambda.3/cm ², and the confluence was reached about 80% after culturing for 3 days in the 37℃incubator (FIG. 7-iMSCs).

Example 3: iMSCs 3D large-scale amplification and identification.

1) IMSCs of cells were observed under a microscope at a confluence of about 80%, collected after digestion of the cells with 0.05% TrypLE ^TM, and then cell viability was calculated to be greater than 90% using trypan blue staining and a cytometer using 3D MiniSPIN bioreactor (Beijing Hua niche organism) combined with 3D/>Cell expansion suit FK01 (Beijing Hua niche organism) is subjected to 3D culture, the rotating speed is controlled to be 30-60 rpm by adopting a magnetic field, the rotating speed is 45rpm in the embodiment, the speed is low, the accuracy is high, the cell growth rate can be effectively improved, and meanwhile, the damage of shearing force to cells in the stirring process is avoided. The method mainly comprises the following steps: taking 50 ten thousand cells corresponding to 1 micro slide as initial input cells, and correspondingly needing 10mL iMSCs amplification culture medium 1, wherein the components of the iMSCs amplification culture medium 1 comprise ：MesenCultACF Plus Medium(STEMCELL technologies)+5ng/mL Human Recombinant GDF5(STEMCELL technologies), or iMSCs amplification culture medium 2, and the components comprise: MSCs medium (Youkang NC 0103) +5ng/mL Human Recombinant GDF (STEMCELL TECHN ologies), in this example iMSCs amplification medium 2. In order to amplify the quantity of a large number of cells, the method selects 250 ten thousand cells to correspond to 5 micro-slides, adds 50mL iMSCs amplification culture medium 1 or 2 (specifically, the amplification culture medium 1 in the embodiment) into a 125mL culture bottle, detects the glucose concentration every 24 hours to determine the liquid supplementing quantity (the glucose concentration needs to reach the initial culture concentration), and after continuous amplification for 4 days, the total number of cells can reach the amplification quantity which is not less than 10 times, namely, more than 2500 ten thousand cells.

2) IMSCs after 3D amplification, the following assays were performed: a) Cell morphology identification; b) Flow cytometry identified MSCS MARKER (CD 73, CD90, CD105, CD34, CD 45) expression; c) Analysis of telomerase activity; d) Single cell sequencing cell subpopulation identification; e) The COL2A1/SOX9 immunofluorescence staining identified iMSCs functional subgroups.

In this example, according to the previous research results, it was found that the specific factor has an important promoting effect on maintaining specific cell subsets of MSCs, for example, SSRP1 promotes highly proliferated MSCs subset, wnt-C59 and GDF5 promote mesoderm and cartilage development, and a iMSCs differentiation system is optimized to obtain MSCs (isscs) derived from iPSCs, which have higher differentiation consistency, significantly improved proliferation capacity, higher telomerase activity, high expression of CD73, CD90, CD105 and low expression of CD34 and CD45, and are superior to international identification standards, and the expression of marker genes is shown in table 1 below. And finally, the 3D micro-slide system is used for completing the large-scale amplification of iMSCs, so that the cell uniformity is good. Laying a good technical foundation for finally establishing clinical application-level human iMSCs cell resources.

TABLE 1

Marker gene	International authentication standard	IMSCs expression ratio
			CD73	95％	99.1％
CD90	95％	98.5％
			CD105	95％	96.8％
CD34	2％	0.06％
			CD45	2％	0.002％

As can be seen from Table 1, iMSCs of the culture method and system of the present example has better quality and application advantages, and compared with the conventional tissue-derived MSCs, the iPSC cell differentiation-derived iMSCs has unlimited source capacity, can be greatly proliferated, and has the advantage of cell reporting medicine.

In an embodiment, the invention establishes a large-scale preparation method of clinical grade iMSCs functional subgroups for the first time, and the whole technical scheme has the advantages of simple operation, easy standard large-scale production, avoiding the use of reagents such as fetal calf serum, heterologous component reagents and the like in the whole process, and having obvious application advantages.

In one embodiment, firstly, mRNA cocktails including multipotency genes Oct-3, oct-4, klf-4, sox2, glis, c-Myc and puromycin (puromycin) resistance gene coding sequences are utilized, activation of a cell endogenous multipotency gene network is triggered by expression of exogenous multipotency genes, and unsuccessfully reprogrammed cells are further removed by screening puromycin drugs, so that a feeder-free serum-free preparation method of human induced multipotency stem cells is established. The method does not need feeder cells and fetal calf serum, has definite culture medium components and simple operation, avoids the integration risk of exogenous genome, and can efficiently and stably prepare the human iPSC cell strain.

The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.

Claims

1. A method of preparing an induced mesenchymal stem cell, comprising:

A stem cell culturing step, including providing mesenchymal stem cells, culturing to obtain non-integrated non-virus induced pluripotent stem cells;

Differentiation step, including that the induced pluripotent stem cells are subjected to primary differentiation culture, secondary differentiation culture and tertiary differentiation culture to obtain mesenchymal stem cells, namely the induced mesenchymal stem cells;

the culture medium for the primary differentiation culture comprises the following components: MESENCHYMAL INDUCTION MEDIUM +1. Mu.M to 5. Mu.M CHIR 99021+0.1. Mu.M to 0.5. Mu.M Wnt-C59;

The culture medium for the secondary differentiation culture comprises the following components: mesenCultACF Plus Medium +5ng/mL Recombinant Structure Specific Recognition Protein 1;

The culture medium composition of the three-time differentiation culture comprises ：MesenCultACF Plus Medium+5ng/mL Recombinant Structure Specific Recognition Protein 1+5ng/mL Human Recombinant GDF5.

2. The method of claim 1, wherein the stem cell culturing step comprises:

A somatic cell preparation step comprising culturing mesenchymal stem cells using a mesenchymal stem cell medium without feeder cells and without animal-derived components;

3. The method of claim 1, wherein in the stem cell culturing step, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

4. The method of claim 3, wherein in the stem cell culturing step, the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells.

5. The method of claim 1, further comprising an expansion step of subjecting the mesenchymal stem cells obtained in the differentiation step to expansion culture using an expansion medium to obtain the mesenchymal stem cells after the expansion culture.

6. The method of claim 5, wherein the medium used in the stem cell culturing step, the differentiating step, and the expanding step is a feeder cell-free, animal-derived component-free cell medium.

7. An induced mesenchymal stem cell prepared by the method of any one of claims 1-6.