Culture and method for indirectly inducing human induced pluripotent stem cells into mesenchymal stem cells

Abstract

The application discloses a culture solution for indirectly inducing human induced pluripotent stem cells into mesenchymal stem cells, which is composed of a neuroectoderm induction culture solution, a mesenchymal stem cell differentiation culture solution and a mesenchymal stem cell maintenance culture solution. The neuroectoderm induction culture solution is composed of a Neurobasal culture medium, a DMEM / F12 culture medium, an N2 additive, a B27 additive, a GSK3 inhibitor and a TGF-beta inhibitor. The application further discloses a method for indirectly inducing human induced pluripotent stem cells into mesenchymal stem cells, which comprises the following steps: taking the induced pluripotent stem cells, culturing the induced pluripotent stem cells in the neuroectoderm induction culture solution for 6 days, then culturing the induced pluripotent stem cells in the mesenchymal stem cell differentiation culture solution for 7 days, and then culturing the induced pluripotent stem cells in the mesenchymal stem cell maintenance culture solution for 3-5 generations. The method can direct the induced pluripotent stem cells to differentiate into mesenchymal stem cells in a short time, and greatly shortens the culture time.

Description

Technical Field

[0001] This invention relates to a culture medium and method for indirectly inducing human induced pluripotent stem cells into mesenchymal stem cells, belonging to the field of cell differentiation technology. Background Technology

[0002] Induced pluripotent stem cells (iPS cells) are somatic cells that have undergone terminal differentiation and been reprogrammed into pluripotent stem cells by introducing specific transcription factors. In 2006, Shinya Yamanaka of Kyoto University in Japan first reported research on induced pluripotent stem cells in the journal *Cell*. They cloned the genes of four transcription factors—Oct3 / 4, Sox2, c-Myc, and Klf4—into a viral vector and introduced it into mouse fibroblasts. They found that this induced transformation, and the resulting iPS cells were similar to embryonic stem cells in morphology, gene and protein expression, epigenetic modification status, cell proliferation capacity, ability to form germ-like bodies and teratomas, and differentiation capacity.

[0003] Mesenchymal stem cells (MSCs) are pluripotent stem cells, first isolated from bone marrow, and later isolated from adipose tissue, umbilical cord, and dental pulp. MSCs possess osteogenic, adipogenic, and chondrogenic differentiation potential, expressing markers such as CD73, CD90, and CD105, but not HLA-DR, thus exhibiting a low immunogenicity. Furthermore, MSCs secrete numerous cytokines, playing important roles in regenerative medicine and immune regulation. Therefore, multiple phase I-III clinical trials have used autologous or allogeneic MSCs to treat degenerative and autoimmune diseases such as neurodegenerative diseases, ischemic heart disease, and graft-versus-host disease (GVHD). In vitro animal experiments and in vivo clinical trials have also demonstrated that transplanted MSCs can home to sites of injury or inflammation, promoting cell survival, repair of damaged cells, or differentiation into functional cells through cell-to-cell contact or paracrine effects.

[0004] MSCs from various adult tissues have been used in clinical trials, but different sources, culture methods, and even cryopreservation conditions all affect cell quality and function. Primary isolated MSCs generally have limited proliferative capacity, and long-term culture affects cell function and differentiation potential. For example, bone marrow-derived MSCs have a doubling time of approximately 3.75 days and can expand to about 10 generations; adipose-derived MSCs have a doubling time of approximately 1.6 days, while umbilical cord-derived MSCs have a shorter doubling time and stronger expansion capacity. In addition, umbilical cord-derived MSCs have lower immunogenicity and stronger immunomodulatory effects, and secrete higher levels of cytokines such as IL-6 and IL-8. Studies have found that not only do MSCs obtained from different tissue sources such as bone marrow, adipose tissue, and umbilical cord blood have different biological characteristics, but bone marrow MSCs from different healthy donors also have different proliferative capacities and osteogenic differentiation potentials, leading to inconsistent data in MSC cell therapy clinical trials. Furthermore, the quality of MSCs obtained from donors of different ages varies. Older donors have limited MSC expansion capacity, differentiation potential, and clinical use due to cell aging, DNA damage accumulation, and metabolic instability. Early studies used co-culturing hESCs with stromal cells such as OP9 cells to induce differentiation into MSCs, but this method has a long culture time and low differentiation efficiency. Embryomorphic bodies (EBs) have also been used for MSC induction; for example, MSCs induced by PDGF-AB and FGF2 can be obtained after flow cytometry sorting. However, these methods are not only time-consuming but also inefficient, making them difficult to use in clinical trials. Currently, there are no reports on inducing induced pluripotent stem cells into mesenchymal stem cells. Summary of the Invention

[0005] In view of the above-mentioned prior art, in order to overcome the problems of long time and low yield in the methods of obtaining mesenchymal stem cells, the present invention provides a culture medium and method for indirectly inducing human induced pluripotent stem cells into mesenchymal stem cells.

[0006] This invention is achieved through the following technical solution:

[0007] A culture medium for indirectly inducing human induced pluripotent stem cells into mesenchymal stem cells is composed of a neuroectodermal induction culture medium, a mesenchymal stem cell differentiation culture medium, and a mesenchymal stem cell maintenance culture medium.

