CN111826348A - In-vitro efficient preparation method and application of mesenchymal stem cells derived from human induced pluripotent stem cells - Google Patents

In-vitro efficient preparation method and application of mesenchymal stem cells derived from human induced pluripotent stem cells Download PDF

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CN111826348A
CN111826348A CN202010536080.8A CN202010536080A CN111826348A CN 111826348 A CN111826348 A CN 111826348A CN 202010536080 A CN202010536080 A CN 202010536080A CN 111826348 A CN111826348 A CN 111826348A
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张磊升
权海宗
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Yunnan Dongsen Biotechnology Co Ltd
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Abstract

The invention discloses an in vitro efficient preparation method and application of mesenchymal stem cells derived from human induced pluripotent stem cells. The preparation method comprises the following steps: 1) culturing and expanding human induced pluripotent stem cells (hiPSCs); 2) by a strategy of screening and optimizing combination of a chemical small molecule library, the hiPSCs are jointly processed by two chemical small molecules LLY-507 and AZD5153 for nine days to obtain mesenchymal precursor cells derived from human induced pluripotent stem cells (hiPSCs) with the proportion of CD73 positive mesenchymal stem cells reaching more than 80%; 3) subculturing the mesenchymal precursor cells twice to obtain further mature CD73+CD105+Human induced pluripotent stem cell (hiPSCs) derived mesenchymal stem cells (hiPSCs-MSCs); 4) identification of C prepared as described above based on immunological means and cell biological analysisD73+CD105+The human induced pluripotent stem cell (hiPSCs) -derived cell of (a) is a mesenchymal stem cell. Experiments show that the strategy of combined treatment of the small chemical molecules can obviously improve the efficiency of differentiating and generating mesenchymal stem cells from human induced pluripotent stem cells (hiPSCs), and the hiPSCs-MSCs low-express pluripotency-related marker molecules, have typical functions of adipogenesis, osteogenesis, chondrogenesis differentiation and low immunogenicity, and can be used for treating immune-related diseases.

Description

In-vitro efficient preparation method and application of mesenchymal stem cells derived from human induced pluripotent stem cells
Technical Field
The invention belongs to the technical field of biological medicines, relates to cell therapy technology and products, and particularly relates to an in vitro efficient preparation method and application of mesenchymal stem cells derived from human induced pluripotent stem cells.
Background
Mesenchymal Stem Cells (MSCs) have unique biological properties of hematopoietic support and immunomodulation and can be directed to adipogenic, osteogenic and chondrogenic differentiation in vitro, participating extensively in a variety of tissue repair and reconstruction, and thus have superior regenerative medical value. MSCs were first isolated from bone marrow in 1960 s and then subsequently isolated and identified from dental pulp, umbilical cord, fat and other tissues. Pre-clinical and clinical studies have shown that MSCs play a therapeutic and ameliorative role in a variety of blood and immune related diseases.
Research has suggested that mesenchymal stem cells may function in disease treatment and tissue repair in a variety of ways. Firstly, mesenchymal stem cells are used as main stromal cells, which can provide stable attachment sites and microenvironment for hematopoiesis; secondly, the mesenchymal stem cells can be stimulated by different cytokines and directionally differentiated to generate specific terminal functional cells, so that the cells can replace damaged or necrotic functional cells to play a role; thirdly, the mesenchymal stem cells have the functions of autocrine and paracrine, can promote the repair of the tissue cells damaged by secreting various cytokines and nutrition, and can inhibit excessive inflammatory response reaction by secreting various inflammation-inhibiting factors so as to protect the tissues and cells of the organism from being damaged; finally, mesenchymal stem cells can exert immunoregulatory effects by interacting with various inflammatory cells (macrophages, leukocytes, etc.), by homing to the site of the lesion and secreting various chemokines (CXCL12, SDF-1, etc.), promoting functional recovery at the site of inflammation. Although the specific mechanism of the MSCs for treating specific diseases is still quite complex, the mesenchymal stem cells can play an important role in treatment and have a wide application prospect in regenerative medicine through the way.
