CN114438037A - Method for preparing inductive mesenchymal stem cells - Google Patents
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Abstract
A method of preparing an induced mesenchymal stem cell, comprising: a stem cell culture step, which comprises providing mesenchymal stem cells for culture to obtain inductive mesenchymal stem cells; and a differentiation step, which comprises the step of differentiating and culturing the induced pluripotent stem cells into mesenchymal stem cells, namely the Induced Mesenchymal Stem Cells (iMSCs). The method does not need feeder cells and fetal calf serum, is simple to operate, avoids exogenous genome integration risk, and can efficiently and stably prepare the human iMSCs cell strain.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for preparing inductive mesenchymal stem cells.
Background
At present, Mesenchymal Stem Cells (MSCs) are applied as cells, and two technical bottlenecks still exist, the first is limited cell number. Mesenchymal stem cells are mainly separated and prepared 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 number of cell amplification processes are required before clinical application, the in vitro amplification processes can also cause different aging states of cells, the proliferation capacity is reduced, and the like, and the number of the harvested cells cannot meet the clinical transplantation requirement. The second is that the preparation of MSCs derived from non-tissue is still a technical challenge, although there is a research on the differentiation of MSCs using Embryonic Stem Cells (ESCs) or Induced Pluripotent Stem Cells (iPSCs), the differentiation of MSCs is still performed by using Embryonic Stem Cells (ESCs) to induce ethical dispute risk, induced pluripotent stem cells often use a virus integration system to have genome integration risk, and moreover, iPSCs differentiation-derived MSCs (iMSCs) depend on the treatment using animal serum and multiple biological small molecules, and have problems of complex procedure, high heterogeneity of 2D amplified cells, and the like, which limits the further clinical application of iMSCs.
Disclosure of Invention
According to a first aspect, in one embodiment, there is provided a method of preparing an induced mesenchymal stem cell, comprising:
a stem cell culture step, which comprises providing mesenchymal stem cells and culturing to obtain induced pluripotent stem cells;
and a differentiation step, which 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 one 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 calf serum are not needed, the operation is simple, the risk of exogenous genome integration is avoided, and the human iMSCs cell strain can be efficiently and stably prepared.
Drawings
FIG. 1 is a flow diagram of one example of the preparation of iMSCs.
FIG. 2 is a graph of MSCs plated microscopically at approximately 25% confluence on 5 million/well cells in example 1.
FIG. 3 is a diagram of clone primitives in example 1.
FIG. 4 is a graph of primary iPSCs in example 1.
FIG. 5 is a diagram of iPSCs in example 2.
Fig. 6 is a diagram of the precursor iMSC in example 2.
FIG. 7 is a graph of iMSCs in example 2.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. Wherein like elements in different embodiments are numbered with like associated elements. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.
Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the description of the methods may be transposed or transposed in order, as will be apparent to a person skilled in the art. Thus, the various sequences in the specification and drawings are for the purpose of clearly describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where a certain sequence must be followed.
The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning.
According to a first aspect, in one embodiment, there is provided a method of preparing an induced mesenchymal stem cell, comprising:
a stem cell culture step, which comprises providing mesenchymal stem cells and 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 mesenchymal stem cells (iMSCs) from which the Induced Pluripotent Stem Cells (iPSCs) are differentiated, which are also called induced mesenchymal stem cells.
In one embodiment, the stem cell culturing step comprises:
a somatic cell preparation step, which comprises the step of culturing mesenchymal stem cells by using a mesenchymal stem cell culture medium without feeder cells and animal-derived components; the mesenchymal stem cell culture medium without feeder cells and animal-derived components can be purchased from the market;
a cell transfection step, which comprises sucking and abandoning the mesenchymal stem cell culture medium and using the mesenchymal stem cell culture medium containing cell factors to culture the mesenchymal stem cells;
adding drugs to screen, including using the mesenchymal stem cell culture medium containing screening reagent, culturing to obtain the screened mesenchymal stem cells;
a drug removing culture step, which comprises the steps of absorbing and discarding the mesenchymal stem cell culture medium containing the screening reagent, adding the drug removing culture medium, and culturing to obtain mesenchymal stem cells;
a step of cloning and screening, which comprises the steps of removing a medicine culture medium by suction, adding a cell culture medium without feeder cells and animal-derived components, and culturing to obtain mesenchymal stem cell clones;
and cloning and selecting, wherein the cloning and selecting step comprises the steps of selecting cell cloning, subculturing and obtaining the induced pluripotent stem cells.
