阅读说明：本技术 一种诱导多能干细胞分化为间充质干细胞的培养基组合及方法 (Culture medium combination and method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells ) 是由 严庆丰 陈虹 贾则晓 齐念民 王皓 于 2019-09-30 设计创作，主要内容包括：本发明公开了一种诱导多能干细胞分化为间充质干细胞的培养基组合及方法。所述培养基组合包括第一阶段的间充质干细胞诱导分化培养基和第二阶段的间充质干细胞支持培养基,所述间充质干细胞诱导分化培养基包括STEMdiff<Sup>TM</Sup>-ACF间充质诱导培养基；所述间充质干细胞支持培养基包括基础培养基、血清和添加剂,所述添加剂包括：L-谷氨酰胺、L-Ascorbic acid、丙酮酸钠、MEM NEEA。所述方法为使用所述培养基组合进行培养,包括：使用间充质干细胞诱导分化培养基诱导培养多能干细胞3天；第4天开始换成间充质干细胞支持培养基进行培养直到获得成熟的间充质干细胞。(The invention discloses a culture medium combination and a method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells. The culture medium combination comprises a first-stage mesenchymal stem cell induced differentiation culture medium and a second-stage mesenchymal stem cell support culture medium, and the mesenchymal stem cell induced differentiation culture medium comprises STEMdiff TM -ACF mesenchymal induction medium; the mesenchymal stem cell support medium comprises a basal medium, serum and additives, wherein the additives comprise: l-glutamine, L-Ascorbic acid, sodium pyruvate, MEM NEEA. The method is to culture by using the culture medium combination, and comprises the following steps: inducing and culturing the pluripotent stem cells for 3 days by using a mesenchymal stem cell induced differentiation culture medium; and starting to change to the mesenchymal stem cell supporting culture medium on the 4 th day until the mature mesenchymal stem cells are obtained.)

1. A culture medium combination for inducing the differentiation of pluripotent stem cells into mesenchymal stem cells is characterized by comprising a first-stage mesenchymal stem cell induced differentiation culture medium and a second-stage mesenchymal stem cell supporting culture medium,

the mesenchymal stem cell induced differentiation medium comprises STEMdiff^TM-ACF mesenchymal induction medium;

the mesenchymal stem cell support medium comprises a basal medium, serum and additives, wherein the additives comprise:

l-glutamine, L-Ascorbic acid, sodium pyruvate, MEM NEEA.

2. The culture medium combination of claim 1, wherein the additives comprise:

l-glutamine with final concentration of 2mmol/L, L-Ascorbic acid with final concentration of 100. mu. mol/L, sodium pyruvate with final concentration of 1mmol/L and MEM NEEA with volume ratio of 1%.

3. The culture medium combination of claim 1, wherein the basal medium is DMEM/F-12 basal medium, the serum is fetal bovine serum, and the basal medium has a fetal bovine serum addition volume of 10%.

4. The culture medium combination of claim 1, wherein 1% by volume of double antibody is added to both the mesenchymal stem cell-inducing differentiation medium and the mesenchymal stem cell-supporting medium.

5. A method for inducing differentiation of pluripotent stem cells into mesenchymal stem cells, wherein the culture medium combination according to any one of claims 1 to 4 is used for culture, and the method comprises the following steps: inducing and culturing the pluripotent stem cells for 3 days by using a mesenchymal stem cell induced differentiation culture medium; and starting to change to the mesenchymal stem cell supporting culture medium on the 4 th day until the mature mesenchymal stem cells are obtained.

6. The method of claim 5, comprising the steps of:

(1) preparing pluripotent stem cells;

(2) performing induction culture by using the mesenchymal stem cell induction differentiation culture medium, replacing the culture medium once a day, and performing induction culture for 3 days in total;

(3) and starting to replace the mesenchymal stem cell support culture medium for induction culture on day 4, replacing the culture medium once every day, carrying out passage when the cell density reaches 70-90% (freezing and storing the rest cells during the first passage), and carrying out passage culture for 5-8 generations to obtain the mature mesenchymal stem cells.

7. The method of claim 6, wherein the pluripotent stem cells used in step (1) are induced pluripotent stem cells.

8. The method of claim 6, wherein the pluripotent stem cells are seeded into a Matrigel-coated culture vessel 2 days before the induction culture in the step (1), and the cell density is 40 to 60% at the induction culture in the step (2).