At present, the application value of mesenchymal stem cells in preclinical and clinical research has attracted wide attention of scholars at home and abroad. Importantly, the novel medicament of the over ten stem cells is sold on the market internationally at present, and 5 novel medicaments of the type 1 stem cells are examined and registered by the national drug administration at home at present. Most of them are adult-derived (e.g., bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells) and perinatal-derived (e.g., umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells). Chinese scholars indicate that mesenchymal Stem cells from the sources often have batch differences, unstable donor sources, weak long-term proliferation capacity, pathogenic microorganisms and ethical risks (Stem cells.2016 Feb; 34(2): 380-91).
In view of the above disadvantages of adult-derived mesenchymal stem cells, which seriously affect large-scale preparation and future large-scale clinical application and disease treatment, researchers at home and abroad begin to search for new mesenchymal stem cells with more stable sources in recent years. Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the properties of self-renewal and multidirectional differentiation, can theoretically differentiate two or more tissue cells of an organism to generate, and can meet the requirements of large-scale and standardized preparation, so that the human pluripotent stem cells (hPSCs) have a wider application prospect in regenerative medicine. Compared with hESCs, hipSCs have no ethical risk and meet the requirement of individualized treatment, and have been applied to natural killer immune cells (NK), in vitro preparation of platelets and preclinical research. Studies on the differentiation of hiPSCs into mesenchymal stem cells in vitro began in 2005, and the major differentiation models included a monolayer differentiation model, an embryoid body differentiation model, and a co-culture differentiation model and a cell programming-mediated differentiation model. The four models can be used for preparing a certain amount of MSCs and meeting the requirements of preclinical research; however, as a whole, the existing research has the disadvantages of long differentiation period, low differentiation efficiency, high differentiation cost, enrichment by sorting, scraping of undifferentiated cells, and virus-mediated gene editing (Stem cells.2016 Feb; 34(2): 380-91; Stem Cell reports.2018 Aug 14; 11(2): 497) 513).
Previous researches show that by utilizing two chemical small molecules JNKi and DAC, human embryonic stem cells can be promoted to be differentiated to generate 60% of CD73 positive mesenchymal precursor cells, and continuous differentiation is carried outMature mesenchymal Stem cells can be prepared by passaging (Stem Cell Res ther.2019 Jun 24; 10(1): 186.). However, the above system has two problems: firstly, the used initial differentiated cells have ethical risks for hESCs, and secondly, a larger space for improving the proportion of differentiated CD73 positive mesenchymal precursor cells still exists. Therefore, how to overcome the defects has important significance in meeting the requirements of future mesenchymal stem cell large-scale and standardized preparation and clinical autologous infusion treatment. In the research, based on a larger-scale strategy of screening and optimizing combination of a chemical small molecule library, the fact that after two small molecules LLY-507 and AZD5153 related to apparent modification inhibition are jointly used for treating human induced pluripotent stem cells for 9 days, mesenchymal precursor cells with the cell expression CD73 positive of more than 80% are prepared, and after the cells are subjected to passage amplification for 2 times, purified CD73 can be further prepared+CD105+A cell. The immunology correlation analysis shows that the prepared hiPSC-MSCs conform to the expression of the relevant immune marker molecules of mesenchymal stem cells (CD73, CD90, CD105, CD31, CD34, CD45, HLA-DR), and present typical long fusiform morphology and low expression of pluripotency associated marker molecules; further, we found, using in vitro cell function experiments, that the prepared human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) have typical in vitro adipogenic, osteogenic, chondrogenic differentiation abilities, and the ability to inhibit proliferation of various lymphocytes (Th1, Th2, and Th 17). Based on the research, the invention establishes an efficient preparation method and application of human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs), and the cells have trilineage differentiation and immunosuppressive activity and are used for preparing and treating mesenchymal stem cell preparations for immunity-related diseases.
Disclosure of Invention
Aiming at the technical defects of long differentiation period, complex differentiation operation and low in-vitro generation efficiency of the mesenchymal stem cells from human induced pluripotent stem cells in the prior art, the invention provides the following solutions.