In one embodiment, the mesenchymal stem cell culture medium further comprises an mRNA in vitro transcription mixture (mRNA cocktails) in the cell transfection step.
In one embodiment, the mRNA in vitro transcription mixture comprises at least one of Oct-3, Oct-4, Klf-4, Sox2, Glis1, c-Myc, puromycin (puromycin) resistance gene coding sequences.
In one embodiment, in the cell transfection step, the cytokine includes, but is not limited to, B18.
In one embodiment, the mesenchymal stem cell culture medium is a mesenchymal stem cell culture medium without feeder cells and animal-derived components in the cell transfection step, the drug adding screening step and the drug removing culture step.
In one embodiment, in the medicated screening step, the screening reagent comprises puromycin. The addition of puromycin was designed to screen for the removal of unsuccessfully reprogrammed cells.
In one embodiment, the drug-removed culture medium in the step of drug-removed culture includes, but is not limited to, mesenchymal stem cell culture medium.
In one embodiment, in the drug-free culture step, the drug-free medium contains cytokines.
In one embodiment, the cytokine comprises, but is not limited to, B18 in the drug-free culture step.
In one embodiment, the culture medium used in the clone screening step and the clone picking step is a cell culture medium without feeder cells and animal-derived components.
In one embodiment, the animal-derived component-free cell culture medium without feeder cells in the clonal selection step includes, but is not limited to, mTeSRTM1, culture medium.
In one embodiment, the culture medium used in the clone picking step includes, but is not limited to, TeSRTM-E8TMAnd (4) a culture medium.
In one embodiment, the differentiation step comprises a primary differentiation culture, a secondary differentiation culture, and a tertiary differentiation 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, PORCN inhibitors in one differentiation culture.
In one embodiment, the PORCN inhibitor includes, but is not limited to, Wnt-C59 at the time of one 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(SSRP1) in secondary differentiation culture. 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 example, three differentiation cultures are performed using a three differentiation medium containing cytokines.
In one embodiment, the cytokines include, but are not limited to, at least one of structure specific recognition protein 1(SSRP1), growth differentiation factor 5(GDF5) in three differentiation cultures. The structure specific recognition protein 1(SSRP1) and the growth differentiation factor 5(GDF5) can be recombinant proteins or natural proteins.
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 an embodiment, the method further comprises an amplification step, including performing amplification culture on the mesenchymal stem cells obtained in the differentiation step by using an amplification culture medium, and obtaining the mesenchymal stem cells after the amplification culture. The step realizes the large-scale amplification of the mesenchymal stem cells.
In one embodiment, the culture medium used in the stem cell culturing step, the differentiation step and the expansion step is a feeder cells-free and animal-derived component-free cell culture medium.
According to a second aspect, in one embodiment, there is provided an induced mesenchymal stem cell produced by the method of the first aspect.
In one embodiment, the method establishes a complete serum-free and double-antibody-free (penicillin streptomycin) -induced pluripotent stem cell model, and differentiates the model into an induced mesenchymal stem cell, so that the method has better clinical application advantages.
In one embodiment, the present invention is directed to a novel method for producing iMSCs by first creating a method for producing human-induced pluripotent stem cells without feeder cells and without animal-derived components using mR NA cocktails. The method does not need feeder cells and fetal calf serum, has definite culture medium components and simple operation, avoids exogenous genome integration risk, and can efficiently and stably prepare the human iPSCs cell strain. And secondly, optimizing an iMSCs differentiation system to obtain iMSCs from iPSCs differentiation sources, wherein the iMSCs 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 CD 45. And finally, the 3D micro-slide system is used for completing the large-scale amplification of the iMSCs, the cell uniformity is better, and a good technical foundation is laid for finally establishing human iMSCs cell resources of clinical application level.
