CN102161980B - Method for culturing induced pluripotent stem cells by using human mesenchymal stem cells as trophoblast - Google Patents

Abstract

The invention provides a method for culturing human induced pluripotent stem cells (iPSCs) by using human bone marrow mesenchymal stem cells as a trophoblast. The method comprises the following steps of: obtaining the human iPSCs from child foreskin fibroblasts by using third to fifth generation human bone marrow mesenchymal stem cells obtained through subculture and culture after thawing from low-temperature freezing, performing amplification culture, and inoculating into trophoblast cells to obtain the human iPSCs. In the method, the human iPSCs are cultured by using the human bone marrow mesenchymal stem cells as the trophoblast cells instead of mouse embryonic fibroblasts in the conventional method, so that the pollution of heterologous cells in a culture system is reduced, that the culture system can make the human iPSCs amplified in vitro is proved, biological characteristics and multipotency of the human iPSCs are maintained for a long time, and the possibility of clinical application of the human iPSCs is provided.

Description

Method for culturing induced pluripotent stem cells using human mesenchymal stem cells as trophoblast

technical field

The invention belongs to the field of biotechnology, and relates to a method for culturing induced pluripotent stem cells using human bone marrow mesenchymal stem cells as a trophoblast.

Background technique

Induced pluripotent stem cells (Induced Pluripotent Stem Cells, iPSCs) are a milestone breakthrough in the field of stem cell research in recent years. It is a pluripotent cell similar to ESCs obtained by ectopically expressing several key nuclear transcription factors related to the maintenance of pluripotency of embryonic stem cells (Embryonic Stem Cells, ESCs). All tissue cells that differentiate into the three germ layers in vitro and in vivo. iPSCs reprogrammed from somatic cells adds a new way to obtain pluripotent stem cells consistent with the patient's own genetic background, and no longer uses human early embryos and oocytes, so the ethical debate will subside. The dilemma of lack of oocytes and complex techniques in transplantation techniques has also been reasonably resolved. It is an ideal seed cell for tissue engineering and regenerative medicine, disease models, and drug screening, so it has a broad clinical application background. At present, researchers have induced the differentiation of human iPSCs into various tissue cells in vitro, such as hematopoietic stem/progenitor cells, red blood cells, T cells, B cells, cardiomyocytes, pancreatic islet β cells, liver cells, etc.; Disease human iPSCs disease model for the study of pathogenesis, and try to treat genetic diseases by genetically modifying iPSCs with genetic defects in animal models; establish a variety of tumor iPSCs disease models for the study of pathogenesis and drug screening.

The in vitro culture of human iPSCs requires cell-cell interactions, various cytokines, substrates, etc. to maintain their self-renewal and pluripotency. The classic culture mode is to use mouse embryonic fibroblasts (Mouse Embryonic fibroblasts, MEF) as the trophoblast, cultured in the culture medium containing bFGF and serum replacement (serum replacement, SR). However, before human iPSCs enter clinical application, the removal of heterologous proteins and cell contamination in the culture system is a problem that must be solved. Therefore, since the emergence of human iPSCs, researchers have begun to try to improve their culture system, while maintaining their self-renewal and pluripotency, while reducing the contamination of heterologous proteins and cells. For example, in 2010, Christian et al. used immortalized human skin fibroblasts as a trophoblast to culture human iPSCs, and found that this humanized trophoblast could produce and maintain the culture of human iPSCs, but the culture time was short (reported In the same year, Kristiina et al. cultured human iPSCs with a well-defined RegES medium without heterologous proteins, and found that it could maintain the self-renewal and pluripotency of iPSCs for a long time (>80 passages), but it did not It has not been demonstrated that the medium can reprogram human cells into iPSCs during the reprogramming process. Ruth et al. used another well-defined mTeSRTM medium to culture human iPSCs in suspension, and found that after 17 generations of culture, they could still maintain their self-renewal and pluripotency, but the culture system was expensive, and it also did not prove that it was in the process of reprogramming. , the culture medium can reprogram human cells into iPSCs. Therefore, further development and study of the humanized culture system of human iPSCs, including the reprogramming process and maintenance culture, as well as the effect on the biological characteristics of iPSCs, is an important prerequisite for the clinical application of human iPSCs.

