Compositions for the differentiation of mesenchymal stem cells into keratinocytes and their applications

Abstract

This invention discloses a composition for the differentiation of mesenchymal stem cells (ADSCs) into keratinocytes and its application. The composition comprises: a Wnt signaling pathway activator, a TGF-β signaling pathway inhibitor, a bone morphogenetic protein pathway activator, and at least one of retinoic acid and a retinoic acid agonist. The small molecule composition provided by this invention is meticulously designed to gradually guide the fate transition of ADSCs by mimicking key signaling events during embryonic development. This strategy is not a simple compound screening, but a rational design based on a deep understanding of developmental biology, achieving precise regulation of cell lineages in vitro by reproducing the natural developmental process. The composition of this invention is not a simple superposition of components, but rather achieves cell fate transition by precisely regulating the balance of signaling pathway networks.

Description

Technical Field

[0001] This invention relates to the fields of cell biology, regenerative medicine and tissue engineering, and particularly to a composition for the differentiation of mesenchymal stem cells into keratinocytes and its application. Background Technology

[0002] 1. Clinical and research needs of keratinocytes Keratinocytes are the main cell type of the epidermis and play a vital role in forming the skin's physical barrier, preventing moisture loss, and resisting external environmental damage. Therefore, in clinical practice, obtaining a large number of functional keratinocytes is crucial for the treatment of large-area burns, chronic wounds (such as diabetic foot ulcers), and hereditary skin diseases (such as bullous epidermolysis). In addition, in vitro cultured keratinocytes and the three-dimensional (3D) skin models and skin organoids they construct are indispensable tools in dermatological research, drug screening, cosmetic efficacy and safety assessment, and toxicological testing [1].

[0003] 2. Current limitations of cell sources Despite the huge demand, existing methods for obtaining keratinocytes have significant limitations that hinder their widespread application. Currently, keratinocytes used in clinical and research settings are mainly derived from human skin biopsy tissue. This method faces many challenges: first, the acquisition process is invasive and causes pain to the donor; second, the source of donors is limited, and there are significant biological differences between cells from different donors; finally, during the in vitro expansion of primary keratinocytes, their stem cell potential is gradually lost, eventually leading to cell differentiation and senescence, making it difficult to obtain a sufficient number of high-quality cells [2].

[0004] To overcome the limitations of primary cells, researchers have developed methods to differentiate keratinocytes from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs). While these methods have shown great potential, they still face serious challenges in clinical translation. For hESCs, their application involves complex ethical issues. For hiPSCs, although ethical controversies are avoided, there is a risk of teratoma formation during differentiation, which is a major safety concern. In addition, the differentiation protocol from pluripotent stem cells to functional keratinocytes is usually multi-stage, long-cycle, and costly, which limits its large-scale application [3][4].

[0005] 3. Existing technologies for cell reprogramming to keratinocyte lineages Direct reprogramming, or transdifferentiation, aims to directly transform a differentiated somatic cell into another type of cell without going through a pluripotent stem cell stage. Early studies have shown that somatic cells (such as fibroblasts) can be directly reprogrammed into induced keratinocytes (iKSCs) by ectopically expressing specific lineage-determining transcription factors. Transcription factors such as p63 and KLF4 have been shown to be key drivers of this process. These studies have demonstrated in principle the feasibility of direct cross-lineage transformation [5][6][7].

[0006] 3.1 Deficiencies of Gene Reprogramming Methods Although transcription factor-mediated reprogramming has made a conceptual breakthrough, its technical means heavily rely on gene delivery vectors, mainly retroviruses or lentiviruses. These viral vectors pose a risk of randomly integrating foreign genes into the host cell genome, which may lead to insertional mutations, activation of proto-oncogenes (such as the commonly used reprogramming factor c-Myc), or triggering an immune response, thus bringing unpredictable long-term safety risks. These inherent safety issues make it difficult for gene modification-based reprogramming technologies to meet strict clinical application standards [8].

