A defined serum-free induction medium and induction method for promoting differentiation of mesenchymal stem cells into dermal papilla cells

Abstract

The present application relates to the technical field of stem cells, and particularly relates to a serum-free induction medium with clear components for promoting differentiation of induced mesenchymal stem cells into dermal papilla cells and an induction method. A small molecule compound composition comprises all-trans retinoic acid, forskolin, CHIR-99021 and minoxidil in a molar ratio of 0.2-5:2-50:0.2-10:0.2-10. The present application adopts multiple small molecule compounds to promote differentiation and function improvement of dermal papilla cells and transformation of mesenchymal stem cells into dermal papilla cells. The first three promote differentiation of dermal papilla cells, and the last one promotes proliferation and inhibits apoptosis of dermal papilla cells. The present application can solve the technical problems of high process cost and unsatisfactory clinical safety caused by the need to add cytokines in the process of inducing differentiation of stem cells into dermal papilla cells, and speeds up the induction process, and has an ideal application prospect.

Description

Technical Field

[0001] This invention relates to the field of stem cell technology, specifically to a serum-free induction culture medium with clearly defined components and an induction method for promoting the differentiation of induced mesenchymal stem cells into dermal papillary cells. Background Technology

[0002] Dermal papillary (DP) cells are a specialized group of mesenchymal cells located at the base of hair follicles and have been shown to regulate hair follicle formation and growth cycles. DP cells have also been shown to work alongside epidermal cells to generate new hair follicles. Furthermore, implanting DP cells alone between the dermis and epidermis can induce new hair follicle growth at the implantation site. Therefore, DP cells are considered an ideal cell therapy for treating hair loss.

[0003] However, the number of dermal papillary stem cells (DP cells) in the human body is limited, their in vitro expansion capacity is limited, and the extraction of DP cells completely damages the original hair follicles. Furthermore, the ability of DP cells to induce hair follicle growth is greatly reduced during culture, making it difficult to obtain a sufficient number of functional DP cells. This seriously hinders the clinical application of DP cells. To obtain a sufficient quantity of functional DP cells, scientists have explored the differentiation of proliferating cells into DP cells. However, cytokines are often used during the induction process to ensure the induction effect. For example, the applicant's prior patent (2024100133312 A culture medium and induction method for promoting the differentiation of mesenchymal stem cells into dermal papillary cells) uses cytokines such as hepatocyte growth factor as inducers. However, cytokines are proteins, generally lacking physicochemical stability, and have high production costs. Small molecule compounds, on the other hand, have several advantages over cytokines: stable physicochemical properties, easy to transport, store, and control quality during use, lower costs for large-scale production, and the resulting cell products do not contain heterologous proteins, are easy to purify, and improve the safety for clinical use. There is an urgent need to study a method for obtaining dermal papillary cells based on the induction of small molecule compounds, in order to improve process stability and reduce preparation costs. Summary of the Invention

[0004] The present invention aims to provide a combination of small molecule compounds that promote the differentiation of induced mesenchymal stem cells into dermal papillary cells, in order to solve the technical problems of existing technologies that require the addition of cytokines in the process of inducing differentiation of stem cells into dermal papillary cells, resulting in high process costs and unsatisfactory clinical safety.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A small molecule compound composition for promoting the differentiation of induced mesenchymal stem cells into dermal papillary cells, comprising all-trans retinoic acid, trichomoniasis, CHIR-99021, and minoxidil in a molar ratio of 0.2-5:2-50:0.2-10:0.2-10.

[0007] Furthermore, the small molecule compound composition is used to be applied to the basal culture medium; the working concentrations of all-trans retinoic acid, salvia miltiorrhiza, CHIR-99021 and minoxidil are 0.2-5 µmol / L, 2-50 µmol / L, 0.2-10 µmol / L and 0.2-10 µmol / L, respectively.

[0008] Furthermore, the small molecule compound composition was added to the basal culture medium; the basal culture medium was DMEM / F-12 medium containing 0.542 mg / mL L-alanyl-L-glutamine dipeptide and 10-20% KnockOut serum substitute by volume; the small molecule compound composition consisted of 1 µmol / L all-trans retinoic acid, 10 µmol / L laryngin, 5 µmol / L CHIR-99021 and 1 µmol / L minoxidil.

[0009] Furthermore, the induced mesenchymal stem cells are prepared by the following method: induced pluripotent stem cells are induced to form brain organoids through induction culture, and the brain organoids are digested and cultured to obtain induced mesenchymal stem cells.

[0010] Furthermore, induced pluripotent stem cells were cultured in suspension for four stages to form brain organoids. During these four stages, the culture medium was supplemented with Dorsomorphin, A83-01, and polyvinyl alcohol in the first stage; SB-431542, CHIR-99021, and polyvinyl alcohol in the second stage; SB-431542, CHIR-99021, Martigel, and polyvinyl alcohol in the third stage; and B27 and insulin in the fourth stage.

[0011] After digestion, brain organoids were cultured in a medium containing B27 and β-FGF.