9. The method of claim 6, wherein the cells are seeded into culture vessels coated with 0.1% gelatin at the time of passaging in step (3).

10. The method of claim 6, wherein the precursor mesenchymal stem cells are obtained at day 6.

Technical Field

The invention relates to the technical field of biomedicine, in particular to a culture medium combination and a method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells.

Background

Induced Pluripotent Stem Cells (iPSCs) are cells which are reprogrammed by introducing somatic cells through transcription factors (Oct4, Sox2, Klf4 and c-Myc) and have similar differentiation capacity to embryonic stem cells, become important cell sources for researching human disease pathogenesis and tissue cell replacement therapy, and have no ethical problem. The iPSCs are used as source cells, and can be amplified in vitro and induced to differentiate into specific tissue cells.

Mesenchymal stem cells are pluripotent stem cells that have all of the commonalities of stem cells, namely self-renewal and multipotential differentiation capacity. Currently available for clinical use to treat a variety of diseases and therefore is of great interest. Mesenchymal stem cells have unique biological properties. Namely, mesenchymal stem cells have the ability to differentiate into various tissues of mesoderm origin, making them very suitable for use in regenerative therapy. Furthermore, mesenchymal stem cells are able to modulate both humoral and cellular immune responses by secreting anti-inflammatory molecules, which means that mesenchymal stem cells are well suited for the treatment of a range of immune system based diseases. Finally, mesenchymal stem cells do not express class II antigens or their costimulatory molecules that allow the cells to evade the host's immune system, making them useful in a xenogeneic transplantation environment. Therefore, the mesenchymal stem cells have great application prospect in clinic. How to effectively obtain the mesenchymal stem cells is a problem to be solved urgently.

At present, the main sources of the domestic mesenchymal stem cells are autologous sources (bone marrow, adipose tissue and the like) and allogeneic sources (umbilical cord, placenta and the like). However, with mesenchymal stem cells of autologous or allogeneic origin, several problems are prevalent: 1. difficult to obtain materials; 2. the preservation period is short, and the taken cells and tissues need to be immediately treated and subjected to in vitro amplification; 3. with the age of the donor, the number and the amplification potential of the mesenchymal stem cells are obviously reduced, and the mesenchymal stem cells can be more accelerated to age by in vitro amplification (the mesenchymal stem cells from the bone marrow can be generally amplified to 5-10 generations); 4. mesenchymal stem cells of different donor ages have different differentiation potentials (the mesenchymal stem cells of young people are easily differentiated into cells such as cartilage, while the mesenchymal stem cells of old people tend to differentiate into fat cells); 5. the quality control is not easy in the preparation process. These drawbacks largely lead to instability in the therapeutic efficacy of adult-derived mesenchymal stem cells.

At present, there are two main methods for inducing differentiation from iPSCs into mesenchymal stem cells. The currently used method is to first induce pluripotent stem cells into embryoid-like bodies (EBs), followed by differentiation into mesenchymal stem cell-like cells by transferring the embryoid-like bodies into a culture dish and inducing using a mesenchymal stem cell induction medium [ Hwang NS, et al 2008 ]. After differentiating into mesenchymal stem cell-like cells, flow sorting is needed, and cells with mesenchymal stem cell surface markers are screened out. The method mainly has the defects of complex induction process, long time, low efficiency and the like.

Direct differentiation method: the direct differentiation method is to differentiate iPSCs into mesenchymal progenitor cells (or precursor mesenchymal stem cells) and then induce the mesenchymal progenitor cells into mature mesenchymal stem cells. This period is about 6 to 10 days (the duration of this period will vary slightly from one laboratory to another due to the different induction media used). This stage is critical for differentiation, and if the induction medium and support medium are not properly configured, it is likely that as the passage time increases, dead cells increase and the differentiation potential of the cells is lost. There is therefore a need for improved media conditions to promote differentiation of cells into homogeneous mesenchymal stem cells.