The invention relates to a high-efficiency preparation and culture system of mesenchymal stem cells from induced pluripotent stem cells, which comprises LLY-507, AZD5153, a human pluripotent stem cell maintaining culture medium (Gibco E8 culture medium) and a human induced pluripotent stem cell differentiation culture medium (Gibco DMEM-F12 culture medium, wherein 3% fetal calf serum and 5nM Y-27632 are added). Human induced pluripotent stem cells are treated for 9 days by the combination of the small chemical molecules discovered by the inventor and then passage and expansion are carried out for 2 times to obtain hiPSC-MSCs, and the biological characteristics of the cells are identified by an immunological method and a cell biological analysis means. Specifically, the proportions of CD73 and CD105 positive cells in the treated hipSC-MSCs are analyzed by a flow cytometry, the expression level of a pluripotency marker molecule is detected by real-time fluorescence quantitative PCR (qRT-PCR), differentiation functions are identified by mesenchymal stem cell in vitro adipogenesis, osteogenesis and chondrogenesis differentiation, and the regulation effect of the mesenchymal stem cell on the proliferation of various lymphocytes (Th1, Th2 and Th17) is evaluated by co-culture with the lymphocytes.
The main operation steps of the invention are as follows:
(1) diluting with DMEM-F12 medium (Gibico) according to the instructions for matrigel GFR product (Corning), adding into a six-well cell culture plate (Corning), and incubating in a cell culture chamber at 37 ℃ for 1 hour for later use;
(2) human induced pluripotent stem cells (hiPSCs) were digested by incubation in a cell culture chamber at 37 ℃ for 5 minutes to single cells according to Dispase enzyme product instructions (StemCell Technology), resuspended in DMEM-F12 medium and centrifuged, and resuspended in human pluripotent stem cell maintenance medium (Gibco E8 medium, supplemented with Y-27632 at a final concentration of 10 nM) and counted for use.
(3) The liquid in the six well cell culture plate was carefully discarded by pipette at 3X 104Inoculating the hiPSCs into a six-hole cell culture plate at a density of one ml, uniformly mixing, and transferring to 37 ℃ and 5% CO2Culturing in a cell culture box for 48-72 hours to obtain a small clone sample.
(4) Human pluripotent stem cell maintenance medium (Gibco E8 medium) in six-well plates was carefully discarded with a pipette and washed twice with 1 XPBS, followed by replacement of human induced pluripotent stem cell differentiation medium (Gibco DMEM-F12 medium, supplemented with 3% fetal bovine serum and 10nM Y-27632), and addition of 10nM LLY-507 and 10nM AZD5153 to initiate induction of differentiation.
(5) The human induced pluripotent stem cell differentiation medium (Gibco DMEM-F12 medium supplemented with 3% fetal bovine serum and 10nM Y-27632) was replaced every 1 day, and 10nM LLY-507 and 10nM AZD5153 were added. Differentiation to day nine, medium was carefully discarded and washed twice with 1 × PBS, followed by digestion to single cells with 0.25% Trypsin (Thermo Fisher Scientific).
(6) According to the following steps of 1: 3-1: 6 in mesenchymal stem cell maintenance medium (DMEM-F12 medium supplemented with 10% fetal bovine serum, 10ng/ml bFGF, 4ng/ml EGF) and 5nM Y-27632, then passaged into new six-well cell culture plates, and cultured and passaged twice.
(7) On the 14 th day after the start of the differentiation culture, the proportion of the generated human induced pluripotent stem cell-derived mesenchymal stem cells (CD73, CD90, CD105, CD31, CD34, CD45, HLA-DR) was examined by a flow cytometer.
(8) On day 0 of differentiation (before changing the differentiation medium), day 9 (before cell passage) and day 14 (before flow cytometry), total mRNA samples were obtained by lysis of the cells with trizol (invitrogen), respectively, for subsequent reverse transcription reaction (RT-PCR) and expression level detection of pluripotency-associated marker molecules.
(9) Respectively detecting the adipogenic, osteogenic and chondrogenic differentiation capacities of mesenchymal stem cells (hiPSC-MSCs) derived from human induced pluripotent stem cells by using an in vitro three-line differentiation experiment and specific staining; human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) were evaluated for their ability to regulate a variety of lymphocytes (Th1, Th2, and Th17) using mesenchymal stem cell in lymphocyte co-culture experiments.