[0012] This technical solution also provides a method for promoting the differentiation of induced mesenchymal stem cells into dermal papillary cells. The method involves using a serum-free induction medium to induce and culture induced mesenchymal stem cells to obtain dermal papillary cells. The serum-free induction medium is a basic culture medium supplemented with a small molecule compound composition. The small molecule compound composition includes all-trans retinoic acid, trichomoniasis, CHIR-99021, and minoxidil.

[0013] Furthermore, the fusion rate of induced mesenchymal stem cells is 80-100%, and the induction culture time is 6-10 days.

[0014] Furthermore, the basal culture medium was DMEM / F-12 medium containing 0.542 mg / mL L-alanyl-L-glutamine dipeptide, and KnockOut serum substitute was added at a volume percentage of 10%-20%; the working concentrations of all-trans retinoic acid, saliva, CHIR-99021 and minoxidil were 0.2-5 µmol / L, 2-50 µmol / L, 0.2-10 µmol / L and 0.2-10 µmol / L, respectively.

[0015] Furthermore, the obtained dermal papillary cells were digested and then resuspended in DP cell maintenance culture medium to obtain a dermal papillary cell suspension; the dermal papillary cell suspension was then cultured and aggregated to obtain dermal papillary cell spheres.

[0016] This technical solution also provides the application of a small molecule compound composition in promoting the differentiation of induced mesenchymal stem cells into dermal papillary cells. The induced mesenchymal stem cells are prepared by the following method: induced pluripotent stem cells are induced to form brain organoids through induction culture, and the brain organoids are digested and cultured to obtain induced mesenchymal stem cells.

[0017] Induced pluripotent stem cells were cultured in suspension for four stages to form brain organoids. During the four stages of suspension culture, the culture medium was supplemented with Dorsomorphin, A83-01, and polyvinyl alcohol in the first stage; SB-431542, CHIR-99021, and polyvinyl alcohol in the second stage; SB-431542, CHIR-99021, Martigel, and polyvinyl alcohol in the third stage; and B27 and insulin in the fourth stage.

[0018] After digestion, brain organoids were cultured in a medium containing B27 and β-FGF in an adherent culture.

[0019] The small molecule compound composition consists of all-trans retinoic acid, saliva, CHIR-99021, and minoxidil in a molar ratio of 0.2-5:2-50:0.2-10:0.2-10.

[0020] In summary, the principle of this technical solution is as follows:

[0021] In this technical solution, a combination of multiple small molecule compounds is used to promote the differentiation and functional enhancement of papillary dermal cells (DP cells). All-trans retinoic acid can activate retinoic acid receptors, thereby promoting the transformation of mesenchymal cells into DP cells; CHIR-99021 can activate the Wnt / β-catenin signaling pathway to promote DP differentiation and activate DP function; the cellular adenylate cyclase activator, trichomoniasis, is used to promote the differentiation of fibroblasts into DP cells. Minoxidil can promote DP cell proliferation and simultaneously activate the ERK and AKT pathways, thereby inhibiting DP cell apoptosis. These four molecules work synergistically to promote the transformation of mesenchymal stem cells into papillary dermal cells.

[0022] The small molecule compound combination in this protocol was obtained through extensive screening and experimentation, and it can effectively promote cell differentiation into DP cells. Compared with cytokines, small molecule compounds have several advantages: stable physicochemical properties, easier transportation, storage, and quality control during use; lower cost for large-scale production; no heterologous proteins in the produced cell products; easier purification; and improved safety for clinical use. The small molecule compound combination includes all-trans retinoic acid, trichoderma, and CHIR-99021 to promote differentiation, and minoxidil to promote proliferation and prevent apoptosis.

[0023] The combination of small molecule compounds used in this protocol effectively promotes the differentiation of induced mesenchymal stem cells (iMSCs) obtained through this protocol, but its effect on other mesenchymal stem cells is not significant. In promoting differentiation while simultaneously preventing apoptosis, CHIR-99021, at a concentration of 5 µmol / L, effectively ensures both cell yield and cell positivity rate. The degree of cell confluence before differentiation induction significantly affects the cell yield (but not the cell positivity rate); using 100% cell confluence yields better results. In selecting proliferative agents to be used in conjunction with differentiation-promoting components, the inventors tried numerous candidate reagents and found that minoxidil was significantly more effective than others. Attached Figure Description

[0024] Figure 1 The diagram shows the dermal papillary cell induction culture process of Example 1, along with typical microscopic images at different stages (A is a schematic diagram of the induction culture process; B is a microscopic image of cells obtained at different culture stages).

[0025] Figure 2 The results of alkaline phosphatase activity assays in dermal papillary cells from Example 2 are as follows: (A: Induced pluripotent stem cells (iPSC); B: Induced mesenchymal stem cells (iMSC); C: Dermal papillary cells induced from induced mesenchymal stem cells (iMSC-iDPC); D: Umbilical cord-derived mesenchymal stem cells (UCMSC); E: Dermal papillary cells induced from umbilical cord-derived mesenchymal stem cells (UCMSC-iDPC)).