Currently, the most used method is the embryoid body method. One of these documents is exemplified by [ dmitric Sheyn et al.2016]: iPSCs were digested and seeded into non-adherent reaction plates, 10000 cells/well, modified Dulbecco's medium (IMDM) (MDM basal medium, 17% KnockOut serum replacement, 1% non-essential amino acids, 110 mmol/L2-mercaptoethanol, and 1% PSA antifungal-antibacterial solution). On day2, EBs were transferred to non-adherent 2.4. mu.g/cm²Poly-HEMA coated flasks and incubated for an additional 3 daysAfter days 8-10, the medium was changed to DMEM medium containing 10% fetal bovine serum, 2mmol/L L-glutamine and 100U/mL penicillin/streptomycin, the medium was changed twice weekly and passaged at 1: 3 ratio at confluence, and finally cells bearing mesenchymal stem cell specific surface markers were screened with flow cells.

Feeder layer cell co-culture method [ Ran Kang, et al.2015]: feeder cells promote the growth of target cells in a culture system by secreting cytokines. iPSCs were co-cultured with feeder layer cells, which secreted some cytokines to support cell differentiation. After 3 days of passaging iPSCs to feeder layer cell culture, KSR medium was replaced with mesenchymal stem cell medium. iPSCs were maintained in mesenchymal stem cell culture medium for 2 weeks, with medium changed every other day. Subsequently, the cells were passaged to gelatin-coated (0.1% gelatin, room temperature, 2 hours) tissue culture vessels by trypsinization (0.25% trypsin/1 mmol/L EDTA). Cells were defined as passage 1 after the first passage (P1). To maintain iPSC-mesenchymal stem cells, cells were passaged at 90% confluence and at 1.6 x10⁴Individual cell/cm²The method is limited in that factors secreted by ① feeder cells are uncertain, and the quality of cells differentiated from different batches is uneven, and ② cells differentiated from one culture dish are mixed with feeder cells, so that the purity of the cells is reduced.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a culture medium combination and a method for inducing pluripotent stem cells to be differentiated into mesenchymal stem cells, and aims to solve the technical problems of high cost, low efficiency, instability and the like of mesenchymal stem cell induced differentiation in the prior art.

The invention firstly provides a culture medium combination for inducing the differentiation of pluripotent stem cells into mesenchymal stem cells, which comprises a first stage mesenchymal stem cell induced differentiation culture medium and a second stage mesenchymal stem cell support culture medium, wherein the mesenchymal stem cell induced differentiation culture medium comprises STEMdiff^TM-ACF mesenchymal induction medium; the mesenchymal stem cell support medium comprises a basal medium, serum and additives, wherein the additives comprise: l-glutamine, L-ascorbic acid, sodium pyruvate, MEM NEEA.

Preferably, the additives include: l-glutamine with final concentration of 2mmol/L, L-Ascorbic acid with final concentration of 100. mu. mol/L, sodium pyruvate with final concentration of 1mmol/L and MEM NEEA with volume ratio of 1%.

Preferably, the basic culture medium is DMEM/F-12 basic culture medium, the serum is fetal calf serum, and the volume of the fetal calf serum added in the basic culture medium is 10%.

Preferably, the mesenchymal stem cell induced differentiation medium and the mesenchymal stem cell support medium are both added with 1% of double antibody by volume ratio. The double-resistant includes penicillin and streptomycin, which can be directly purchased as finished products for use, and the antibiotic is added mainly for preventing cell contamination.

The invention also provides a method for inducing the differentiation of pluripotent stem cells into mesenchymal stem cells, which uses the culture medium combination for culture and comprises the following steps: inducing and culturing the pluripotent stem cells for 3 days by using a mesenchymal stem cell induced differentiation culture medium; and starting to change to the mesenchymal stem cell supporting culture medium on the 4 th day until the mature mesenchymal stem cells are obtained.

Preferably, the method comprises the following steps:

(1) preparing pluripotent stem cells;

(2) performing induction culture by using the mesenchymal stem cell induction differentiation culture medium, replacing the culture medium once a day, and performing induction culture for 3 days in total;

(3) and starting to replace the mesenchymal stem cell support culture medium for induction culture on day 4, replacing the culture medium once every day, carrying out passage when the cell density reaches 70-90% (freezing and storing the rest cells during the first passage), and carrying out passage culture for 5-8 generations to obtain the mature mesenchymal stem cells.

More preferably, the pluripotent stem cells used in step (1) are induced pluripotent stem cells.

