CN111826348B - 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 PDFInfo
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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) Through a strategy of chemical small molecule library screening and optimized combination, the hiPSCs are jointly processed for nine days by utilizing two chemical small molecules LLY-507 and AZD5153, and mesenchymal precursor cells derived from human induced pluripotent stem cells (hiPSCs) with the proportion of CD73 positive mesenchymal stem cells reaching more than 80% are obtained; 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 the CD73 prepared as described above based on immunological means and cell biological analysis + 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
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. Existing preclinical and clinical studies have shown that MSCs play a therapeutic and ameliorative role in a variety of blood and immune related diseases.
Studies have 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 to directionally differentiate to generate specific terminal functional cells, so that the mesenchymal stem 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 (CXCL 12, SDF-1, etc.), promoting functional recovery at the site of inflammation. Although the specific mechanism of MSCs for treating specific diseases is still quite complex, 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 the extensive 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 mesenchymal stem cells, which seriously affect large-scale preparation and future large-scale clinical application and disease treatment, in recent years, scholars at home and abroad begin to search for new mesenchymal stem cells with more stable sources. 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 regenerative medicine application prospect. 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. Research 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, on the whole, the existing research has the defects of long differentiation period, low differentiation efficiency, high differentiation cost, enrichment by sorting, scraping of undifferentiated cells, virus-mediated gene editing and the like (Stem cells.2016 Feb;34 (2): 380-91 Stem Cell reports.2018 Aug 11 (2): 497-513..
Our earlier studies found that human embryonic Stem cells could be promoted to differentiate to give 60% proportion of CD73 positive mesenchymal precursor cells by using two chemical small molecules, JNKi and DAC, and mature mesenchymal Stem cells could be prepared by serial passages (Stem Cell Res ther.2019 Jun 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 the differentiated CD73 positive mesenchymal precursor cells still exists. Therefore, how to overcome the above-mentioned disadvantagesThe method has important significance in meeting the requirements of future mesenchymal stem cells on scale, 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 adopted to jointly treat human induced pluripotent stem cells for 9 days, mesenchymal precursor cells with more than 80% of cell expression CD73 positive 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 accord with the expression of related immune marker molecules of mesenchymal stem cells (CD 73, CD90, CD105, CD31, CD34, CD45, HLA-DR), and present typical long fusiform morphology and low expression pluripotency related 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 capacity and the ability to inhibit proliferation of various lymphocytes (Th 1, 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 solution.
The invention provides 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 using the combination of the small chemical molecules found 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 flow cytometry is adopted to analyze the proportion of CD73 and CD105 positive cells in the treated hiPSC-MSCs, the expression level of the pluripotency marker molecules is detected through real-time fluorescence quantitative PCR (qRT-PCR), differentiation function identification is carried out through in vitro adipogenesis, osteogenesis and chondrogenesis of mesenchymal stem cells, and the regulation effect of the mesenchymal stem cells on the proliferation of various lymphocytes (Th 1, th2 and Th 17) is evaluated through co-culture with the lymphocytes.
The main operation steps of the invention are as follows:
(1) Adding a six-well cell culture plate (Corning) after diluting with DMEM-F12 medium (Gibico) according to the matrigel GFR product instruction (Corning), and transferring the cell culture plate into a cell culture box at 37 ℃ for incubation for 1 hour for later use;
(2) Human induced pluripotent stem cells (hiPSCs) were digested by incubation in a 37 ℃ cell culture box for 5 minutes to single cells according to Dispase enzyme product instructions (StemCell Technology), resuspended in DMEM-F12 medium and centrifuged, and then resuspended in human pluripotent stem cell maintenance medium (Gibco E8 medium, further supplemented with Y-27632 to a final concentration of 10 nM) and counted for use.
(3) The liquid in the six-well cell culture plate was carefully discarded by a pipette at 3X 10 4 Inoculating the above hiPSCs at a density of/ml into a six-well cell culture plate, mixing well, then transferring to 37 ℃,5% CO 2 Culturing 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 a six-well plate was carefully discarded by 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 start induction of differentiation.
(5) 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 0.25% trypsinase (Thermo Fisher Scientific) digestion to single cells.
(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 (CD 73, 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), cells were lysed with TRIZol (Invitrogen) to obtain total mRNA samples for subsequent reverse transcription reaction (RT-PCR) and expression level detection of pluripotency-associated marker molecules, respectively.