[0026] Figure 3 Cell immunofluorescence identification of dermal papillary cells in Example 3 (A: α-SMA; B: α-SMA / DAPI; C: Versican; D: Versican / DAPI).

[0027] Figure 4 Microscopic images showing the screening effect of small molecule compound combinations in Example 4 (A: cell induction effect of high concentrations of the three molecules (day 3); B: cell induction effect of low concentrations of the three molecules (day 8); B-1: control group; B-2: low concentration all-trans retinoic acid group; B-3: low concentration of saliva extract group; B-4: low concentration of CHIR-99021 group).

[0028] Figure 5 Microscopic images showing the screening effect of small molecule compound combinations in Example 4 (screening for CHIR-99021 concentration).

[0029] Figure 6 Microscopic images showing the screening effect of induced initial confluence in Example 5 (A: Cell microscopic images at 80% and 100% initial confluence; B: Alkaline phosphatase activity detection results at 80% and 100% initial confluence).

[0030] Figure 7 Immunofluorescence images showing the screening effect of induced initial confluence in Example 5 (A: α-SMA; B: α-SMA / DAPI; C: Versican; D: Versican / DAPI).

[0031] Figure 8 Microscopic images and alkaline phosphatase detection results of the screening effect of different small molecule compounds in Example 6.

[0032] Figure 9 Immunofluorescence images of the screening effects of different small molecule compounds in Example 6 (A: α-SMA; B: α-SMA / DAPI; C: Versican; D: Versican / DAPI). Detailed Implementation

[0033] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0034] Example 1: Dermal papillary (DP) cell induction and culture process

[0035] Before induction begins, the induced mesenchymal stem cells (iMSCs, specifically human induced mesenchymal stem cells in this protocol) or umbilical cord-derived mesenchymal stem cells (UCMSCs) are cultured and maintained in a 37°C, 5% CO2 incubator. Using conventional methods, the appropriate amount of mesenchymal stem cell culture medium (Nuwacell® ncMission hMSCMedium) is replaced every 3 days to expand the cells until the cell confluence reaches 80%-100% (preferably 100% in this embodiment). Then, the induction culture of dermal papilla (DP) cells begins (the obtained DP cells are called iDPCs, and the induced dermal papilla cells are shown in the process diagram). Figure 1 The specific process is described below (for the convenience of describing Example 1, iMSC and UCMSC are collectively referred to as MSC).

[0036] The method for preparing induced mesenchymal stem cells (iMSCs) is detailed in patent CN113025569B. In short: iMSCs are cultured in suspension using brain organoid stage 1 culture medium; the culture medium is sequentially replaced with different stages of brain organoid culture medium at different time points; the cells are transferred to a miniature organoid bioreactor, cultured for 3-4 days, and then seeded into gelatin-coated culture containers for adherent culture using MSC stage 1 culture medium to obtain induced mesenchymal stem cells. For specific details, please refer to Example 1 of the patent (CN113025569B). Furthermore, the iMSCs in this method are obtained by inducing the initial cells, iPSCs. A more detailed description of the above process is as follows:

[0037] (1) Induced pluripotent stem cells were cultured in suspension using brain organoid stage 1 culture medium;

[0038] (2) Induced pluripotent stem cells were cultured in suspension using brain organoid stage 2 medium, brain organoid stage 3 medium and brain organoid stage 4 medium in sequence;

[0039] (3) The cells cultured in (2) were transferred into a micro organoid bioreactor, and then seeded into a gelatin-coated culture container. Mesenchymal stem cells were obtained by adhering to the culture using N-MSCs first-stage culture medium.

[0040] The brain organoid stage 1 culture medium includes: GMEM basal medium, NEAA 1%-2%, Glumax 1%-2%, β-mercaptoethanol 80-120 nM, KnockOut serum substitute 12-18%, Dorsomorphin 0.8-1.5 μM, A83-01 0.8-1.2 μM, and polyvinyl alcohol 0.5-1.5%.

[0041] The brain organoid stage 2 culture medium includes: GMEM basal medium, NEAA 1%-2%, Glumax 1%-2%, β-mercaptoethanol 80-120 nM, N2 1%-2%, SB-431542 0.8-1.2 μM, CHIR-99021 0.8-1.2 μM, and polyvinyl alcohol 0.8-1.5%.

[0042] The brain organoid three-stage culture medium includes: MEM basal medium, NEAA 1%-2%, Glumax 1%-2%, β-mercaptoethanol 80-120 nM, N2 1%-2%, SB-431542 0.8-1.2 μM, CHIR99021 0.8-1.2 μM, Martigel 0.8-1.5%, and polyvinyl alcohol 0.8-1.5%.

[0043] The brain organoid four-stage culture medium includes: GMEM basal medium, NEAA 1%-2%, Glumax 1%-2%, β-mercaptoethanol 80-120nM, N2 1%-2%, B27 2%-3%, and insulin 2μg / mL;

[0044] The first-stage culture medium for N-MSCs includes:

[0045] GMEM basal medium, NEAA 1-2%, Glumax 1%-2%, β-mercaptoethanol 100nM, N2 1%-2%, B27 2%-3%, β-FGF 5-6ng / ml.