Aiming at the technical problem of the existing preparation of the MSCs, in one embodiment, the invention establishes a set of iMSCs preparation system which is simple to operate, good in stability, infinite in amplification capacity and high in quality.
In one embodiment, as shown in FIG. 1, the overall process includes the following steps:
(1) establishing a non-integrated non-virus, serum-free, medium-ingredient-clear and feeder-layer-cell-free iPSCs cell strain.
(2) The iPSCs are efficiently differentiated into iMSCs.
(3) 3D large-scale amplification of iMSCs.
In one embodiment, the specific steps are as follows:
(1) establishing a non-integrated non-virus, serum-free, medium-ingredient-clear and feeder-layer-cell-free iPSCs cell strain. The method comprises the following specific steps:
1) preparing somatic cells: resuscitating and culturing umbilical cord-derived Mesenchymal Stem Cells (MSCs) by using MSCs culture medium (Youkang NC0103) with definite chemical components, no need of feeder cells and no animal-derived components, observing the cell confluency under microscope to be about 70%, and using 0.05% trypLETMExpress (thermo Fisher scientific) digestion followed by cell collection, followed by trypan utilizationBlue staining (STEMCELL technologies) and a cell Counter (Counter star) calculate the cell viability to be greater than 90%, then the cells are plated into 6-well plates previously coated with 5. mu.g/well Vitronectin (Vitronectin), 5-10 ten thousand cells per well, preferably 5 ten thousand cells are plated, and then transferred to a 37 ℃ incubator for 24 hours.
2) Cell transfection: the confluency of the cells was observed at about 25% under a microscope, the MSCs medium was discarded, and 1mLD-PBS (containing no Ca) was added2+And Mg2+) Washing is carried out for 1 time, and then 1mL of MSCs culture medium with 100ng/mL factor B18 is added and placed back into the incubator at 37 ℃ for 20 minutes. Then, mRNA co cktails reagent combination (specifically including 1. mu.L of ReProRNA) after being left at room temperature for 5 minutes was uniformly dropped into each 6-well plateTM-OKSGM、100μL
I Reduced-Ser um Medium、2μL ReproRNATMTransfection Supplement and 2. mu.L of ReProRNATMTransfection Reagent) and then transferred to a 37 ℃ incubator for 24 hours.
3) Adding drugs and screening: the medium in the 6-well plate was discarded, 1.5mL of screening medium (MSCs medium +100ng/mL B18+ 0.8. mu.g/mL Puromycin) was added, and the mixture was transferred to a 37 ℃ incubator and cultured for 24 hours, and the medium was changed every day, and the operation was repeated for 2 days.
4) Removing the medicine and culturing: the culture medium in the 6-well plate is removed, 1.5mL of drug-removing culture medium (MSCs culture medium +100ng/mL B18) is added, then the culture medium is transferred to a 37 ℃ incubator for continuous culture, the change of cell morphology is observed, the cobblestone-shaped epithelial-like cells can be observed, the liquid change operation is carried out every day, and the operation is repeated for 2 days.
5) Cloning and screening: the medium in the 6-well plate was aspirated away and 2mL mTeSR was addedTM1 medium (STEMCELL techno cells), transferred to a 37 ℃ incubator for further culture, and the change of cell morphology was followed under a microscope by changing liquid every day until iPSCs clone appeared (after about 2 weeks).
mTeSRTM1 is a defined, feeder-free, serum-free cell culture medium.
The effect of each medium on the cells is different; wherein mTeSRTM1, the culture medium is used for culturing induced pluripotent stem cells, and is used for cloning and screening in the process; while MSCs media is the media required for the steps prior to clonal selection.
6) Cloning, picking and identifying: recording clone shape under microscope and taking picture to mark, transferring single clone to 12-hole plate coated with 2 mug vitronectin in advance by 10 muL liquid-transfering gun, adding 1mL TeSRTM-E8TMThe medium (STEMCE LL technologies) was pipetted 10 times with a 1mL pipette tip, labeled passage 1, and then transferred to a 37 ℃ incubator for further culture. After 3 to 5 days, the confluence of iPSCs was around 80%, and after digestion of iPSCs with ReLeSR digest (STEMCELL technologies), the ratio of iPSCs was adjusted to a value of not less than 1:10 cell number ratio for subculture. After the iPSCs are cultured to passage 8, performing quality identification, including: a) analyzing cell morphology; b) alkaline phosphatase staining; c) immunofluorescent staining includes, but is not limited to OCT4, SSEA-4 and TRA-1-60, etc.; d) differentiating the three germ layers of the embryoid body; e) and (4) identifying teratoma and other experiments, and determining a non-integrated non-viral iPSCs cell model with higher quality.