(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 (Th 1, th2, and Th 17) 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 proportion of cells exceeding 80%, the cells conforming to the immunophenotype of mesenchymal stem cells (CD 73) + CD90 + CD105 + CD31 - CD34 - CD45 + HLA-DR - ) (ii) a (2) The hiPSC-MSCs with typical mesenchymal stem cell long spindle morphology and trilineage differentiation capacity (adipogenic, osteogenic, chondrogenic differentiation) are efficiently prepared; (3) The hiPSC-MSCs with the regulation capacity of various lymphocytes (Th 1, th2 and Th 17) 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, and the aim of the invention is to better meet the requirements of large-scale standardized preparation of the human induced pluripotent stem cell-derived mesenchymal stem cells for the 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, detecting CD73 generated by inducing differentiation without adding or independently adding different chemical small molecules by flow cytometry + CD105 + Mesenchymal precursor cells, ordinate CD73 production + 、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 is a schematic diagram: 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 is a schematic view of: 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: the regulation and control effect of human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) on proliferation of Th1 (left), th2 (middle) and Th17 (right) lymphocytes (upper part is lymphocyte culture alone, lower part is lymphocyte culture together with hiPSC-MSCs).
Detailed description of the preferred embodiment
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: according to the GFR matrigel (Corning) use instruction, after being diluted by DMEM-F12 medium, the medium is transferred to a six-well cell culture plate (1 mL/well) by a pipette, and is transferred to a cell culture box at 37 ℃ for incubation for 1 hour for standby;
2. human induced pluripotent stem cell digestion: human induced pluripotent stem cells (hipSCs) were subjected to CO-reduction at 37 ℃ and 5% according to the Dispase enzyme product Specification (StemCell Technology) 2 The 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 and centrifuged (5 minutes at room temperature at 300 g), and then 3mL human pluripotent stem cell maintenance medium (Gibco E8 medium, supplemented with Y-27632 to a final concentration of 10 nM) was resuspended in the above hipSCs and counted for use.
Inoculation of human induced pluripotent stem cells: the liquid in the six-well cell culture plate was carefully discarded by a 10mL pipette at 3X 10 4 The number of hiPSCs used for the inoculation was calculated at a density of/mL, the volume of the human pluripotent stem cell maintenance medium (Gibco E8 medium, with the addition of Y-27632 at a final concentration of 10 nM) used was calculated at a volume of 2 mL/well, the hiPSCs were resuspended in the E8 medium and inoculated into six-well cell culture plates, after mixing well, the plates were transferred to 37 ℃ and 5% CO 2 Culturing 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, EED 226) at final concentrations of 10nM/mL, and control groups were differentiation-inducing basal media (no small chemical molecule addition).
2. And (3) cell treatment: observing the morphology and density of the hiPSCs under a mirror, when the cells grow into a small clone morphology, discarding the original old human pluripotent stem cell maintaining medium (Gibco E8 medium, additionally added with Y-27632 with a final concentration of 10 nM) by a pipette according to experimental groups, washing twice with 2mL of 1 XPBS, then replacing the hiPSCs prepared above with the MSCs induced differentiation medium, adding 10nM different small chemical molecules to each well of the experimental group to start induced differentiation, and adding a control group of induced differentiation basic medium (without adding small chemical molecules).
3. Cell liquid change and subculture inoculation: the differentiation culture medium (3% fetal bovine serum, DMEM-F12 culture medium, 5nM Y-27632) of the 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 the chemical micromolecules). Differentiation to day nine, medium carefully discarded and washed twice with 2mL 1 XPBS, followed by digestion to single cells with 0.25% Trypsin enzyme (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 collected 5 The cell sample was resuspended to 150. Mu.L in phosphate buffer (1 XPBS). Labeling CD73 and CD105, respectively, and performing CD73 analysis with a FACS Calibur flow cytometer (BD Co.) + CD105 + Detection and analysis of mesenchymal precursor cells.
5. Screening according to the method to obtain the CD73 capable of efficiently promoting + 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 human induced pluripotent stem cells to induce hipSCs to differentiate into MSCs till the 9 th day; subsequently, as 1:3-1:6, resuspended 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 further.
6. On day 14 after the start of the above differentiation, immunophenotype of hiPSC-MSCs generated by differentiation was examined using flow cytometry and marker molecules (CD 73, CD90, CD105, CD31, CD34, CD45, HLA-DR) identified by MSCs commonly used internationally.