[0046] It should also be noted that iMSCs are obtained through the induction and culture of induced pluripotent stem cells (iPSCs). For example, iPSCs can be induced using reagents from the Erythroid Progenitor Reprogramming Kit (product number 05924) from STEMCELL. Using this kit and following the instructions, erythroid progenitor cells obtained through conventional methods are induced to produce stable human induced pluripotent stem cells derived from erythroid progenitor cells. The method for obtaining iPSCs described above is also described in the applicant's prior patent CN113025569B and represents a conventional method in the prior art.

[0047] Day 0: Microscopic observation confirmed MSC growth and cell confluence (100%). The culture medium was removed from the cells, and 0.1 mL / 1 cm⁻¹ water was used to treat the cells. 2 Wash with DPBS to remove dead cells and residual culture medium. Add 0.2 mL / cm³ of DPBS. 2Serum-free induction medium for promoting MSC differentiation into DP cells was prepared by spreading the medium evenly in culture dishes and incubating for 24 hours. The serum-free induction medium consisted of a base component and additives. The base component (basal medium) was DMEM / F-12 medium containing 0.542 mg / mL L-alanyl-L-glutamine dipeptide. Additives included 10%-20% KnockOut serum substitute and other supplementary factors. The added factors include: 0.2-5 µmol / L all-trans retinoic acid (ATRA, CAS No.: 302-79-4; manufacturer: Taoshu Biotechnology; catalog number: T1051), 2-50 µmol / L forskolin (CAS No.: 66575-29-9; manufacturer: Taoshu Biotechnology; catalog number: T2939), 0.2-10 µmol / L CHIR-99021 (CAS No.: 252917-06-9; manufacturer: Taoshu Biotechnology; catalog number: T2310), and 0.2-5 µmol / L minoxidil (CAS No.: 38304-91-5; manufacturer: Taoshu Biotechnology; catalog number: T0451). In the specific experimental process of this embodiment, the composition of the serum-free induction medium used was as follows: DMEM / F-12 basal medium containing 0.542 mg / mL L-alanyl-L-glutamine dipeptide, supplemented with 15% KnockOut serum substitute, 1 µmol / L all-trans retinoic acid, 10 µmol / L salamiin, 5 µmol / L CHIR-99021 and 1 µmol / L minoxidil.

[0048] Day 1-8: Cells were cultured for another 8 days in serum-free induction medium to promote MSC differentiation into DP cells. Microscopic observation was used to confirm iDPC growth and cell confluence. Cells were cultured at a concentration of 0.2 mL / 1 cm². 2 Change the culture medium daily.

[0049] By day 8 of culture, the iMSC-iDPC cells (dermal papillary cells induced by induced mesenchymal stem cells) and UCMSC-iDPC cells (dermal papillary cells induced by umbilical cord-derived mesenchymal stem cells) transformed into polygonal sheets, with cells tightly connected and aggregated into a monolayer structure. The nuclei of iMSC-iDPC cells were more prominent, while the nuclei of UCMSC-iDPC cells were not prominent.

[0050] Day 8: Microscopic observation to confirm cell state and cell density, removal of culture medium from cells, and treatment with 0.1 mL / 1 cm⁻¹ 2 Wash twice with DPBS to remove dead cells. Wash at 37°C with 0.1 mL / 1 cm⁻¹ water. 2Cells were treated with tryple for 3 minutes, then digestion was stopped with DP cell maintenance medium. Cells were collected by centrifugation at 950 rpm for 5 minutes, and the supernatant was removed to eliminate digestion fluid. Cells were resuspended in DP cell maintenance medium (DMEM / F-12 basal medium + 15% KOSR + 1% insulin-transferrin-selenium supplement). Cells were collected in centrifuge tubes and gently pipetted until single-celled (a portion was observed under a microscope). Cells were counted and diluted to 2 × 10⁶ cells with DP cell maintenance medium. 5 50 μL / well of cell suspension was seeded into a U-bottom ultra-low adsorption 96-well plate and incubated for 24 h.

[0051] Day 9: Microscopic observation was used to confirm the aggregation of DP cells, the state of DP spheres, and the size of DP spheres in the 96-well plate. 50 μL of solution was added to each well. DP cells have a strong self-aggregation ability. The cells obtained by induction will self-aggregate into spheres (forming DPLTs, dermal papillary cell spheres) within 6-24 hours.

[0052] Example 2: Detection of alkaline phosphatase activity in dermal papillary cells

[0053] The iMSC-iDPC cells and UCMSC-iDPC cells obtained in Example 1 were analyzed. Alkaline phosphatase (AKP) activity has been used as a biomarker for detecting the presence of hair follicles (DPs) and is considered an indicator of DP cell hair induction. Research by Japanese scientist Takashi Matsuzaki showed the dynamic changes in AKP activity in DP cells. AKP activity in DP cells reaches its highest level in the early growth phase and declines after the middle growth phase. The temporal and spatial variations of AKP activity are consistent with the hair induction characteristics of DP cells. Furthermore, research by Korean scientist Mi Hee Kwack showed that overexpression of alkaline phosphatase in cultured human DP cells improved the hair induction ability of cultured human DP cells.