TeSRTM-E8TMIs a human embryonic stem cell culture medium without feeder cells and animal-derived components, and is used for culturing human Embryonic Stem (ES) cells and human induced pluripotent stem (hiPS) cells.
TeSRTM-E8TMThe culture medium is used for cloning culture after cloning and screening; and mTeSRTM1 for clone screening. TeSR from the clinical application level perspectiveTM-E8TMCompared to mTeSRTM1, the pH is more stable, and the added factors are more suitable for cell growth and have the advantage of large-scale preparation.
(2) The iPSCs are efficiently differentiated into iMSCs. The method mainly comprises the following steps:
1) recovering the non-integrated non-viral iPSCs into a 6-well plate pre-coated with 5 mu g of vitronectin, and culturing the iPSCs in an incubator at 37 ℃ for 3-5 days to obtain about 80% confluency of iPSCs cells.
2) Absorbent TeSRTM-E8TMCulture medium, 1mL of D-PBS (Ca-free) was added2+And Mg2+) Washing for 1 time, and adding a 3mLiMSCs differentiation culture medium 1, wherein the components comprise: mesenchymal indication Medium (STEMCELL technologies es) + 1. mu.M-5. mu.M CHIR99021(STEMCELL technologies) + 0.1. mu.M-0.5. mu.M Wnt-C59(Selleck), preferably 1. mu.M CHIR99021, 0.1. mu.M Wnt-C59, and then transferred to a 37 ℃ incubator for 24 hours, followed by repeating the liquid change operation every day up to 72 hours.
3) The iMSC differentiation medium 1 in the 6-well plate was discarded and 1mL of D-PBS (Ca-free) was added2+And Mg2+) Wash 1 pass, add 2mL of iMSC differentiation medium 2, ingredients include: MesenCultACF Plus Medium (STEMCELL tech nologic) +5ng/mL Recombinant structural specificity Recognition Protein 1(SSRP1, Recombinant structural specificity Recognition Protein 1), then transferring to a 37 ℃ incubator for 24 hours, repeating the liquid change operation for 1 time each day, observing the cell reaching a confluency of more than 80% by a microscope, using 0.05% TrypeTMExpress to digest cells, collect cell suspension into 15mL centrifuge tube, centrifuge at 300 × g for 5 minutes, discard supernatant, resuspend cell pellet with 2mL ims differentiation medium 3, the composition includes: MesenCultACF Plus Medium (STEMCELL technologies) +5ng/mL Recombinant structural Recognition Protein 1(SSRP1) +5ng/mL Human Recombinant GDF 5. Then the concentration is 1.5-6X10^3/cm2The mixture is spread into a 6-well plate which is pre-coated with 5 mu g/well of vitronectin, and the confluency reaches about 80 percent after the mixture is cultured in an incubator at 37 ℃ for 3 days.
(3) And (3) carrying out large-scale amplification and identification on the iMSCs.
1) The iMSCs with about 80% confluency of cells were observed under a microscope using 0.05% TrypLETMExpress cells were collected after digestion, then cell viability was greater than 90% using trypan blue staining and cell counter, using 3D
MiniSPIN bioreactor (Beijing Hua niche organisms) combined with 3D
The cell amplification kit FK01 (Beijing Hua niche organisms) is used for 3D culture, the rotating speed is controlled by a magnetic field to be 30-60 rpm, 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 1 micro slide corresponding to 50 ten thousand cells as initial input cells and correspondingly needing 10mL of iMSCs amplification culture medium 1, the components of which comprise: mes enCultACF Plus Medium (stem cell technologies) +5ng/mL Human Recombinant GDF5(stem cell technologies), or iMSCs amplification Medium 2, the composition of which comprises: MSCs nutrient (friend NC0103) +5ng/mL Human Recombinant GDF5(stem cell technologies), preferably iMSCs expansion medium 2. In order to amplify a large number of cells, 250 ten thousand cells are selected to correspond to 5 micro-slides, 50mL of iMSCs amplification culture medium 1 or 2 is added into a 125mL culture bottle, glucose concentration is detected every 24 hours to determine the fluid infusion amount (the glucose concentration needs to reach the initial culture concentration), and after continuous amplification is carried out for 4 days, the amplification number of 1 time or more than 10 times, namely 2500 ten thousand cells can be achieved.