7. Cell lysis and total mRNA sample extraction were performed on day 0 (before changing the differentiation medium), day 9 (before cell passage) and day 14 (before flow cytometry) of the above differentiation, respectively, with TRIZol (Invitrogen) reagent instructions. The above-mentioned experimental procedures for synthesizing cDNA by reverse transcription of mRNA were performed according to Beijing Quanji corporation
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 the amplification data according to ABI Q5 software, use 2 -ΔΔCT The 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 10 4 And 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, and 4ng/ml EGF), the adipogenic differentiation medium of MensenCult from Stem Cell was replaced, and the adipogenic 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 the 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 condition of the red dyeing lipid drops under an inverted phase contrast microscope, 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 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 was replaced with MensenCult from Stem Cell, and the osteogenic differentiation medium was replaced with fresh one at intervals of 3.5 days, and the cells were continuously cultured until day 18. According to the following formula: acetone =1, fixing the 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 with 1 XPBS for 3-4 times. The cells were observed under an inverted phase contrast microscope and red-stained mineral nodules were visualized 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/ml bFGF, 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 chondrogenic differentiation medium was replaced with MensenCult from Stem Cell, and the chondrogenic differentiation medium was replaced with a fresh one every 3.5 days, and the culture was continued until day 18. Fixing the differentiated chondroblasts with 4% paraformaldehyde at room temperature for 30-45 min, and adding deionized water (ddH) 2 O) cells were washed 3 times. Adding 2mL of alcian blue staining solution, sealing and staining the mixture in an incubator at 37 ℃ for 12 hours, discarding the alcian blue staining solution, and deionized water (ddH) 2 O) wash cells 3 times. The cells were observed under an inverted phase contrast microscope and blue-stained mineral nodules were visualized 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/ml bFGF, 4ng/ml EGF) without undergoing chondrogenic differentiation were used as a negative control group.
(4) Markers associated with adipogenic, osteogenic and chondrogenic differentiation of hiPSC-MSCs in vitroFor the detection of molecular expression, cell lysis and total mRNA sample extraction were performed on the adipogenic, osteogenic, and chondrogenic differentiation-to-day 18 cell samples and negative control (non-differentiation-induced) cell samples, respectively, according to TRIZol (Invitrogen) reagent instructions. Then, according to Beijing Quanjin Co
The cDNA kit instructions were used to perform the above-described procedures for reverse transcription of mRNA to synthesize cDNA. The expression detection of marker molecules related to adipogenic (ADIPOQ, PPAR-gamma), osteogenic (RUNX 2, BGLAP) and chondrogenic (ACAN, SOX 9) in the above samples was carried out by a relative real-time fluorescent quantitative PCR (qRT-PCR) using SYBR Green PCR Master Mix, international ABI. Analysis of the amplification data according to ABI Q5 software, use 2 -ΔΔCT The method carries out relative expression quantitative analysis.
hipsc-MSCs were analyzed in vitro for various lymphocyte immunomodulatory functions: according to 2X 10 5 Cells/well were seeded in twelve well cell adherent culture plates (Corning) for future use. From mononuclear cells (PBMCs) prepared by peripheral blood separation, CD4 was enriched by flow cytometric separation (BD Co.) + T lymphocytes. In the control group, the above 1X 10 6 CD4 + 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 group 6 CD4 + T lymphocyte inoculation by adding 2X 10 cells in advance 5 Twelve-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, CD4 in the two groups was carried out using FACS Canto II (BD Biosciences) flow cytometers of BD company using flow cytometric analysis and marker molecules (CD 73, CD90, CD105, CD31, CD34, CD45, HLA-DR) for identification of different lymphocyte subpopulations that are common internationally + Immunophenotypic testing of T lymphocyte subpopulations. Results analysis was analyzed using TreeStar corporation FlowJo software.
As a result: differentiation of hips from non-chemical small molecule-induced hipsThe chemical small molecules related to the regulation of a plurality of apparent modifications can be independently added to a chemical control group (Ctr) to promote CD73 to different degrees + CD105 + The production rate of mesenchymal precursor cells. Among them, addition of LLY-50 and AZD5153 alone has a stronger effect of promoting differentiation into mesenchymal precursor cells (FIG. 1). After the LLY-50 and AZD5153 are treated with the hipSCs 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 (CD 73, CD90 and CD 105) by combining flow cytometry analysis (figure 2), and the differentiated hipSC-MSCs show typical fusiform 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 showed that the prepared hiPSC-MSCs were effective in inhibiting proliferation of Th1 and Th2 lymphocyte subsets in vitro and promoting proliferation of Th17 lymphocytes (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 (1)
1. An in vitro preparation method of mesenchymal stem cells derived from human induced pluripotent stem cells is characterized by comprising the following steps:
1) Culturing and expanding human induced pluripotent stem cells (hiPSCs);
2) The human induced pluripotent stem cells are treated by the combination of two chemical micromolecules LLY-507 and AZD5153 for one week to obtain mesenchymal stem cell precursor cells with the proportion of CD73 positive cells reaching more than 80 percent, and then the precursor cells are enriched and inoculated for culture and amplification 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) Based on immunological means and cell biological analysis, the prepared cell is identified as mesenchymal stem cell.
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