[0054] Following the instructions for the AKP detection kit, AKP activity was measured in iPSCs, iMSCs, DP cells derived from iMSCs (iMSC-iDPC), UCMSCs, and DP cells derived from UCSMCs (UCMSC-iDPC). The results are as follows: Figure 2As shown, iPSCs, iMSC-iDPCs, and UCMSC-iDPCs all exhibited AKP activity (AKP-positive cell rates of 100%, 89%, and 24%, respectively), while iMSCs and UCMSCs showed poorer AKP activity (positive rates of 13% and 2%, respectively). This demonstrates that the combination of small molecule compounds effectively promotes MSC differentiation into functional DP cells. The experimental results also show that iMSCs and UCMSCs responded differently to the serum-free induction medium used in this regimen for promoting MSC differentiation into DP cells. The AKP positivity rate of iMSCs and UCMSCs differed by only 11%. However, after induction with the same inducing factors, the difference in positivity rate between iMSC-iDPCs and UCMSC-iDPCs reached 65%, indicating that the medium containing four factors in this regimen had a significantly stronger effect on iMSCs than the others, which was unexpected by the inventors before the experiment.

[0055] Example 3: Immunofluorescence detection of dermal papillary cells

[0056] The iMSC-iDPC cells and UCMSC-iDPC cells obtained in Example 1 were analyzed. α-Smooth muscle actin (α-SMA) expression was found in the lower and middle dermal sheath of rat and human hair follicles, but not in DP cells. However, DP cells became α-SMA positive in culture. Therefore, α-SMA is a marker for dermal sheath cells in vivo, as well as for DP cells and dermal sheath cells in vitro. Furthermore, in human hair follicles, pluripotent proteoglycan (Versican) is expressed in DP cells during the anagen phase. Weak Versican expression was also observed in the dermal sheath outside the follicular protuberance. In mice, Versican is expressed in anagen-phase hair follicles but not in telogen-phase follicles. Therefore, Versican may play an important role in the induction and maintenance of early hair growth. Ascorbic acid-2-phosphate can induce Versican expression in human DP cells, thereby enhancing hair follicle initiation and growth. Japanese scientists Kishimoto and colleagues used GFP, driven by a Versican promoter, as a method for enriching DP cells by flow cytometry. These GFP-positive cells exhibited behavior and morphology consistent with DP cells. When bound to epidermal cells, they induced hair regrowth in implantation assays, while GFP-negative cells did not induce hair follicle formation.

[0057] Immunofluorescence was performed on induced DP cells to detect the expression of α-SMA and Versican. The results are as follows: Figure 3As shown, Versican was expressed in all iDP cells, and the expression level was increased compared to before induction. α-SMA was expressed in most iMSC-iDPC cells (α-SMA positive cell rate 81%), but only a small number of cells in UCMSC-iDPC expressed α-SMA (α-SMA positive cell rate 22%); the α-SMA positive cell rate was 0% in both iMSC and UCMSC. These results indicate that this culture medium is more effective in promoting iMSC differentiation into DP cells than in promoting UCMSC differentiation, and iMSCs show a stronger response to the culture medium in this protocol, making this medium more suitable for iMSCs. Furthermore, the Versican positive cell rate was 0% in iMSC-iDPC, UCMSC-iDPC, iMSC, and UCMSC.

[0058] Example 4: Determination of CHIR-99021 concentration (cell confluence rate of 80%)

[0059] (1) Experiment 1:

[0060] Three small molecule compounds that promote DP differentiation were added to DP culture medium for iMSC differentiation: the retinoic acid receptor agonist trans-retinoic acid (all-trans-retinoic acid), the Wnt / β-catenin signaling pathway inhibitor (CHIR-99021), and the adenylate cyclase activator (trichosamine). Experimental results showed that this combination of small molecules promoted apoptosis during iMSC differentiation. Figure 4 As shown in Figure A (the basal medium was DMEM / F12 containing 0.542 mg / mL L-alanyl-L-glutamine dipeptide, supplemented with 15% KOSR, 1 µmol / L all-trans retinoic acid, 10 µmol / L salamiin, and 10 µmol / L CHIR-99021). Due to apoptosis and other factors, the number of DP cells harvested per 35 mm culture dish was limited to 5.57 × 10⁻⁶ cells. 4 (Day 3)

[0061] The concentrations of each small molecule compound were reduced to identify the main components that promote apoptosis.