2) 3D amplified iMSCs, including but not limited to, the following identification, a) cell morphology identification; b) flow cytometry identified MSCs marker (CD73, CD90, CD105, CD34, CD45) expression; c) analyzing the activity of telomerase; d) identifying the single cell sequencing cell subset; e) COL2A1/SOX9 immunofluorescent staining identified a functional subset of iMSCs.
In one embodiment, the invention establishes a preparation method of clinical-grade iMSCs for the first time, and firstly utilizes mRNA cocktails comprising pluripotent gene Oct-3/4, Klf-4, Sox2, Glis1, c-Myc and resistance gene puromycin (puromycin) protein coding sequences, triggers activation of a cell endogenous pluripotent gene network through expression of exogenous pluripotent genes, and further screens and removes cells which are not successfully reprogrammed through puromycin drugs, thereby establishing a preparation method of feeder-layer-free and serum-free human induced pluripotent stem cells. The method does not need feeder cells and fetal calf serum, has definite culture medium components and simple operation, avoids exogenous genome integration risk, and can efficiently and stably prepare the human iPSCs cell strain. Secondly, specific factors are found to have important promotion and maintenance effects on maintaining specific cell subsets of the MSCs according to previous research results, for example, SSR P1 (structure specific recognition protein 1) promotes highly-proliferated MSCs subsets, Wnt-C59 and GDF5 promote mesoderm and cartilage development effects, an iMSCs differentiation system is optimized, and MSCs (iMSCs) derived from iPSCs differentiation are obtained, and the differentiation consistency is high, the proliferation capacity is remarkably improved, the telomerase activity is high, and high-expression CD73, CD90 and CD105, low-expression CD34 and CD45 are achieved. And finally, the 3D micro-slide system is used for completing the large-scale amplification of the iMSCs, and the cell uniformity is good. And a good technical foundation is laid for finally establishing the clinical application level human iMSCs cell resources.
Example 1: establishing a clinical-grade human iPSCs cell strain.
1) Preparing somatic cells: resuscitating and culturing Human Umbilical Cord-derived Mesenchymal Stem Cells (Human Umbilical Cord Mesenchymal Stem Cells, hUC-MSCs) by using a serum-free MSCs culture medium (Youkang NC0103) with clear chemical components, observing the confluency of the Cells to be about 70% under a microscope, and utilizing 0.05% TrypLETMExpress digestion followed by cell collection, followed by cell viability calculation of greater than 90% using trypan blue staining and a cell counter, followed by plating of cells into 6-well plates previously coated with 5 μ g/well Vitronectin (Vitronectin) at 5 and 10 million different test conditions per well, followed by transfer to a 37 ℃ incubator for 24 hours.
2) Cell transfection: the confluency of 5-ten-thousand/well plated cells was observed under a microscope at about 25% (FIG. 2-MSCs), the MSCs medium (Youkang NC0103) was discarded, and 1mL of D-PBS (containing no Ca) was added2+And Mg2+) After washing 1 time, 1mL of MSCs medium (commercial serum-free MSCs medium) with a concentration of 100ng/mL factor B18 was added and the mixture was returned to the 37 ℃ incubator for 20 minutes. Then, mRNA cocktails reagent combination (specifically including 1. mu.L of ReProRNA) after being left at room temperature for 5 minutes is uniformly dropped into each 6-well plateTM-OKSGM，100μL
I Reduced-Serum Me dium，2μL ReproRNATMTransfection SupplementAnd 2. mu.L of ReProRNATMTransfection Reagent) and then transferred to a 37 ℃ incubator for 24 hours.