According to the above-mentioned research method, we achieved the following effects (1) we obtained CD73 within 14 days+CD105+Human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) having a cell proportion of more than 80%, which cells conform to the immunophenotype of mesenchymal stem cells (CD 73)+CD90+CD105+CD31-CD34-CD45+HLA-DR-) (ii) a (2) Efficiently preparing the long fusiform shape and trilinear differentiation of typical mesenchymal stem cellshiPSC-MSCs of capacity (adipogenic, osteogenic, chondrogenic differentiation); (3) the hiPSC-MSCs with the regulation capacity of various lymphocytes (Th1, Th2 and Th17) are obtained and can be used for treating diseases related to immune abnormality.
The human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) induced to differentiate and prepared by adding the two chemical small molecules are considered to be a new alternative source of adult-derived mesenchymal stem cells or perinatal-derived mesenchymal stem cells, so as to better meet the requirements of large-scale standardized preparation of human induced pluripotent stem cell-derived mesenchymal stem cells for preclinical and clinical research of immune-related diseases in the future.
Drawings
FIG. 1: differentiating the human induced pluripotent stem cells to the 9 th day, and detecting CD73 generated by inducing differentiation without adding or independently adding different chemical small molecules by flow cytometry+CD105+Mesenchymal precursor cells, production of CD73 on the ordinate+、CD105+The ratio of cells, the abscissa is different treatment groups (small molecule treatment not added: Ctr; the rest is chemical small molecule treatment group added);
FIG. 2: a human-induced pluripotent stem cell-derived mesenchymal stem cell flow cytophenotype;
FIG. 3: morphological changes (upper) and expression changes (lower) of pluripotency-associated marker molecules during differentiation of human induced pluripotent stem cells into mesenchymal stem cells (hipscs-MSCs);
FIG. 4: differentiation staining of human induced pluripotent stem cell-derived mesenchymal stem cells (undifferentiated on the left, differentiated on the right) into adipogenic (upper), osteogenic (middle) and chondrogenic (lower); the adipogenic differentiation adopts oil red staining, the osteogenic differentiation adopts alizarin red staining, and the chondrogenic differentiation adopts alcian blue staining; real-time fluorescent quantitative PCR (qRT-PCR) detects the expression of adipogenic differentiation marker molecules ADIPOQ and PPAR-gamma, the expression of osteogenic differentiation marker molecules RUNX2 and BGLAP, and the expression of chondrogenic differentiation marker molecules ACAN and SOX 9.
FIG. 5: human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) have a regulatory effect on proliferation of Th1 (left), Th2 (middle) and Th17 (right) lymphocytes (upper part is lymphocyte culture alone, and lower part is lymphocyte coculture with hiPSC-MSCs).
Detailed description of the invention
The invention is illustrated and described in detail with respect to the specific embodiments listed below.
Example 1: culturing human induced pluripotent stem cells.
GFR matrigel planking: after diluted with DMEM-F12 medium according to the GFR matrigel (Corning) instructions, the cells were transferred to a six-well cell culture plate (1 mL/well) by a pipette and incubated in a cell incubator at 37 ℃ for 1 hour for further use;
2. human induced pluripotent stem cell digestion: human induced pluripotent stem cells (hiPSCs) were cultured at 37 ℃ in 5% CO according to Dispase enzyme product instructions (stemcell technology)2The cell culture chamber was incubated to digest the cells for 5 minutes, resuspended in 5mL DMEM-F12 medium and blown to the cells with 10mL pipette, centrifuged (5 minutes at room temperature at 300 g), resuspended in 3mL human pluripotent stem cell maintenance medium (Gibco E8 medium, supplemented with Y-27632 to a final concentration of 10 nM) and counted for use.
Inoculation of human induced pluripotent stem cells: the liquid in the six well cell culture plate was carefully discarded with a 10mL pipette at 3X 104The number of hiPSCs used for inoculation was calculated at a density of/mL, the volume of human pluripotent stem cell maintenance medium (Gibco E8 medium, with the addition of Y-27632 at a final concentration of 10 nM) was calculated in terms of a volume of 2 mL/well, the hiPSCs were resuspended in the E8 medium and inoculated into six-well cell culture plates, mixed well and transferred to 37 ℃ with 5% CO2Culturing in a cell culture box for 48-72 hours to obtain a small clone sample.
Example 2: human induced differentiation of pluripotent stem cells into mesenchymal stem cells.