[0007] 3.2 Small molecule-mediated cell differentiation As a safer alternative, the use of small molecule compounds to regulate cell fate has attracted widespread attention. Small molecules have advantages such as non-integration, reversible action, controllable dosage, low cost, and ease of standardized production. Currently, studies have successfully induced mesenchymal stem cells (MSCs) into other cell lineages such as neurons and hepatocytes using combinations of small molecules. However, there is currently a lack of a scheme for efficiently and stably reprogramming MSCs into functional keratinocytes that is entirely based on small molecules with well-defined chemical compositions and does not depend on co-culture systems or culture medium additives with undefined components [9]

[10]

[11] .

[0008] In summary, a significant "safety-efficiency gap" exists in the field of regenerative dermatology. Transcription factor reprogramming demonstrates the feasibility of direct conversion, but its genetic risks hinder its clinical application. Differentiation from pluripotent stem cells, on the other hand, cannot bypass the risks of teratoma and complex procedures. This invention bridges this gap with a novel strategy: employing a safer tool—small molecule compounds—to achieve direct lineage conversion from one type of adult stem cell (adipose-derived mesenchymal stem cell) to another type of functional cell (keratinocyte). This method bypasses the pluripotent intermediate state with tumorigenic risks, providing a more direct, safer, and more efficient pathway to meet the urgent clinical and research needs for large quantities of functional keratinocytes.

[0009] References: [1] Fundamental in vitro 3Dhuman skin equivalent tool development forassessing biological safety and biocompatibility–towards alternative foranimal experiments.Ayesha Idrees, Inge Schmitz, Alice Zoso, Dierk Gruhn,Sandra Pacharra, Siegfried Shah, Gianluca Ciardelli,Richard Viebahn, ValeriaChiono, Jochen Salber. open 4 1 (2021) DOI: 10.1051 / fopen / 2021001 [2] Shahin,H.; Abdallah, S.; Das, J.; He, W.; El-Serafi, I.;Steinvall, I.; Sjöberg, F.; Elmasry, M.; El-Serafi, A.T. miRNome and ProteomeProfiling of HumanKeratinocytes and Adipose Derived Stem Cells ProposedmiRNA-Mediated Regulations of Epidermal Growth Factor and Interleukin 1-Alpha. Int. J.Mol. Sci. 2023, 24, 4956. https: / / doi.org / 10.3390 / ijms24054956 [3] Sah SK,Kanaujiya JK, Chen IP, Reichenberger EJ. Generation ofKeratinocytes from Human Induced Pluripotent Stem Cells Under Defined CultureConditions. CellReprogram. 2021 Feb;23(1):1-13. doi: 10.1089 / cell.2020.0046.Epub 2020 Dec 29. PMID: 33373529; PMCID: PMC8665820. [4] Pavathuparambil Abdul Manaph N, Sivanathan KN, Nitschke J, ZhouXF, Coates PT, Drogemuller CJ. An overview on small molecule-induceddifferentiation ofmesenchymal stem cells into beta cells for diabetictherapy. Stem Cell Res Ther. 2019 Sep 23;10(1):293. doi: 10.1186 / s13287-019-1396-5. PMID: 31547868; PMCID:PMC6757413. [5] Zhu F, NieG. Cell reprogramming: methods, mechanisms andapplications. Cell Regen. 2025 Mar 27;14(1):12. doi: 10.1186 / s13619-025-00229-x. PMID: 40140235; PMCID:PMC11947411. [6] Lin H, PanY, Cai S. Direct Lineage Reprogramming for InducedKeratinocyte Stem Cells: A Potential Approach for Skin Repair. Stem CellsTransl Med. 2023 May15;12(5):245-257. doi: 10.1093 / stcltm / szad019. PMID:37018467; PMCID: PMC10184702. [7] Chen Y,Mistry DS, Sen GL. Highly rapid and efficient conversionof human fibroblasts to keratinocyte-like cells. J Invest Dermatol. 2014 Feb;134(2):335-344. doi: 10.1038 / jid.2013.327.Epub 2013 Aug 6. PMID: 23921950;PMCID: PMC3875612. [8] Jiang H,Li G. Transcription factors direct epigeneticreprogramming at specific loci in human cancers. Front Genet. 2023 Oct 9;14:1234515. doi:10.3389 / fgene.2023.1234515. PMID: 37876590; PMCID: PMC10591108. [9] Yu Y,Huang H, Ye J, Li Y, Xie R, Zeng L, Huang Y, Zeng T, Luo D,Zhong J, Peng W. 3D Spheroids Facilitate Differentiation of Human Adipose-Derived Mesenchymal StemCells into Hepatocyte-Like Cells via p300-MediatedH3K56 Acetylation. StemCells Transl Med. 2024 Feb 14;13(2):151-165. doi:10.1093 / stcltm / szad076. PMID: 37936499; PMCID: PMC10872693.