Contents of the invention

The purpose of the present invention is to provide a method for culturing human induced pluripotent stem cells (iPSCs) using human bone marrow mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) as a trophoblast, which is achieved through the following technical solutions:

(1) Preparation of trophoblast cells: The third to fifth passage human bone marrow mesenchymal stem cells cultured after subculture, cryopreservation and recovery were used as trophoblast cells. Human bone marrow mesenchymal stem cells were isolated and cultured by the commonly used Ficoll-paque density gradient centrifugation combined with adherence screening. When used as a feeder layer, the cells were seeded on a six-well plate treated with 0.1% gelatin, and the cell confluence reached 80%-90% (about 2×10 ⁴ /cm ² ), and treated with 10ug/ml mitomycin C Treat for 2 hours to inactivate, and use the inactivated cells within 24 hours;

(2) Obtaining human iPSCs: Foreskin fibroblasts from 2-3-year-old children were taken and transfected twice with retroviruses carrying Klf4, Sox2, Oct4 and c-Myc transcription factors. Add vitamin C 25ug/ml to the culture system on the second day, add VPA2 μM on the fifth day (used for 5 days in total), inoculate the transfected fibroblasts on the trophoblast cells on the sixth day, and replace the Human ESCs culture medium containing bFGF and replacing serum was used. Then change the medium every day until ESCs-like clones appear, select, expand and culture;

(3) Expansion culture of human iPSCs: ESCs-like clones were selected under a microscope, digested with type IV collagenase for 5-10 minutes, blown gently into small clumps, and inoculated on the prepared trophoblast cells. Change fluid daily. Subculture every 7-8 days. The amplified cells are passaged mechanically, that is, the cells are gently scraped off the trophoblast with a 5ml glass dropper, and then gently blown into small clumps, and inoculated on the prepared trophoblast cells;

(4) Biological characteristics and maintenance of pluripotency of human iPSCs: after 14 passages of expansion and culture on human bone marrow mesenchymal stem cells, RT-PCR method was used to identify the ESCs specificity of human iPSCs after expansion and culture. Gene expression; at the same time, the EB formation method and the immunodeficiency mouse teratoma formation method were used to detect whether it could differentiate into three germ layers in vitro and in vivo.

The present invention provides a method for obtaining and culturing human iPSCs using humanized cells, that is, human bone marrow mesenchymal stem cells, as a trophoblast. The humanized trophoblast cells can maintain the biological characteristics and pluripotency of human iPSCs for a long time; The human iPSCs thus obtained are free from contamination by heterologous cells, which opens up the possibility of their clinical application. The present invention uses human bone marrow mesenchymal stem cells instead of using mouse embryonic fibroblasts as trophoblast cells in the traditional method to cultivate human induced pluripotent stem cells, which reduces the contamination of heterologous cells in the culture system, and proves that the culture system can Human induced pluripotent stem cells are expanded in vitro (see Example 4), and their biological characteristics and pluripotency are maintained (see Example 5), which provides the possibility for human induced pluripotent stem cells to enter clinical applications.

Description of drawings

Figure 1 is the technology roadmap.

Figure 2 is the 4th passage of human bone marrow mesenchymal stem cells. A is before inactivation; B is after mitomycin C inactivation.

Figure 3 shows human iPSCs obtained using human bone marrow mesenchymal stem cells as a feeder layer. A is the granuloma-like colony that appeared 21-25 days after transfection of fibroblasts (the scale bar is 100 μm), B is the iPSCs clone formed after expansion and culture (the scale bar is 100 μm), and C is the local cell morphology of the clone (the scale bar is 20 μm ).

Figure 4 is the growth curve of human iPSCs at passage 8-11 in a culture system using human bone marrow mesenchymal stem cells at passage 4 as a feeder layer.

Figure 5 shows the expansion multiples of human iPSCs at the 9th and 10th passages in the culture system of the 4th passage human bone marrow mesenchymal stem cells from different donor sources (hMSC1 and hMSC2 are from female donors, and hMSC3 is from male donors source).

Figure 6 shows the expression of human ESCs-specific genes detected by RT-PCR after human iPS/MSC7 was amplified and cultured for 14 generations in a culture system in which human bone marrow mesenchymal stem cells were used as trophoblast.

Figure 7 shows the differentiation of human iPS/MSC7 from EB to three germ layers in vitro after being expanded and cultured for 14 passages in a culture system in which human bone marrow mesenchymal stem cells are trophoblasts. A is the suspension EB formed after EB cultured for 8 days (the scale is 50 μm); B-D is the various types of cells differentiated from the adherent EB after 14 days of culture (the scale is 100 μm); E is the PT-PCT detection of pluripotency genes after EB differentiation ( SOX2 and OCT4) and three germ layer marker genes (FOXA2 and AFP are endoderm marker genes; MSX1 and BRACHYURY are mesoderm marker genes; MAP2 and PAX6 are ectoderm marker genes) expression (U means undifferentiated, D means differentiated ).