[0008] The neuroectodermal induction culture medium consists of Neurobasal medium, DMEM / F12 medium, N2 additive, B27 additive, GSK3 inhibitor, and TGF-β inhibitor. The proportions of each component are as follows: Neurobasal medium accounts for 47%–50% (volume percentage, the same below), N2 additive accounts for 0.8%–1.2%, B27 additive accounts for 1.8%–2.2%, GSK3 inhibitor concentration is 8–12 μM, TGF-β inhibitor concentration is 2.5–3.5 μM, and the remainder is DMEM / F12 medium.

[0009] The mesenchymal stem cell differentiation culture medium is composed of EGF (epidermal growth factor), bFGF (basic fibroblast growth factor) and mesenchymal stem cell maintenance culture medium, wherein the concentration of EGF is 4-6 ng / ml, preferably 5 ng / ml, and the concentration of bFGF is 4-6 ng / ml, preferably 5 ng / ml.

[0010] The mesenchymal stem cell maintenance culture medium is YinFengBIO serum-free medium, which is a culture medium that is already available in the prior art and can be purchased from the market. It is a culture medium independently developed by Beijing YinFeng Dingcheng Biotechnology Co., Ltd., and consists of 400ml of Mesenchy Stem Cell Basal Medium and 100ml of Mesenchy Stem Cell Supplement.

[0011] Preferably, the proportions of the components in the neuroectodermal induction culture medium are as follows: Neurobasal medium accounts for 48.5%, N2 additive accounts for 1.0%, B27 additive accounts for 2.0%, the concentration of GSK3 inhibitor is 10 μM, the concentration of TGF-β inhibitor is 3.0 μM, and the remainder is DMEM / F12 medium.

[0012] The Neurobasal medium and DMEM / F12 medium are commonly used cell culture media and can be purchased from the market. The N2 additive and B27 additive are commonly used nerve cell culture additives and can be purchased from the market.

[0013] The TGF-β inhibitor is selected from SB431542 and is available on the market. It can induce stem cells to differentiate into multiple cell types, including endothelial cells and nerve cells, and can also be used directly for the differentiation of embryonic stem cells into mesenchymal-like cells.

[0014] The GSK3 inhibitor is selected from CHIR99021, which is available on the market. It is an activator of WNT signaling and induces the differentiation of nerve cells when used in combination with reagents such as Forskolin / ISX-9 / SB431542.

[0015] The application of the culture medium for indirectly inducing human induced pluripotent stem cells into mesenchymal stem cells in the process of indirectly inducing human induced pluripotent stem cells into mesenchymal stem cells.

[0016] A method for indirectly inducing human induced pluripotent stem cells into mesenchymal stem cells includes the following steps:

[0017] (1) Take induced pluripotent stem cells and culture them in a directional differentiation culture medium at 37℃ and 5% CO2 for 6 days to obtain the culture medium; change the culture medium every 1 to 3 days (preferably 1 day) during the directional differentiation culture;

[0018] (2) The culture medium obtained in step (1) (containing neuroectodermal cells) is cultured in a mesenchymal stem cell differentiation culture medium at 37°C and 5% CO2 for 7 days to obtain a culture medium; the culture medium is changed every 1 to 3 days (preferably 1 day) during the directed differentiation culture; under the induction of the mesenchymal stem cell differentiation culture medium, the neuroectodermal cells transform into mesenchymal stem cells, and growth factors can promote the transformation and rapid growth of cells and maintain the good condition of cells during the growth process;

[0019] (3) The culture medium obtained in step (2) is cultured for 3 to 5 generations in a mesenchymal stem cell maintenance culture medium at 37°C and 5% CO2 to obtain mesenchymal stem cells; the culture medium is changed every 2 to 4 days (preferably 2 days) during the culture period.

[0020] The culture medium of this invention uses Neurobasal and DMEM / F12 basal media, and is supplemented with key components essential for cell growth, such as N2 and B27, as well as GSK3 inhibitors, TGF-β inhibitors, EGF, and other factors. Each component works in a targeted manner to synergistically induce iPSCs to differentiate into mesenchymal stem cells. The culture medium contains specific signal inhibitors, which can improve the purity and yield of mesenchymal stem cells.

[0021] The method of this invention can directionally differentiate induced pluripotent stem cells into mesenchymal stem cells in a relatively short time. Starting with induced pluripotent stem cells, the quantity of raw materials is unlimited, and induced pluripotent stem cells can be expanded indefinitely, avoiding the ethical controversies surrounding the use of embryonic stem cells. The culture cycle is approximately two weeks, significantly shortening the culture time and reducing production costs. This invention uses serum-free culture medium to culture mesenchymal stem cells, and the resulting mesenchymal stem cells can be rapidly translated into clinical applications. Attached Figure Description

[0022] Figure 1 : Schematic diagram of iPS cell pluripotency identification results, where a, b, and c represent endoderm, mesoderm, and ectoderm, respectively.

[0023] Figure 2 : Schematic diagram of cell morphology at each stage.