[10] Pavathuparambil Abdul Manaph N, Sivanathan KN, Nitschke J, ZhouXF, Coates PT, Drogemuller CJ. An overview on small molecule-induceddifferentiation ofmesenchymal stem cells into beta cells for diabetictherapy. Stem Cell Res Ther. 2019 Sep 23;10(1):293. doi: 10.1186 / s13287-019-1396-5. PMID: 31547868;PMCID: PMC6757413.

[11] Willems E, Spiering S, Davidovics H, Lanier M, Xia Z, Dawson M,Cashman J, Mercola M. Small-molecule inhibitors of the Wnt pathway potentlypromotecardiomyocytes from human embryonic stem cell-derived mesoderm. CircRes. 2011 Aug 5;109(4):360-4. doi: 10.1161 / CIRCRESAHA.111.249540. Epub 2011Jul 7. PMID:21737789; PMCID: PMC3327303. Summary of the Invention

[0010] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a composition for the differentiation of mesenchymal stem cells into keratinocytes and its application. This invention provides a combination of small molecule compounds with clearly defined chemical compositions to guide the directed induction of mesenchymal stem cells, particularly adipose-derived mesenchymal stem cells (ADSCs), into keratinocytes (KCs) or keratinocyte-like cells.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a composition for the differentiation of mesenchymal stem cells into keratinocytes, the composition comprising: a histone deacetylase (HDAC) inhibitor, a Wnt signaling pathway activator, a TGF-β signaling pathway inhibitor, a bone morphogenetic protein (BMP) pathway activator, and at least one of retinoic acid (RA) and a retinoic acid agonist.

[0012] Preferably, the histone deacetylase inhibitor is VPA.

[0013] Preferably, the Wnt pathway activator is CHIR99021.

[0014] Preferably, the TGF-β signaling pathway inhibitor is SB431542.

[0015] Preferably, the bone morphogenetic protein (BMP) pathway activator is recombinant human BMP4.

[0016] Preferably, the retinoic acid agonist is all-trans retinoic acid.

[0017] Preferably, the composition for the differentiation of mesenchymal stem cells into keratinocytes further includes at least one of a cAMP signaling pathway activator and a ROCK inhibitor. Preferably, the cAMP signaling pathway activator is Forskolin and the ROCK inhibitor is Y-27632.

[0018] Preferably, the concentration range of the histone deacetylase inhibitor is 200 µM to 800 µM.

[0019] Preferably, the concentration range of the Wnt pathway activator is 0.1 µM to 15 µM; The concentration range of TGF-β signaling pathway inhibitors is 1 µM to 50 µM; The concentration range of bone morphogenetic protein (BMP) pathway activators is 2.5 ng / mL to 25 ng / mL; The concentration range of retinoic acid agonists is 1 µM to 10 µM; The concentration range of cAMP signaling pathway activators is 0.5-5 µM; The concentration range of ROCK inhibitors is 5 µM to 20 µM.

[0020] A second aspect of the invention also provides the use of the composition described above in the production of keratinocytes.

[0021] A third aspect of the present invention also provides a method for producing a population of keratinocyte-like cells, characterized in that the method comprises contacting a population of mesenchymal stem cells with the composition described above for a sufficient period of time to induce the expression of one or more keratinocyte markers and changes in cell morphology.