3) Adding drugs and screening: the medium in the 6-well plate was discarded, 1.5mL of screening medium (MSCs medium +100ng/mL B18+ 0.8. mu.g/mL Puromycin) was added, and the mixture was transferred to a 37 ℃ incubator and cultured for 24 hours, and the medium exchange operation was repeated every day.
4) Removing the medicine and culturing: the 6-well plate medium was discarded, 1.5mL of drug-free medium (MSCs medium +100ng/mL B18) was added, and the mixture was transferred to a 37 ℃ incubator to continue culturing, and the morphology of the cells was observed to change, at which time the appearance of colonies of cobblestone-like epithelial cells was observed (FIG. 3-clonal nestle).
5) And (3) clone screening: the culture medium in the 6-well plate is discarded, 2mL of mTeSR culture medium is added, the culture is continued after being transferred to a 37 ℃ incubator for culture, and the morphological change of the cells is tracked and observed under a microscope until iPSCs clone appears (figure 4-primary iPSCs).
6) Cloning, picking and identifying: recording clone shape under microscope and taking picture to mark, transferring single clone to 12-hole plate coated with 2 mug vitronectin in advance by 10 muL liquid-transfering gun, adding 1mL TeSRTM-E8TMThe culture medium is blown and beaten 10 times by a 1mL pipette tip, marked as passage 1 and then transferred to a 37 ℃ incubator for culture and continued culture. After 3 to 5 days, the confluence of iPSCs is about 80%, and iPSCs are subjected to passage expansion culture at a cell number ratio of not less than 1:10 after being digested by ReLeSR digestive fluid (STEMCELL technologies). After the iPSCs are cultured to passage 8, performing quality identification, including: a) identifying cell morphology; b) alkaline phosphatase staining; c) immunofluorescent staining includes, but is not limited to OCT4, SSEA-4 and TRA-1-60, etc.; d) differentiating the three germ layers of the embryoid body; e) and (4) identifying teratoma and other experiments, and determining a non-integrated non-viral iPSCs cell model with higher quality.
Example 2: the iPSCs are efficiently differentiated into iMSCs.
1) Recovering non-integrated non-viral iPSCs into a 6-well plate pre-coated with 5 mu g of vitronectin, and culturing the iPSCs in an incubator at 37 ℃ for 3-5 days until the iPSCs reach the confluency of about 80% (shown in figure 5-iPSCs).
2) Suction and disposalTeSRTM-E8TMCulture medium, 1mL of D-PBS (Ca-free) was added2+And Mg2+) Washing for 1 time, and adding a 3mLiMSCs differentiation medium 1, wherein the differentiation medium 1 comprises the following components: mesenchym industry Medium + 1. mu.M CHIR99021+ 0.1. mu.M Wnt-C59; then transferred to a 37 ℃ incubator for 24 hours, and then the liquid change operation was repeated every day for 72 hours.
3) The iMSC differentiation medium 1 in the 6-well plate was discarded and 1mL of D-PBS (Ca-free) was added2+And Mg2+) Washing for 1 time, adding 2mL of iMSCs differentiation medium 2, wherein the differentiation medium 2 comprises the following components: MesenCultACF Plus Medium +5ng/mL Recombinant structural registration Protein 1(SSRP1) (10ng/mL SSRP1 conditions also work), then transferring to a 37 ℃ incubator to culture for 24 hours, repeating the liquid change operation 1 time each day, observing the cell reaching the confluence degree of more than 80% by a microscope (figure 6-precursor IMSC), and using 0.05% TrypeTMExpress to digest cells, collect cell suspension into 15mL centrifuge tube, centrifuge for 5 minutes at 300 × g, discard supernatant, resuspend cell pellet with 2mL ims differentiation medium 3, the composition of differentiation medium 3 includes: MesenCultACF Plus Medium (STEMCELL technical gies) +5ng/mL Recombinant structural Recognition Protein 1(SSRP1) +5ng/mL Huma n Recombinant GDF 5. Then, the ratio of the concentration of the water to the concentration of the water is 1.5-6x10^3/cm2Plating the plates into 6-well plates previously coated with 5. mu.g/well of vitronectin, culturing in an incubator at 37 ℃ for 3 days to reach about 80% confluency (shown in figure 7-iMSCs).