Figure 8 shows the formation of teratomas in NOD-SCID mice after human iPS/MSC7 was expanded and cultured for 14 passages in a culture system in which human bone marrow mesenchymal stem cells were used as trophoblast. A is the mouse with teratoma; B is the adenoid tissue formed in the teratoma (HE staining, the scale bar is 20 μm); C is the muscle-like tissue formed in the teratoma (HE staining, the scale bar is 20 μm) .

Detailed ways

The present invention will be further described in conjunction with drawings and embodiments.

Example 1: Isolation and culture of human bone marrow mesenchymal stem cells, cryopreservation and recovery

(1) Isolation and culture of human bone marrow mesenchymal stem cells

The bone marrow of 3 healthy donors was collected under sterile conditions, anticoagulated with heparin, mononuclear cells were collected by density gradient centrifugation with Ficoll-paque (specific gravity 1.077), seeded at a density of 5×10 ⁵ cells/cm ² , and the culture medium contained 10% (V/V) fetal bovine serum and 1% glutamine low-glucose DMEM (LG-DMEM) culture solution, placed in a 37 ⁰ C, 5% CO2 incubator for culture. After 24 hours, the culture medium was replaced, the non-adherent cells were discarded, and there were spindle-shaped adherent cells. After that, the medium was changed every 3 days, and the adhesion and fusion of the primary cells reached 90%-95% in about 14 days. They were digested with 0.25% trypsin-1 mmol EDTA, subcultured in bottles at a ratio of 1:3, and labeled as P1 cells. During the subculture process, the medium was changed every 3 days. After the cells reached 90% confluence, they were digested and passaged, and marked as P2, and so on.

(2) Cryopreservation and recovery of human bone marrow mesenchymal stem cells

During cryopreservation, digest the mesenchymal stem cells in the logarithmic growth phase, and resuspend the cells in cryopreservation solution (60% LG-DMEM, 30% fetal bovine serum, 10% DMSO), and the final concentration of cells is 5× 10 ⁶ /ml, divided into cryopreservation tubes, marked with the cell name, generation number, cryopreservation time and cryopreserver. Immediately put the cryopreservation tube into the isopropanol freezing box, put it at -80 ⁰ C, and transfer it to a -196 ⁰ C liquid nitrogen tank for storage overnight. When the cells were resuscitated, the cells were taken out of the liquid nitrogen and placed in a 37 ⁰ C constant temperature water bath to quickly thaw. Under sterile conditions, transfer the cell suspension into a 15ml centrifuge tube, add 10ml of pre-cooled culture solution dropwise, centrifuge at 200g for 5min, and discard the supernatant. Resuspend the cell mass with an appropriate amount of preheated culture medium. The revived surviving cells were completely attached to the wall 6 hours after inoculation, and the cell shape gradually became spindle-shaped. Cells began to proliferate rapidly after a rest period of 1-2 days. The cell morphology after recovery was basically the same as that before cryopreservation.

Example 2: Preparation of human bone marrow mesenchymal stem cells as trophoblast cells

Human bone marrow mesenchymal stem cells of the 3rd to 5th generation were used as the trophoblast cells. The 6-well plate was pre-incubated with 0.1% gelatin at 37 ^° C for 1h to overnight. Human bone marrow mesenchymal stem cells were seeded on gelatin-treated six-well plates at a density of 2×10 ⁴ /cm ² , so that the cell confluency reached 80%-90% the next day. Treated with 10ug/ml mitomycin C for 2 hours to inactivate, the cell morphology after inactivation was basically the same as that before inactivation (see Figure 2). The inactivated cells were used within 24 hours.