Preparation of culture medium for inducing differentiation of hiPSCs into MSCs: differentiation-inducing basal media (3% fetal bovine serum, DMEM-F12 medium, 5nM Y-27632) were prepared in advance, wherein experimental groups were supplemented with the corresponding small chemical molecules (PCI-24781, EPZ011989, J147, UNC1999, TG101348, CUDC-101, MS-275, BI 2536, LLY-507, Ginkgolide C, BI-7273, CPI-637, OTX015, UNC1215, EPZ6438, LBH589, GSK126, UNC0379, SP2509, (+) -JQ-1, PFI-3, UNC669, CPI-455, OICR-9429, MS023, AZD5153, EED226) to a final concentration of 10nM/mL, and control group was differentiation-inducing basal media (no small chemical molecule addition).
2. Cell treatment: the morphology and density of hiPSCs were observed under a mirror, and when the cells grew into a small clone, the original old human pluripotent stem cell maintenance medium (Gibco E8 medium, additionally supplemented with Y-27632 at a final concentration of 10 nM) was discarded by a pipette according to experimental groups, washed twice with 2mL of 1 × PBS, and then replaced with the hiPSCs prepared above to MSCs differentiation induction medium, and the experimental groups were supplemented with different chemical small molecules at 10nM per well to initiate differentiation induction, and the control group was differentiation induction medium (without chemical small molecules).
3. Cell liquid change and subculture inoculation: the differentiation culture medium (3% fetal bovine serum, DMEM-F12 culture medium, 5nM Y-27632) of human induced pluripotent stem cells is replaced every 1 day, wherein the experimental group is added with corresponding chemical micromolecules, and the control group is the induction differentiation basal culture medium (without chemical micromolecules). Differentiation to day nine, medium was carefully discarded and washed twice with 2mL of 1 × PBS, followed by digestion to single cells with 0.25% Trypsin (Thermo Fisher Scientific).
4. Identification of cellular immunophenotyping by differentiation by flow cytometry: 2X 10 of control (Ctr) and experimental groups cultured for 9 days were collected5The cell sample was resuspended to 150. mu.L in phosphate buffer (1 XPBS). Labeling CD73 and CD105, respectively, and performing CD73 with a FACS Calibur flow cytometer (BD Co., Ltd.)+CD105+Detection and analysis of mesenchymal precursor cells.
5. Screening according to the method to obtain the CD73 capable of being efficiently promoted+CD105+Chemical micromolecules of mesenchymal precursor cells, namely 10nM LLY-507 and 10nM AZD5153, are jointly added into a differentiation culture medium (3% fetal calf serum, DMEM-F12 culture medium and 5nM Y-27632) of the human induced pluripotent stem cells to induce the hipSCs to differentiate into the MSCs till the 9 th day; subsequently, as 1: 3-1: 6, in mesenchymal stem cell maintenance medium (DMEM-F12 medium,10% fetal bovine serum, 10ng/mlbFGF, 4ng/ml EGF) and 5nM Y-27632, followed by passaging to a new six-well cell culture plate, continued culturing and passaging twice.
6. On day 14 after the start of the above differentiation, immunophenotype of hiPSC-MSCs produced by differentiation was examined using flow cytometry and marker molecules identified by internationally common MSCs (CD73, CD90, CD105, CD31, CD34, CD45, HLA-DR).
7. Cell lysis and total mRNA sample extraction were performed on day 0 (before the change of differentiation medium), day 9 (before cell passage) and day 14 (before flow cytometry) of the above differentiation, respectively, using trizol (invitrogen) reagent instructions. The above-mentioned experimental procedures for synthesizing cDNA by reverse transcription of mRNA were performed according to Beijing Quanji corporation
Figure BDA0002537101850000041
cDNA kit instructions. Detection of the pluripotency-associated marker molecules expressed in the samples at the three time points (day 0, day 9, and day 14) was performed by relative real-time fluorescent quantitative PCR (qRT-PCR) using SYBR Green PCR Master Mix, International ABI. Analysis of amplification data according to ABI Q5 software, use 2-ΔΔCTThe method carries out relative expression quantitative analysis.
Example 3: and (3) carrying out in-vitro functional identification on mesenchymal stem cells (hiPSC-MSCs) generated by differentiation of the human induced pluripotent stem cells.