[0062] Experimental Groups: The experiment was divided into 4 groups: control group (1 µmol / L all-trans retinoic acid, 10 µmol / L forskolin, 10 µmol / L CHIR-99021), low-concentration all-trans retinoic acid group (low-concentration ATRA group, 0.5 µmol / L all-trans retinoic acid, 10 µmol / L forskolin, 10 µmol / L CHIR-99021), low-concentration forskolin group (low-concentration forskolin group, 1 µmol / L all-trans retinoic acid, 1 µmol / L forskolin, 10 µmol / L CHIR-99021), and low-concentration CHIR-99021 group (1 µmol / L all-trans retinoic acid, 10 µmol / L forskolin, 1 µmol / L CHIR-99021). The basal culture medium was DMEM / F12 containing 0.542 mg / mL L-alanyl-L-glutamine dipeptide, with 15% KOSR (KnockOut serum substitute, Gibco™) added, followed by the addition of the aforementioned small molecule compounds.

[0063] Experimental methods: Induced mesenchymal stem cells (iMSCs) were uniformly grouped and induced according to the method in Example 1. During the induction process, the corresponding culture medium for each group was used instead of the serum-free induction culture medium in Example 1.

[0064] Experimental results are as follows Figure 4 As shown in Figure B, apoptosis was assessed on day 8 of culture. The control group yielded 9.11 × 10⁻⁶ DP cells per 35 mm culture dish. 4 The low-concentration all-trans retinoic acid group yielded 1.03 × 10⁻⁶ DP cells per 35 mm culture dish. 5 The low-concentration trichosin group yielded 6.03 × 10⁶ DP cells per 35 mm culture dish. 4 The low-concentration CHIR-99021 group yielded 3.11 × 10⁻⁶ DP cells per 35 mm culture dish. 5 Decreasing the concentrations of all-trans retinoic acid and trichodin had a certain promoting effect on apoptosis of DP cells induced by iMSCs. However, lowering the concentration of CHIR-99021 significantly reduced the number of apoptotic cells, resulting in nearly a three-fold increase in the number of harvested DP cells compared to the control group. These results indicate that reducing the concentration of CHIR-99021 significantly reduces apoptosis during iMSC differentiation, thus concluding that high concentrations of CHIR-99021 induce apoptosis during iMSC-to-DP induction. The working concentrations of the three factors used in this technique effectively increased the yield of DP cells.

[0065] (2) Experiment 2:

[0066] A concentration gradient experiment with CHIR-99021 was set up to screen for a suitable concentration of CHIR-99021 for inducing iMSC-iDP.

[0067] Experimental groups: The experiment was divided into 4 groups: 1 µmol / L CHIR-99021 group, 2 µmol / L CHIR-99021 group, 5 µmol / L CHIR-99021 group, and 10 µmol / L CHIR-99021 group. The addition of other substances was the same as that of the low concentration CHIR-99021 group in Experiment 1.

[0068] Experimental methods:

[0069] The induced mesenchymal stem cells (iMSCs) were uniformly grouped and induced according to the method described in Example 1. During the induction process, the corresponding culture medium for each group was used instead of the serum-free induction medium used in Example 1. After 8 days of induction, the AKP activity of the induced cells was detected according to the method described in Example 2.

[0070] Experimental results are as follows Figure 5 As shown, the results indicate that as the concentration of CHIR99021 increases, the proportion of induced apoptosis increases, but AKP activity and the proportion of positive cells also increase. 5 µmol / L CHIR-99021 is preferred for promoting iMSC differentiation into iDPCs. Using working concentrations of CHIR-99021 of 1 µmol / L, 2 µmol / L, 5 µmol / L, and 10 µmol / L to induce iMSC differentiation, the AKP-positive cell rates obtained were 10%, 39%, 58%, and 73%, respectively. The AKP-positive cell rate was calculated as: (number of cells with alkaline phosphatase activity / total number of cells) × 100%. The increasing working concentration of CHIR-99021 showed an opposite trend in the apoptosis rate and the AKP-positive cell rate, which is a novel discovery by the inventors regarding the specific process of iMSC-induced differentiation into iDPCs. Based on these findings, a working concentration of 5 µmol / L CHIR-99021 is most suitable for controlling the quality and yield of iMSC-iDPCs.

[0071] Therefore, the optimal combination of small molecule compounds for promoting the differentiation of iMSCs into iMSC-iDPCs is "1 µmol / L all-trans retinoic acid + 10 µmol / L trichodin + 5 µmol / L CHIR-99021". After 8 days of induction, the number of DP cells harvested per 35 mm culture dish is 1.45 × 10⁻⁶. 5The number of DP cells exceeded 58%, and the AKP-positive cell rate reached 58%. Based on this, how to further ensure DP cell yield while simultaneously increasing the AKP-positive cell rate is a problem that needs to be addressed in subsequent studies. The initial culture target in this protocol is iMSC, which, unlike other cell types, cannot be adequately addressed by simply applying the aforementioned small molecule compounds that promote differentiation, as it is difficult to simultaneously ensure DP cell yield and iMSC-iDPC content (AKP-positive cell rate).

[0072] Example 5: Study on the effect of different initial confluence degrees on the differentiation of iMSCs into iDPCs

[0073] Experimental grouping: The experiment was divided into two groups: the 80% confluence group and the 100% confluence group.