Example 3: Induction of human iPSCs using human bone marrow mesenchymal stem cells as trophoblast

Foreskin fibroblasts from children of the third generation (2-3 years old) were transfected twice with retrovirus carrying Klf4, Sox2, Oct4 and c-Myc transcription factors. After transfection, vitamin C 25ug/ml was added to the culture system until clones were selected, VPA2 μM was added on the fifth day (5 days in total), and the transfected fibroblasts were inoculated into human bone marrow mesenchyme on the sixth day On the stem cell trophoblast cells, on the seventh day, replace with human ESCs culture medium containing 8ng/ml bFGF, 1% glutamine, 1% non-essential amino acids, 100μM and 20% serum replacement. Then change the medium every day until ESCs-like clones appear (see Figure 3A) with clear borders and dense cells. Select one by one under the microscope, amplify and cultivate. The technical roadmap is shown in Figure 1

Example 4: Expansion and culture of human iPSCs using human bone marrow mesenchymal stem cells as trophoblast

(1) Cell morphology of human iPSCs expanded and cultured in a culture system using human bone marrow mesenchymal stem cells as a feeder layer

Select ESCs-like clones under a microscope, digest them with 1mg/ml type IV collagenase for 5-10 minutes, blow them gently into small clumps, inoculate them on the prepared trophoblast cells, and use human ESCs culture medium at 37 ^0° C, 5% _CO2 culture. Change fluid daily. iPSCs were passaged every 7-8 days. The amplified cells are mechanically passaged, that is, the cells are gently scraped off the feeder layer with a 5ml glass dropper, and then gently blown into small clumps, and inoculated at a ratio of 1:2-1:3 until ready on the trophoblast cells. Human iPSCs were expanded in a culture system using human bone marrow mesenchymal stem cells as a feeder layer to form typical human ESCs-like clones: clear boundaries, dense cells, most of the cells are round, with obvious nucleoli, and the border is fibroblast-like Like cells (see Figure 3B, C).

(2) Growth kinetics of human iPSCs in a culture system using human bone marrow mesenchymal stem cells as feeder layer

Three lines of human iPSCs (iPS/MSC1, iPS/MSC7 and iPS/MSC10) at passage 8 were inoculated at 5×10 ⁴ cells/well (inoculated in small clumps) into a culture medium lined with human bone marrow mesenchymal stem cells as feeder layer. In the 12-well plate, three replicate wells were set. After 7 days of culture, human iPSCs were scraped off from the trophoblast, a part was counted, and the expansion factor was calculated; at the same time, part of it was inoculated into a new 12-well plate covered with human bone marrow mesenchymal stem cells as a trophoblast, and three Duplicate wells were cultured for 7 days, counted and calculated the amplification factor. By analogy, three generations were co-cultured, and the amplification factor was calculated. Human iPSCs can be continuously expanded in a culture system using human bone marrow mesenchymal stem cells as a feeder layer, with an average expansion of 2.32±0.05 times per generation (see Figure 4).

(3) The growth of human iPSCs in the culture system of human bone marrow mesenchymal stem cells from different donors as trophoblast

Three lines of human iPSCs (iPS/MSC1, iPS/MSC7 and iPS/MSC10) at passage 9 and 10 were seeded at 5× ¹⁰⁴ cells/well (inoculated in small clumps) onto human bone marrow from different donor sources Mesenchymal stem cells (hMSC1, hMSC2, hMSC3, where hMSC1 and hMSC2 are female donors, and hMSC3 is male donors) are used as trophoblasts in a 12-well plate, and three replicate wells are set. After 7 days of culture, the human iPSCs were scraped off from the feeder layer, a part was counted, and the expansion factor was calculated. There was no significant difference in the expansion of human iPSCs in culture systems in which bone marrow mesenchymal stem cells from different donors were used as trophoblasts (see Figure 5).

Example 5: Maintenance of biological characteristics and pluripotency of human iPSCs after 14 generations of expansion and culture in a culture system in which human bone marrow mesenchymal stem cells are used as feeder layer

(1) Human iPSCs express human ESCs-specific genes

The human iPSC/MSC7 cell aggregates expanded and cultured in the culture system of human bone marrow mesenchymal stem cells for 14 passages were collected, and the total RNA was extracted with TRIZOL, followed by RT-PCR to detect the expression of human ESCs-specific genes : Including endogenous KLF4, SOX2, c-Myc, OCT4, NANOG, DNMT3B, DPPA4, hTERT, NODAL, REX13, TDGF1, etc. At the same time, H1 human ESCs and children's foreskin fibroblasts were used as controls. The detection results showed that the expression levels of these genes in human iPSCs were similar to H1 human ESCs.