Adipogenic, osteogenic and chondrogenic differentiation of hiPSC-MSCs in vitro: according to 6X 104And inoculating the cells/hole on a six-hole cell adherent culture plate (Corning), and discarding the hiPSC-MSCs maintenance culture medium to perform in-vitro directional induced differentiation respectively as follows when the hiPSC-MSCs grow adherently until the cell fusion degree reaches 70% -80%.
(1) In vitro induced adipogenic differentiation culture system: after discarding the mesenchymal Stem Cell maintenance medium (DMEM-F12 medium supplemented with 10% fetal bovine serum, 10ng/ml bFGF, 4ng/ml EGF), the lipogenic differentiation medium of MensenCult from Stem Cell was replaced, and the above lipogenic differentiation medium was replaced every 3.5 days and continuously cultured until day 18. Differentiated adipocytes were fixed with 10% neutral formaldehyde at room temperature for 20 minutes, and cells were washed 3-4 times with 1 XPBS. Adding 2mL of oil red O staining solution, and carrying out sealed staining for 45-60 minutes in an incubator at 37 ℃; the oil red O staining solution was aspirated by a pipette, and the cells were washed 3 to 4 times with 1 XPBS. And observing the red-stained lipid droplets under an inverted phase contrast microscope, and photographing and recording. Among them, hiPSC-MSCs cultured in a mesenchymal stem cell maintenance medium (DMEM-F12 medium supplemented with 10% fetal bovine serum, 10ng/ml bFGF, 4ng/ml EGF) without adipogenic differentiation were used as a negative control group.
(2) In vitro induced osteogenic differentiation culture system: after discarding the mesenchymal Stem Cell maintenance medium (DMEM-F12 medium supplemented with 10% fetal bovine serum, 10ng/ml bFGF, and 4ng/ml EGF), the osteogenic differentiation medium of the Stem Cell company was replaced with MensenCult, and the fresh osteogenic differentiation medium was replaced every 3.5 days, followed by continuous culture until day 18. According to the following formula: preparing a fixing solution according to the proportion of 1:1, fixing differentiated osteoblasts for 10-15 minutes at room temperature, adding 2mL of 0.5% alizarin red staining solution, sealing and staining in an incubator at 37 ℃ for 30-60 minutes, discarding the alizarin red staining solution, and washing the cells for 3-4 times by using 1 XPBS. Cells were observed under an inverted phase contrast microscope and red-stained mineral nodules were visible and recorded by photography. Among them, hiPSC-MSCs cultured in a mesenchymal stem cell maintenance medium (DMEM-F12 medium supplemented with 10% fetal bovine serum, 10ng/mlbFGF, 4ng/ml EGF) without osteogenic differentiation were used as a negative control group.
(3) Inducing in vitro to form a cartilage differentiation culture system: after discarding the mesenchymal Stem Cell maintenance medium (DMEM-F12 medium supplemented with 10% fetal bovine serum, 10ng/ml bFGF, and 4ng/ml EGF), the Mensencult chondrogenic differentiation medium from Stem Cell was replaced, and the chondrogenic differentiation medium was replaced with fresh one every 3.5 days and continuously cultured until day 18. Fixing the differentiated chondroblasts with 4% paraformaldehyde at room temperature for 30-45 min, and adding deionized water (ddH)2O) cells were washed 3 times. Adding 2mL of alcian blue staining solution, sealing and staining for 12 hours in an incubator at 37 ℃, discarding the alcian blue staining solution, and deionized water (ddH)2O) cells were washed 3 times. When the cells were observed under an inverted phase contrast microscope, blue-stained mineral nodules were observed,and taking a picture for recording. Among them, hiPSC-MSCs cultured in a mesenchymal stem cell maintenance medium (DMEM-F12 medium supplemented with 10% fetal bovine serum, 10ng/ml bFGF, 4ng/ml EGF) without undergoing chondrogenic differentiation were used as a negative control group.