Example 3: and (3) carrying out large-scale amplification and identification on the iMSCs.
1) Cells were observed microscopically for iMSCs at around 80% confluency using 0.05% TrypLETMExpress cells were collected after digestion, then cell viability was greater than 90% using trypan blue staining and cell counter, using 3D
MiniSPIN bioreactor (Beijing Hua niche organisms) combined with 3D
Cell amplification suit FK01 (Beijing Hua niche organism) carries out 3D culture, adopts magnetic field control rotational speed 30 ~ 60rpm, and this embodiment is 45rpm, and speed is low and the precision is high, can effectively improve cell growth rate, when avoid the damage of shearing force to the cell in the stirring process. The method mainly comprises the following steps: taking 1 micro slide corresponding to 50 ten thousand cells as an initial input cell, and correspondingly needing 10mL of iMSCs amplification culture medium 1, wherein the iMSCs amplification culture medium 1 comprises the following components: MesenCultACF Plus Medium (STEMCELL technologies) +5ng/mL Human Recombinant GDF5(STEMCELL technologies), or iMSCs amplification Medium 2, the composition comprising: MSCs medium (friend NC0103) +5ng/mL Human Recombinant GDF5(stem cell technologies), specifically iMSCs amplification medium 2 in this example. In order to amplify a large number of cells, 250 ten thousand cells are selected to correspond to 5 micro-slides, 50mL of iMSCs amplification culture medium 1 or 2 (specifically, amplification culture medium 1 in this embodiment) is added into a 125mL culture flask, glucose concentration is detected every 24 hours to determine the fluid infusion amount (the glucose concentration needs to reach the initial culture concentration), and after continuous amplification is carried out for 4 days, the total number of cells can reach not less than 10 times of the amplification number, namely more than 2500 ten thousand cells.
2) And (3) carrying out the following identification on the iMSCs after 3D amplification: a) identifying cell morphology; b) flow cytometry identified MSCs marker (CD73, CD90, CD105, CD34, CD45) expression; c) analyzing the activity of telomerase; d) identifying the single cell sequencing cell subset; e) COL2A1/SOX9 immunofluorescent staining identified a functional subset of iMSCs.
In this embodiment, it is found from previous research results that specific factors have important promotion and maintenance effects on maintaining specific cell subsets of MSCs, for example, SSRP1 promotes highly proliferating MSCs subsets, Wnt-C59 and GDF5 promote mesoderm and cartilage development, and an ims differentiation system is optimized to obtain MSCs (ismcs) derived from iPSCs differentiation, which have higher differentiation consistency, significantly improved proliferation capacity, higher telomerase activity, high expression of CD73, CD90, and CD105, and low expression of CD34 and CD45, and are superior to international identification standards, and the expression of marker genes is detailed in table 1 below. And finally, the 3D micro-slide system is used for completing the large-scale amplification of the iMSCs, and the cell uniformity is good. And a good technical foundation is laid for finally establishing human iMSCs cell resources of clinical application levels.
TABLE 1
| Marker gene | International standard of identity | Expression ratio of iMSCs |
| 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, the iMSCs cultured by the culture method and system of the present embodiment have better quality and application advantages, and compared with the MSCs derived from traditional tissues, the iMSCs derived from iPSC cell differentiation have unlimited sourcing ability, can proliferate in large quantities, and have the advantage of cell drug reporting.
In one embodiment, the invention establishes a large-scale preparation method of clinical-grade iMSCs functional subsets for the first time, the whole technical scheme has the advantages of simple operation and easy standard large-scale production, avoids the use of reagents such as fetal calf serum and heterogeneous component reagents in the whole process, and has obvious application advantages.