[0022] Preferably, the contacting step is carried out in multiple stages with different components.

[0023] A fourth aspect of the invention also provides keratinocyte-like cells produced by the above-described method, and the use of these cells in the preparation of engineered skin tissues for therapeutic or research purposes.

[0024] The beneficial effects of this invention are: The small molecule compositions provided by this invention are meticulously designed to gradually guide the fate transition of ADSCs by simulating key signaling events during embryonic development. This strategy is not a simple compound screening, but a rational design based on a deep understanding of developmental biology, achieving precise regulation of cell lineages in vitro by reproducing natural developmental processes. The compositions of this invention are not simply a superposition of components, but utilize synergistic crosstalk between signaling pathways to achieve their effects. For example, inhibiting TGF-β signaling not only directly promotes MET, but may also relieve its inhibitory effect on the Wnt pathway, thereby enhancing the epithelial-promoting effect of Wnt signaling. Simultaneous activation of the Wnt and BMP pathways simulates the synergistic effect of these two signals in embryonic development to determine cell fate. This composition achieves cell fate transition by precisely regulating the balance of signaling pathway networks.

[0025] This invention overcomes the shortcomings of existing technologies that involve the unavoidable risk of teratoma and complex procedures in the differentiation of pluripotent stem cells through a novel strategy. This invention employs a safer tool—small molecule compounds—to achieve direct lineage conversion from one type of adult stem cell (adipose-derived mesenchymal stem cell) to another type of functional cell (keratinocyte). This method bypasses the pluripotent intermediate state with tumorigenic risks and provides a more direct, safer, and more efficient pathway to meet the urgent clinical and research needs for a large number of functional keratinocytes. Attached Figure Description

[0026] Figure 1 The results of cell morphology changes during the reprogramming process in Example 2; Figure 2 The results are the qPCR detection results from Example 2; Figure 3 The results of WB protein detection in Example 2; Figure 4 The results of immunofluorescence staining in Example 2; Figure 5 The results of HE staining and thickness statistics of the epidermal model in Example 2; Figure 6 The results are statistical results of the sodium fluorescein permeation experiment in Example 2. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0028] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0030] 1. Terminology Definition For ease of understanding of this invention, the following terms are defined as follows: Mesenchymal stem cells (MSCs) and adipose-derived mesenchymal stem cells (ADSCs) refer to a type of adult stem cells with self-renewal capacity and multi-lineage differentiation potential. Their characteristics include: adherent growth under standard culture conditions; differentiation into osteoblasts, chondrocytes, and adipocytes; and expression of a specific combination of cell surface markers, typically positive for CD44, CD90, and CD105, while negative for hematopoietic lineage markers such as CD45 and CD34.

[0031] Keratinocytes and keratinocyte-like cells (KLCs) are cells with typical epithelial cell morphology (such as a "pebble" shape) and express specific molecular markers. Markers for basal keratinocytes include keratin 5 (K5), keratin 14 (K14), and the transcription factor p63. Markers for terminally differentiated keratinocytes include keratin 1 (K1), keratin 10 (K10), involucrin (IVL), and loricrin (LOR).

[0032] "Small molecule combination" refers to a composition containing two or more synthetic or naturally derived organic compounds, which typically have a molecular weight of less than 900 Daltons and are capable of modulating specific biological pathways.

[0033] 2. Mechanism of action of the composition The small molecule compositions provided by this invention are meticulously designed to gradually guide the fate transition of ADSCs by mimicking key signaling events during embryonic development. This strategy is not a simple compound screening, but a rational design based on a deep understanding of developmental biology, achieving precise regulation of cell lineages in vitro by reproducing the natural developmental process.