(4) In the expression detection of adipogenic, osteogenic and chondrogenic differentiation related marker molecules of hiPSC-MSCs in vitro, firstly, cell samples of adipogenic, osteogenic and chondrogenic differentiation to day 18 and negative control group (not subjected to differentiation induction) cell samples are subjected to cell lysis and total mRNA sample extraction by trizol (invitrogen) reagent instructions respectively. Then, according to Beijing Quanjin Co
Figure BDA0002537101850000051
The cDNA kit instructions were used to perform the above-described procedures for reverse transcription of mRNA to synthesize cDNA. The expression of marker molecules related to adipogenic (ADIPOQ, PPAR-gamma), osteogenic (RUNX2, BGLAP) and chondrogenic (ACAN, SOX9) in the above samples was detected, and the relative real-time fluorescent quantitative PCR (qRT-PCR) was performed using SYBR Green PCR Master Mix, International ABI. Analysis of amplification data according to ABI Q5 software, use 2-ΔΔCTThe method carries out relative expression quantitative analysis.
hiPSC-MSCs were analyzed in vitro for various lymphocyte immunoregulatory functions: according to 2X 105Cells/well were seeded in twelve well cell adherent culture plates (Corning) for future use. The mononuclear cells (PBMCs) prepared by separation from peripheral blood were enriched with CD4 by flow cytometry (BD Co.)+T lymphocytes. In the control group, the above 1X 106CD4+T lymphocytes were seeded on twelve-well cell-adherent culture plates (Corning) and cultured alone, and the above 1X 10 cells were used in the experimental group6CD4+T lymphocyte inoculation by adding 2X 10 cells in advance5Twelve-well cell adherent culture plates (Corning) of hipSC-MSCs were co-cultured. After 3 days, separately cultured CD4 in the supernatant was collected+T lymphocytes, co-cultured CD4+T lymphocytes were washed twice with 1 XPBS. Subsequently, flow cytometry and the identification of different lymphocyte subsets in common internationally (CD73, CD90, CD105, CD31, C) were used as marker moleculesD34, CD45, HLA-DR), CD4 in the above two groups was performed with a FACS Canto II (BD Biosciences) flow cytometer of BD Co+Immunophenotypic testing of T lymphocyte subpopulations. Results analysis was analyzed using TreeStar corporation FlowJo software.
As a result: compared with a control group (Ctr) without adding the chemical small molecules to induce the differentiation of the hiPSCs into the MSCs, the CD73 can be promoted to different degrees by independently adding a plurality of chemical small molecules related to the appearance modification regulation+CD105+The production rate of mesenchymal precursor cells. Among them, addition of LLY-50 and AZD5153 alone had a stronger effect of promoting differentiation to generate mesenchymal precursor cells (FIG. 1). After the LLY-50 and AZD5153 are treated with the hipSC for 9 days and are enriched by two successive passages, more than 80 percent of the hipSC-MSCs are found to highly express MSCs related marker molecules (CD73, CD90 and CD105) by combining flow cytometry analysis (figure 2), and the differentiated hipSC-MSCs show typical spindle cell morphology and low expression pluripotency related marker molecules (figure 3). Experiments on the function of inducing the trilineage differentiation in vitro show that the hiPSC-MSCs have the characteristics of typical specific staining of adipogenic, osteogenic and chondrogenic differentiation and respectively highly express the trilineage differentiation related marker molecules (figure 4). Meanwhile, in vitro co-culture experiments with lymphocytes show that the prepared hiPSC-MSCs can effectively inhibit proliferation of subpopulations of Th1 and Th2 lymphocytes and promote proliferation of Th17 lymphocytes in vitro (fig. 5).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (3)

1. An in vitro efficient preparation method and application of mesenchymal stem cells derived from human induced pluripotent stem cells are characterized by comprising the following steps:
1) culturing and amplifying human induced pluripotent stem cells (hiPSCs);
2) processing the human induced pluripotent stem by the combination of two chemical small molecules through the strategy of screening and optimizing the combination of the chemical small molecule libraryOne week of cells, obtaining mesenchymal stem cell precursor cells with the proportion of CD73 positive cells reaching more than 80 percent, then enriching the precursor cells, inoculating, culturing and amplifying for 1 week to obtain CD73+CD90+CD105+CD31-CD34-CD45-HLA-DR-Human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs);
3) identification of CD73 prepared as described above based on immunological means and cell biological analysis+CD105+The cells are mesenchymal stem cells.
2. A human induced pluripotent stem cell-derived mesenchymal stem cell (hiPSC-MSCs) prepared by the method of claim 1.
3. Use of the mesenchymal stem cell derived human induced pluripotent stem cell of claim 2 in the preparation of a stem cell medicament for the treatment of an immune disease.
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