In one embodiment, mRNA cocktails, including coding sequences of pluripotent genes Oct-3, Oct-4, Klf-4, Sox2, Glis1, c-Myc and puromycin (puromycin) resistance genes, are used for triggering activation of a cell endogenous pluripotent gene network through expression of exogenous pluripotent genes, and puromycin drugs are further used for screening and removing cells which are not successfully reprogrammed, so that a feeder-layer-free and serum-free preparation method of the human induced pluripotent stem cells is established. The method does not need feeder cells and fetal calf serum, has definite culture medium components and simple operation, avoids exogenous genome integration risk, and can efficiently and stably prepare the human iPSC cell strain.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (10)
1. A method of preparing an induced mesenchymal stem cell, comprising:
a stem cell culture step, which comprises providing mesenchymal stem cells and culturing to obtain induced pluripotent stem cells;
and a differentiation step, which comprises the step of differentiating and culturing the induced pluripotent stem cells into mesenchymal stem cells, namely the induced mesenchymal stem cells.
2. The method of claim 1, wherein the stem cell culturing step comprises:
a somatic cell preparation step, which comprises the step of culturing mesenchymal stem cells by using a mesenchymal stem cell culture medium without feeder cells and animal-derived components;
a cell transfection step, which comprises sucking and abandoning the mesenchymal stem cell culture medium and using the mesenchymal stem cell culture medium containing cell factors to culture the mesenchymal stem cells;
adding medicine to screen, including using mesenchymal stem cell culture medium containing screening reagent, culturing to obtain screened mesenchymal stem cells;
a drug-removing culture step, which comprises the steps of absorbing and discarding the mesenchymal stem cell culture medium containing the screening reagent, adding the drug-removing culture medium, and culturing to obtain mesenchymal stem cells;
a step of cloning and screening, which comprises the steps of removing a medicine culture medium by suction, adding a cell culture medium without feeder cells and animal-derived components, and culturing to obtain mesenchymal stem cell clones;
and cloning and selecting, wherein the cloning and selecting step comprises the steps of selecting cell cloning, subculturing and obtaining the induced pluripotent stem cells.
3. The method of claim 2, wherein in the cell transfection step, the mesenchymal stem cell culture medium further comprises an in vitro transcription mixture of mRNA;
preferably, the mRNA in vitro transcription mixture comprises at least one of Oct-3, Oct-4, Klf-4, Sox2, Glis1, c-Myc, puromycin resistance gene coding sequences.
4. The method of claim 2, wherein in the cell transfection step, the cytokine comprises B18.
5. The method of claim 2, wherein in the medicated screening step, the screening agent comprises puromycin.
6. The method of claim 2, wherein in the drug-removal culturing step, the drug-removal medium comprises a mesenchymal stem cell medium;
preferably, in the drug-removing culture step, the drug-removing culture medium contains cytokines;
preferably, in the drug-free culturing step, the cytokine comprises B18.
7. The method of claim 2, wherein in the clone screening step, the feeder cells-free, animal-derived component-free cell culture medium comprises mTeSRTM1, culture medium;
preferably, in the clone picking step, the medium used comprises TeSRTM-E8TMAnd (4) a culture medium.
8. The method of claim 1, wherein the differentiating step comprises a primary differentiation culture, a secondary differentiation culture, a tertiary differentiation culture;
preferably, in the first differentiation culture, the inhibitor is contained in the first differentiation culture medium;
preferably, the inhibitor comprises PORCN inhibitor at one differentiation culture;
preferably, said PORCN inhibitor comprises Wnt-C59;
preferably, in the secondary differentiation culture, a secondary differentiation culture medium containing cytokines is used;
preferably, in the second differentiation culture, the cytokine comprises a structure-specific recognition protein 1;
preferably, in the third differentiation culture, the third differentiation culture medium contains cytokines;
preferably, the cell factor comprises at least one of a structure-specific recognition protein 1 and a growth differentiation factor 5 in three differentiation cultures;
preferably, in the stem cell culturing step, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells;
preferably, in the stem cell culturing step, the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells.
9. The method of claim 1, further comprising an expansion step, comprising performing expansion culture on the mesenchymal stem cells obtained in the differentiation step by using an expansion medium to obtain the mesenchymal stem cells after the expansion culture;
preferably, the culture medium used in the stem cell culture step, the differentiation step and the amplification step is a feeder layer-free cell culture medium without animal-derived components.
10. Induced mesenchymal stem cells produced by the method of any one of claims 1 to 9.
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