More preferably, the pluripotent stem cells are inoculated into a culture vessel coated with Matrigel 2 days before the induction culture in the step (1), and the cell density reaches 40-60% when the induction culture in the step (2) is carried out.

More preferably, the cells at the time of passaging in step (3) are seeded into culture vessels coated with 0.1% gelatin.

More preferably, the precursor mesenchymal stem cells are obtained at day 6.

Compared with the prior art, the invention has the following gain effects:

(1) according to the invention, the two culture mediums of the mesenchymal stem cell induced differentiation culture medium and the mesenchymal stem cell support culture medium are adopted to sequentially induce and differentiate the pluripotent stem cells into the precursor mesenchymal stem cells and the mature mesenchymal stem cells, so that the efficiency of inducing the pluripotent stem cells into the mesenchymal stem cells is improved.

(2) After the precursor mesenchymal stem cells are induced and differentiated (namely differentiation day 6), the cells can be frozen, and the frozen cells can be induced and differentiated for the next time, so that the differentiation time is shortened.

Drawings

FIG. 1: the cell growth status at different stages of the two induced differentiation modes was compared: the cells proliferated well in example 1, and the morphology and proliferation potency of the P6 generation cells remained good. The control group had more dead cells per passage and the proliferative capacity began to decline after P3-P6 passages.

FIG. 2: the differentiation efficiency of example 1 and the comparative group were compared. The cells were counted at different stages of differentiation, in contrast to example 1, which showed a significantly greater efficiency of cell differentiation than the control.

FIG. 3: the iPSCs differentiated mesenchymal stem cells (induced and cultured for 27 days) highly express mesenchymal stem cell specific marker: CD73, CD105, CD90, and CD 146; (induction culture day 10 and day27) low expression of embryonic stem cell-specific marker: 4-Oct, Nanog, Sox2, and EPCAM. The above results indicate that the iPSCs of example 1 successfully induced mesenchymal stem cells.

FIG. 4: immunofluorescence of CD73 was performed on mesenchymal stem cells differentiated on day 27. The results show that CD73 is highly expressed in mesenchymal stem cells. The positive of the mesenchymal stem cell CD73 differentiated by the method can be seen through immunofluorescence staining, and the success of the differentiation of the mesenchymal stem cell is proved.

Detailed Description

The culture medium and method for inducing differentiation of pluripotent stem cells into mesenchymal stem cells according to the present invention will be described in further detail with reference to the following examples. The media and supplements used in this example are commercially available.

The invention aims to provide a mesenchymal stem cell induced differentiation medium and a mesenchymal stem cell induced differentiation method.

The mesenchymal stem cell induced differentiation culture medium of the first stage is STEMdiff^TM-ACF MesenchymalInduction Medium(STEMdiff^TMMesenchymal induction medium, cat #05241), 1% diabody (including penicillin and streptomycin, GIBCO, cat # 15140122).

The mesenchymal stem cell supporting culture medium at the second stage comprises 90 percent of basal culture medium, 10 percent of fetal calf serum and additives; wherein the basic culture medium is DMEM/F-12 culture medium; additives were 2mmol/L L-glutamine (GIBCO, cat #25030081), 100. mu. mol/L L-Ascorbic acid (sigma, cat # A92902), 1mmol/L sodium pyruvate (GIBCO, cat #11360070), 1% MEM NEEA (MEM non-essential amino acids, GIBCO, cat #11140050) and 1% diabase (including penicillin and streptomycin, GIBCO, cat # 15140122).

The content of the additives and the components refers to the concentration content or the volume ratio of the additives in the mesenchymal stem cell induced differentiation culture medium.

STEMdiff^TM-ACF Mesenchymal Induction Medium(STEMdiff^TMMesenchymal stem cell induced differentiation medium is unfrozen at room temperature (15-25 ℃) or unfrozen at 2-8 ℃ overnight. Thoroughly mixed before use or stored in sub-package at-20 ℃. After thawing, the cells were stored at 4 ℃ for no more than 2 weeks.

And (3) storing the basic culture medium and each additive in the second-stage mesenchymal stem cell support culture medium at 4 ℃, diluting the L-Ascorbic acid according to the proportion of 1000X of final concentration, subpackaging, and storing at-20 ℃. The mesenchymal stem cell supporting culture medium is prepared and then stored for no more than 4 weeks at 4 ℃.