[0074] Experimental methods: The optimal induction medium of Example 4 was prepared (differentiation promoting factors: 1 µmol / L all-trans retinoic acid, 10 µmol / L salvia miltiorrhiza, 5 µmol / L CHIR-99021; minoxidil was not added compared to Example 1); the induced mesenchymal stem cells (iMSCs) were uniformly divided into groups, and induction was started when the confluence reached 80% or 100%, respectively, and induction was carried out in the manner of Example 1. After 8 days of induction, the AKP activity of the induced cells was detected in Example 2, and the immunofluorescence detection of the induced cells was performed in Example 3.

[0075] Experimental results are as follows Figure 6 and Figure 7 As shown, the results indicate that after 8 days of induction, the cell count at 100% confluence was 3.62 × 10⁻⁶. 5 The confluence of each 35mm dish is significantly greater than that of the 80% confluence group (1.46 × 10⁻⁶). 5 (per 35mm dish). The initial cell count at 100% confluence was approximately 1.25:1 compared to the initial cell count at 80% confluence. After 8 days of culture, the cell count at 100% confluence was approximately 2.5:1 compared to the initial cell count at 80% confluence. The increase in cell count ratio from 1.25:1 to 2.5:1 was not solely due to the initial cell number advantage of the 100% confluence group. The inventors explained that the cell-cell contact in the culture environment of this protocol leads to cell signaling, thereby inhibiting apoptosis. Therefore, the 100% confluence group yielded more induced cells, and the difference in cell count after 8 days of culture was not simply due to the difference in initial cell count.

[0076] In addition, the expression levels of α-SMA and Versican were not significantly correlated with cell confluence. AKP activity was slightly increased in the 100% confluence group, indicating superior confluence. Therefore, 100% confluence of iMSCs was preferred as the initial confluence level for induction. More specifically, for iMSC-iDPC: the AKP-positive cell rate was 55.6% in the initial 80% confluence group and 57.1% in the 100% confluence group; the α-SMA-positive cell rate was 70% in the initial 80% confluence group and 70% in the 100% confluence group; the Versican-positive cell rate was 100% in both the initial 80% confluence group and the 100% confluence group. The α-SMA-positive cell rate was calculated as: (α-SMA-positive cells / total cells) × 100%; the Versican-positive cell rate was calculated as: (Versican-positive cells / total cells) × 100%.

[0077] Example 6: Screening for small molecule compounds that promote iMSC differentiation into DP cells.

[0078] Experimental groups: The experiment was divided into 4 groups, namely the control group, minoxidil group, dexamethasone group, and Z-VAD-FMK group.

[0079] Experimental methods:

[0080] The most preferred induction medium for Example 4 was the control medium (differentiation promoting factors: 1 µmol / L all-trans retinoic acid, 10 µmol / L salvia miltiorrhiza, 5 µmol / L CHIR-99021). In addition, the minoxidil group medium was the control medium with 1 µmol / L minoxidil added, the dexamethasone group medium was the control medium with 1 µmol / L dexamethasone added, and the Z-VAD-FMK group medium was the control medium with 5 µmol / L Z-VAD-FMK (cysteine ​​protease inhibitor) added.

[0081] The induced mesenchymal stem cells (iMSCs) used were uniformly grouped, and induction was started when the confluence reached 100%. Induction was carried out according to the method of Example 1. After 8 days of induction, the AKP activity of the induced cells was detected according to Example 2, and the immunofluorescence detection of the induced cells was carried out according to Example 3.

[0082] Experimental results are as follows Figure 8 and 9As shown, the experimental results indicate that the minoxidil group had higher AKP activity and a higher proportion of positive cells than the control group, and the expression levels of Versican and α-SMA were also higher than those of the control group. However, the dexamethasone group and the Z-VAD-FMK group did not show a significant increase in AKP activity, the proportion of positive cells, and the expression levels of Versican and α-SMA compared to the control group. Therefore, minoxidil is the preferred choice for promoting the differentiation of iMSCs into iDPCs.

[0083] More specifically: In the control group, the AKP-positive cell rate was 55.3%, the α-SMA-positive cell rate was 66.7%, and the Versican-positive cell rate was 100%; in the minoxidil group, the AKP-positive cell rate was 87.8%, the α-SMA-positive cell rate was 82.5%, and the Versican-positive cell rate was 100%; in the dexamethasone group, the AKP-positive cell rate was 53.5%, the α-SMA-positive cell rate was 82.5%, and the Versican-positive cell rate was 100%; and in the Z-VAD-FMK group, the AKP-positive cell rate was 40.5%, the α-SMA-positive cell rate was 54.5%, and the Versican-positive cell rate was 100%. Minoxidil, dexamethasone, and Z-VAD-FMK all promote cell proliferation and inhibit apoptosis. However, in the specific target group (iMSCs) and application environment of this regimen (when used in combination with other differentiation-promoting cytokines), minoxidil showed superior efficacy compared to the others.