(2) Human iPSCs formed EBs in vitro and differentiated into three germ layers

Human iPSC/MSC7 expanded and cultured for 14 passages in a culture system in which human bone marrow mesenchymal stem cells were used as a trophoblast were scraped off from the trophoblast, and inoculated in ultra-low adhesion 6-well plates as cell clumps, and the culture medium was Human ESCs culture medium without bFGF was cultured at 37 ⁰ C, 5% CO ₂ , and the medium was changed every other day; after 8 days of culture, inoculated into 6-well plates treated with 0.1% gelatin, and continued to be cultured with human ESCs without bFGF ESCs culture medium was adherently cultured at 37 ^° C and 5% _CO2 for 8 days. Experimental results show that the expanded human iPSCs can form EBs in vitro (see Figure 7A), and these EBs can differentiate into various types of cells after adherent culture (see Figure 7B-D). These differentiated cells were collected, and the expression of three germ layer marker genes were detected by RT-PCR. The results showed that the differentiated cells had three germ layer marker genes (FOXA2 and AFP were endoderm marker genes; MSX1 and BRACHYURY were mesoderm marker genes; MAP2 and PAX6 are ectoderm marker genes) expression was significantly increased. It shows that human iPSCs after 14 passages in the culture system in which human bone marrow mesenchymal stem cells are used as trophoblast still maintain their multilineage differentiation potential.

(3) Human iPSCs formed teratomas in vivo and differentiated into three germ layers

The human iPSC/MSC7 expanded and cultured in the culture system of human bone marrow mesenchymal stem cells for 14 passages was scraped from the feeder layer, washed twice with PBS buffer without calcium and magnesium ions, and washed with PBS. Human iPSCs were resuspended, and 200ul was injected into the root muscle of the hind legs of NOD-SCID mice, and the number of cells at each injection point was 2-3×10 ⁶ . Tumor formation was observed after injection. After the teratoma was formed, the teratoma was taken out for paraffin section and HE staining to observe its differentiation into three germ layers. The experimental results showed that the expanded human iPSCs could form teratomas in immunodeficient mice (see Figure 8A), and after HE staining, muscle-like and adenoid tissues were found in the tumor (see Figure 8B, C). It indicated that human iPSCs after 14 passages in the culture system in which human bone marrow mesenchymal stem cells were used as trophoblast still maintained their multidirectional differentiation potential.

Claims (2)

1. one kind with the method for human marrow mesenchymal stem cell as trophoderm cultivator inductive pluripotent stem cells, is achieved through the following technical solutions:

(1) trophocyte's preparation: the human marrow mesenchymal stem cell of using the 3rd ~ the 5th generation of cultivating behind go down to posterity cultivation, the low temperature cryopreservation resuscitation is as the trophocyte, on six orifice plates of cell inoculation after handle with 0.1%gelatin, cytogamy degree 80%-90%, and handle deactivation in 2 hours with the ametycin of 10 μ g/ml, the cell after the deactivation used in 24 hours;

(2) acquisition of people's inductive pluripotent stem cells: get 2-3 year children's foreskin inoblast, with twice of the retrovirus transfection that carries Klf4, Sox2, Oct4 and c-Myc transcription factor, second day vitaminize C 25 μ g/ml in culture system, added VPA 2 μ M on the 5th day, inoblast after the transfection was inoculated on the trophocyte in the 6th day, the 7th day, use the people ESCs nutrient solution that contains bFGF, alternative serum instead, change liquid subsequently every day, clone up to class ESCs occurring selects, amplification cultivation;

(3) amplification cultivation of people's inductive pluripotent stem cells: microscopically is selected the clone of class ESCs, with the enzymic digestion of IV Collagen Type VI after 5-10 minute, dispel into little agglomerate gently, be inoculated on the ready trophocyte, change liquid every day, went down to posterity once in every 7-8 days, the cell after the amplification goes down to posterity with mechanical process;

(4) keeping of the biological characteristics of people's inductive pluripotent stem cells and multipotency: with the human marrow mesenchymal stem cell be amplification cultivation after 14 generations on the trophoderm, whether the ESCs specific gene expression with RT-PCR method evaluation amplification cultivation descendant inductive pluripotent stem cells detects it with EB forming method and immunodeficient mouse teratoma forming method simultaneously and can break up to triploblastica in vivo and in vitro.

2. according to claim 1 a kind of with the method for human marrow mesenchymal stem cell as trophoderm cultivator inductive pluripotent stem cells, it is characterized in that, it is that glass dropper with 5ml scrapes cell from trophoderm gently that cell after the described amplification of step (3) goes down to posterity with mechanical process, dispel into little agglomerate subsequently gently, be inoculated on the ready trophocyte.

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