[0034] Core components and their mechanisms of action Histone deacetylase (HDAC) inhibitors: This component regulates the epigenetic modification state of cells and is a key inducing factor in overcoming epigenetic barriers during chemical reprogramming. In cell fate remodeling, the openness of chromatin directly determines the activation efficiency of reprogramming-related genes. A preferred embodiment of this invention uses small-molecule histone deacetylase inhibitors, such as valproic acid (VPA). Its mechanism of action is through competitive inhibition of HDAC catalytic activity, leading to increased acetylation levels of core histones (especially H3 and H4). Increased histone acetylation levels reduce the electrostatic attraction between histones and DNA, prompting a shift in chromatin structure from a compact heterochromatin state to a looser euchromatin state. This "open" epigenetic map significantly increases the accessibility of binding sites for endogenous pluripotent genes or lineage-specific transcription factors, thereby lowering the reprogramming barrier and greatly improving the efficiency of chemically induced reprogramming.

[0035] Wnt signaling pathway activator: This component is used to activate the classical Wnt / β-catenin signaling pathway. In early embryonic development, Wnt signaling is crucial for ectoderm fate determination and skin formation. A preferred embodiment of this invention uses an inhibitor of glycogen synthase kinase 3 (GSK3), such as CHIR99021. Its mechanism of action is to inhibit GSK3 phosphorylation of β-catenin, thereby preventing its degradation by the proteasome, leading to the accumulation of β-catenin in the nucleus, which in turn activates the transcription of downstream target genes, initiating the conversion program to the epithelial lineage.

[0036] TGF-β signaling pathway inhibitors: This component is key to inducing mesenchymal-to-epithelial transition (MET). As mesenchymal-derived cells, ADSCs' phenotypic maintenance largely depends on the TGF-β signaling pathway, a potent driver of epithelial-to-mesenchymal transition (EMT). Therefore, inhibiting the TGF-β signaling pathway (e.g., using SB431542, which specifically targets type I receptors ALK4 / 5 / 7) can effectively reverse the mesenchymal characteristics of ADSCs, leading to an epithelial phenotype—a crucial step in overcoming germline barriers and achieving lineage reprogramming.

[0037] Bone morphogenetic protein (BMP) pathway activators: Although the TGF-β signaling pathway is inhibited, the BMP signaling pathway, which belongs to the TGF-β superfamily, is essential for epidermal fate determination. BMP signaling (such as BMP4) is a potent epidermal inducer, and one of its mechanisms of action is the activation of p63, the dominant transcription factor for epidermal development. Using inhibitors that specifically inhibit ALK4 / 5 / 7, such as SB431542, it is possible to block mesenchymal-promoting TGF-β signaling while allowing normal transduction of epidermal-promoting BMP signaling, thereby achieving precise regulation of the signaling pathway.

[0038] Retinoic acid (RA) or its agonists: RA is an important regulator of epithelial cell maturation and differentiation. In the later stages of induced differentiation of epithelial progenitor cells into mature keratinocytes, the addition of RA or its agonists (such as all-trans retinoic acid, ATRA) can effectively promote the expression of terminal differentiation markers.

[0039] ROCK inhibitors: Rho-associated kinase (ROCK) inhibitors, such as Y-27632, can significantly improve cell survival in the early stages of culture, passage, or reprogramming process by inhibiting cytoskeleton contraction and apoptosis (anorexia), ensuring a sufficient number of starting cells to enter the differentiation process and reducing cell death during differentiation.

[0040] cAMP signaling pathway activator: This component is used to increase intracellular cyclic adenosine monophosphate (cAMP) levels and synergistically enhance the effects of other signaling pathways. This invention preferably uses Forskolin, which increases intracellular cAMP concentration by activating adenylate cyclase. cAMP, as an important second messenger, can broadly regulate cellular metabolism, proliferation, and differentiation. In this combination, low concentrations of Forskolin can synergistically interact with other small molecules, contributing to epithelial cell phenotypic transformation and improving the overall efficiency of the reprogramming process.

[0041] The combination of components in this invention is not a simple superposition, but rather utilizes synergistic crosstalk between signaling pathways. For example, inhibiting TGF-β signaling not only directly promotes MET, but may also relieve its inhibitory effect on the Wnt pathway, thereby enhancing the epithelial-promoting effect of Wnt signaling. Simultaneous activation of the Wnt and BMP pathways simulates the synergistic effect of these two signals in embryonic development to determine cell fate. This design, which achieves cell fate transition through precise regulation of the signaling pathway network balance, embodies the non-obviousness of this invention.