[0084] In addition, the number of DP cells harvested per 35 mm culture dish in the minoxidil group was 3.4 × 10⁻⁶. 5 More than one, which indicates that the use of minoxidil can effectively ensure both the yield of DP cells and the proportion of active iMSC-iDPC.

[0085] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A small molecule compound composition that promotes the differentiation of induced mesenchymal stem cells into dermal papillary cells, characterized in that: It consists of all-trans retinoic acid, saliva, CHIR-99021, and minoxidil at working concentrations of 0.2-5µmol / L, 2-50µmol / L, 5µmol / L, and 0.2-10µmol / L, respectively.

2. The small molecule compound composition for promoting the differentiation of induced mesenchymal stem cells into dermal papillary cells according to claim 1, characterized in that: The small molecule compound composition consists of 1 µmol / L all-trans retinoic acid, 10 µmol / L saliva extract, 5 µmol / L CHIR-99021 and 1 µmol / L minoxidil.

3. A method for promoting the differentiation of induced mesenchymal stem cells into dermal papillary cells, characterized in that, Induced mesenchymal stem cells were cultured using serum-free induction medium to obtain dermal papillary cells; the serum-free induction medium was a basic culture medium supplemented with a small molecule compound composition. The small molecule compound composition consists of all-trans retinoic acid, pilosula, CHIR-99021 and minoxidil; The working concentrations of all-trans retinoic acid, saliva, CHIR-99021 and minoxidil are 0.2-5 µmol / L, 2-50 µmol / L, 5 µmol / L and 0.2-10 µmol / L, respectively.

4. The method for promoting the differentiation of induced mesenchymal stem cells into dermal papillary cells according to claim 3, characterized in that: The fusion rate of induced mesenchymal stem cells is 80-100%, and the induction culture time is 6-10 days.

5. The method for promoting the differentiation of induced mesenchymal stem cells into dermal papillary cells according to claim 4, characterized in that: The basal medium was DMEM / F-12 medium containing 0.542 mg / mL L-alanyl-L-glutamine dipeptide, with 10%-20% KnockOut serum substitute added by volume.

6. The method for promoting the differentiation of induced mesenchymal stem cells into dermal papillary cells according to claim 5, characterized in that: After digestion, the obtained dermal papillary cells were resuspended in DP cell maintenance culture medium to obtain a dermal papillary cell suspension; the dermal papillary cell suspension was then cultured and aggregated to obtain dermal papillary cell spheres.

7. The application of a small molecule compound composition in promoting the differentiation of induced mesenchymal stem cells into dermal papillary cells, characterized in that, The induced mesenchymal stem cells were prepared by the following method: (1) Induced pluripotent stem cells were cultured in suspension using brain organoid stage 1 culture medium; (2) Induced pluripotent stem cells were cultured in suspension using brain organoid stage 2 medium, brain organoid stage 3 medium and brain organoid stage 4 medium in sequence; (3) The cells cultured in (2) were transferred into a micro organoid bioreactor, and after culture, they were seeded into a gelatin-coated culture container and cultured in the first stage of N-MSCs medium to obtain mesenchymal stem cells. The brain organoid stage 1 culture medium includes: GMEM basal medium, NEAA 1%-2%, Glumax 1%-2%, β-mercaptoethanol 80-120 nM, KnockOut serum substitute 12-18%, Dorsomorphin 0.8-1.5 μM, A83-01 0.8-1.2 μM, and polyvinyl alcohol 0.5-1.5%. The brain organoid stage 2 culture medium includes: GMEM basal medium, NEAA 1%-2%, Glumax 1%-2%, β-mercaptoethanol 80-120 nM, N2 1%-2%, SB-431542 0.8-1.2 μM, CHIR-99021 0.8-1.2 μM, and polyvinyl alcohol 0.8 μM. 1.5%; The brain organoid three-stage culture medium includes: MEM basal medium, NEAA 1%-2%, Glumax 1%-2%, β-mercaptoethanol 80-120 nM, N2 1%-2%, SB-431542 0.8-1.2 μM, CHIR99021 0.8-1.2 μM, Martigel 0.8-1.5%, and polyvinyl alcohol 0.8-1.5%. The brain organoid four-stage culture medium includes: GMEM basal medium, NEAA 1%-2%, Glumax 1%-2%, β-mercaptoethanol 80-120nM, N2 1%-2%, B27 2%-3%, and insulin 2μg / mL; The first-stage culture medium for N-MSCs includes: GMEM basal medium, NEAA 1-2%, Glumax 1%-2%, β-mercaptoethanol 100nM, N2 1%-2%, B27 2%-3%, β-FGF 5-6ng / ml; The small molecule compound composition consists of all-trans retinoic acid, pilosula, CHIR-99021 and minoxidil; The small molecule compound composition is used for application to the basal culture medium; the working concentrations of all-trans retinoic acid, saliva, CHIR-99021 and minoxidil are 0.2-5 µmol / L, 2-50 µmol / L, 5 µmol / L and 0.2-10 µmol / L, respectively.

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