[0024] Figure 3 Schematic diagram of flow cytometry identification results. Detailed Implementation

[0025] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0026] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0027] The induced pluripotent stem cells used in this invention were obtained through conventional methods: using the Sendai virus reprogramming kit (Invitrogen). TM CytoTune TM -iPS2.0 Sendai Virus Reprogramming Kit (Catalog No.: A16517) is used to obtain high-frequency HLA homozygous umbilical cord blood-derived induced pluripotent stem cells by standard reprogramming methods according to the instructions.

[0028] Example 1: Indirect induction of human induced pluripotent stem cells into mesenchymal stem cells

[0029] The steps are as follows:

[0030] (1) Take induced pluripotent stem cells and culture them in a six-well plate according to the stem cell culture method. Digest and passage them with Versene every 3 to 4 days, and culture them in mTeSR-1 complete medium in a 37°C, 5% CO2 incubator.

[0031] Before proceeding with mesenchymal stem cell differentiation, Versene was used to passage iPS cells and identify their pluripotency expression (results are shown below). Figure 1 As shown, by Figure 1 It is evident that the iPS cells used for induced differentiation have the ability to differentiate into tissues of the three germ layers in vitro. Differentiation experiments can be performed once identification is complete and cell density reaches 70%.

[0032] (2) The cells passaged into Matrigel were cultured at 37°C and 5% CO2. When the cell density reached 80%, the neuroectodermal induction culture medium was added (2 mL / well of a six-well plate; SB431542 and CHIR99021 were added fresh for use; the basal culture medium was stored at 4°C for no more than 7 days and was taken out and warmed to room temperature 30 minutes before use). The cells were cultured at 37°C and 5% CO2 for 6 days, and the culture medium was changed once a day.

[0033] (3) At the end of 6 days of induction, plasma cells were digested with TryplE and 8 × 10⁻⁶ g / mL. 4 / cm 2 The cells were seeded at a density in Matrigel-coated six-well plates and induced to differentiate for 7 days using mesenchymal stem cell differentiation medium (growth factors were prepared fresh for use, and the basal medium could be stored at 4°C for 30 days after preparation). The culture medium was changed daily at 37°C and 5% CO2.

[0034] (4) After differentiation induction, cells were digested with TryplE and then 4×10 4 / cm 2 The cells were seeded at a density in Matrigel-coated six-well plates and cultured in mesenchymal stem cell maintenance medium at 37°C and 5% CO2, denoted as P0; the culture medium was changed every 2 days.

[0035] When P0 generation cells reach 90% confluence, they are passaged and cultured at a concentration of 1×10⁶ cells / year. 4 / cm 2 The cells were densely seeded in six-well plates and cultured using mesenchymal stem cell maintenance medium. P2 and P3 passages were obtained.

[0036] Cell morphology was identified at each stage, and the results are as follows: Figure 2 As shown, from left to right and from top to bottom, the cells represent iPS cells, cells obtained in step (2), cells obtained in step (3), P0 generation cells, P1 generation cells, and P2 generation cells. Figure 2 It is evident that iMSCs obtained through staged induced differentiation exhibit a typical fibroblast-like morphology.

[0037] Characteristic protein expression was identified in P0 generation cells using flow cytometry, and the results are as follows: Figure 3 As shown. By Figure 3 It is evident that the induced differentiated iMSCs express the MSC cell hallmark marker.

[0038] Conclusion: The method of the present invention is simple to operate, has high differentiation efficiency, and is highly feasible. It can be well used for the induction and differentiation of mesenchymal stem cells derived from pluripotent stem cells.

[0039] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for indirectly inducing human induced pluripotent stem cells into mesenchymal stem cells, characterized in that, Includes the following steps: (1) Take induced pluripotent stem cells and culture them in a directional differentiation culture medium at 37℃ and 5% CO2 for 6 days to obtain the culture medium; change the culture medium every 1 to 3 days during the directional differentiation culture. (2) The culture medium obtained in step (1) is used to differentiate and culture mesenchymal stem cells for 7 days at 37℃ and 5% CO2. The culture medium is changed every 1 to 3 days during the directed differentiation culture. (3) The culture medium obtained in step (2) is cultured in mesenchymal stem cell maintenance culture medium at 37℃ and 5% CO2 for 3 to 5 generations to obtain mesenchymal stem cells; the culture medium is changed every 2 to 4 days during the culture period. The proportions of the components in the neuroectodermal induction culture medium are as follows: Neurobasal medium 48.5%, N2 additive 1.0%, B27 additive 2.0%, GSK3 inhibitor 10 μM, TGF-β inhibitor 3.0 μM, and the remainder is DMEM / F12 medium; the TGF-β inhibitor is selected from SB431542; the GSK3 inhibitor is selected from CHIR99021. The mesenchymal stem cell differentiation culture medium is composed of EGF, bFGF and mesenchymal stem cell maintenance culture medium, wherein the concentration of EGF is 4-6 ng / ml and the concentration of bFGF is 4-6 ng / ml. The mesenchymal stem cell maintenance culture medium is YinFengBIO serum-free medium.

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