[0042] In a preferred embodiment, the specific selection and corresponding concentration of the components in the composition for the differentiation of mesenchymal stem cells into keratinocytes provided by the present invention are as follows: Histone deacetylase inhibitor: VPA, with a concentration range of 200 µM to 800 µM, more preferably 500 µM; Wnt pathway activator: CHIR99021, with a concentration range of 0.1 µM to 15 µM, more preferably 10 µM; TGF-β signaling pathway inhibitor: SB431542, with a concentration range of 1 µM to 50 µM, more preferably 20 µM; Bone morphogenetic protein (BMP) pathway activator: recombinant human BMP4, in the range of 2.5 ng / mL to 25 ng / mL, more preferably 20 ng / mL; Retinyl agonist: all-trans retinoic acid (ATRA), in the concentration range of 1 µM to 10 µM, more preferably 5 µM; cAMP signaling pathway activator: Forskolin, with a concentration range of 0.5-5 µM, more preferably 1 µM; ROCK inhibitor: Y-27632, with a concentration range of 5 µM to 20 µM, more preferably 10 µM.

[0043] The above is the general concept of the present invention. Based on this, detailed embodiments and comparative examples are provided below to further illustrate the present invention.

[0044] Example 1 This embodiment provides a composition for the differentiation of mesenchymal stem cells into keratinocytes, comprising the following components at the following concentrations: Histone deacetylase inhibitor: VPA, at a concentration of 500 µM; Wnt pathway activator: CHIR99021, at a concentration of 10 µM; TGF-β signaling pathway inhibitor: SB431542, at a concentration of 20 µM; Bone morphogenetic protein (BMP) pathway activator: recombinant human BMP4, at a concentration of 20 ng / mL; Retinyl agonist: all-trans retinoic acid (ATRA), concentration 5 µM; cAMP signaling pathway activator: Forskolin, at a concentration of 1 µM; ROCK inhibitor: Y-27632, concentration range 10 µM.

[0045] This embodiment also provides a method for producing keratinocytes using the above composition, which includes cell acquisition, culture, and a multi-stage directed differentiation process, the specific steps of which are as follows: 1. Isolation and culture of mouse ADSCs The procedure was performed according to the standard protocol, specifically as follows: White adipose tissue from the groin of C57BL / 6 mice was aseptically harvested, washed three times in 0.01 M PBS buffer containing 1% (v / v) penicillin-streptomycin solution, and after removing blood vessels and connective tissue, it was minced to approximately 1 mm. 3 Tissue blocks were collected. 1 mg / mL type I collagenase (dissolved in PBS, pH 7.2-7.4, free of calcium and magnesium ions) was added at a tissue-to-enzyme ratio of 5:1, and the mixture was digested at 37°C and 120 rpm for 30-60 minutes with constant temperature shaking. After digestion, an equal volume of DMEM complete medium containing 10% (v / v) fetal bovine serum (FBS) was added to terminate the digestion. The resulting cell suspension was filtered through a 100 μm nylon cell strainer. The filtrate was centrifuged at 300×g for 8 minutes at 4°C, and the supernatant was discarded. The cell pellet was resuspended in DMEM / F12 basal medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin (containing 100 U / mL penicillin and 100 μg / mL streptomycin). After adjusting the cell concentration, the cells were seeded into T75 culture flasks and cultured in a constant temperature incubator at 37°C, 5% (v / v) CO2, and saturated humidity. When the cell confluence reaches 80-90%, passage is performed using 0.25% (w / v) trypsin-0.02% (w / v) EDTA digestion solution. Cells from the 2nd and 3rd generations with cell viability of over 95% are selected for subsequent differentiation experiments.

[0046] 2. Multi-stage differentiation plan This invention employs a sequential addition of different small molecule combinations to simulate the time sequence of development and gradually guide cell fate.

[0047] Phase 1 (Reprogramming Initiation, Days 1-7): After digestion, logarithmically growing ADSCs were seeded at an appropriate density in culture dishes. The basal medium (DMEM / F12 + 2% B27 + 50 nM dexamethasone + 50 μM β-mercaptoethanol) was supplemented with the Wnt pathway activator CHIR-99021 (10 μM), the TGF-β signaling pathway inhibitor SB431542 (20 μM), and the synergistic components VPA (500 μM), cAMP signaling pathway activator Forskolin (1 μM), and the ROCK inhibitor Y-27632 (10 μM). This phase aimed to suppress mesenchymal characteristics and initiate the reprogramming process to epithelial-like cells.

[0048] Phase II (Directed Differentiation, Days 7-14): The medium was changed to KSFM basal medium, and the small molecule combination was adjusted. CHIR-99021, SB431542, VPA, and Forskolin were removed. All-trans retinoic acid (ATRA) (5 μM) and the neural differentiation inhibitor BMP4 (20 ng / mL) were added, while epidermal growth factor (EGF) (20 ng / mL) was added. This phase promotes the directed differentiation of the initially transformed epithelial progenitor cells into the epidermal keratinocyte lineage.

[0049] Phase 3 (functional maturation, after day 14): Continue culturing in KSFM medium and further promote the formation of intercellular connections and terminal functional maturation of keratinocytes by adjusting culture conditions (increasing calcium ion concentration to >1.0 mM).

[0050] Example 2: Identification and application of keratinocytes induced by the method in Example 1 1. Verification of reprogramming success To confirm successful cell reprogramming, comprehensive identification at both the morphological and molecular levels is required.

[0051] Morphological analysis: Cell morphology was observed under a microscope. Successful reprogramming will manifest as a transformation of cells from the typical elongated spindle-shaped, fibroblast-like morphology of ADSCs to the tightly packed, "paving stone"-like, polygonal epithelial morphology characteristic of keratinocytes. Results are as follows: Figure 1 As shown, the cell clone morphology tends to be stable, indicating that it has completed the morphological transformation into a keratinocyte.

[0052] Molecular marker analysis: Quantitative real-time PCR (qPCR), Western blotting, and immunofluorescence were used to detect changes in the expression of a series of lineage-specific markers. Results are as follows: Figures 2 to 4 As shown, qPCR results indicated that the relative expression level of the key gene KRT14 in the induced group (BCEFRSVY) was more than 100-fold higher than that in the control group, and key epithelial genes such as CDH1 and Trp63 were also significantly highly expressed (P<0.001). The increase in the maturation marker KRT10 suggested that the cells possessed complete differentiation potential. Western blotting clearly showed that TP63, CDH1, and KRT14 proteins were significantly expressed in the induced group, which was highly consistent with the qPCR results. Immunofluorescence staining further confirmed that the induced miKCs showed high abundance of KRT14, Trp63, and CDH1 positive signals on day 14, and CDH1 was continuously distributed in the cell membrane (indicating the formation of complete cell junctions). Its fluorescence distribution characteristics were basically consistent with those of the primary KCs.

[0053] 2. Functional assessment using 3D skin equivalents Demonstrating that the generated KLCs possess biological functions is key to this invention. Constructing 3D skin equivalents is the gold standard for verifying their ability to form layered epidermis and barrier functions.

[0054] Skin model construction: Histological analysis: After 10-14 days of ALI culture, the epidermal model was harvested, fixed with PFA, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The formation of a multi-layered structure similar to natural skin was observed under a microscope. Results are as follows: Figure 5 As shown, the control group (mADSCs) could not form a layered structure, exhibiting only a single-layered, scattered distribution; while the miKCs group induced by this invention constructed epidermal tissue with a typical multi-layered structure.

[0055] Barrier function test: Permeability assay: A fluorescent tracer (such as sodium fluorescein) was added to the top of the chamber (cell surface), and the fluorescence intensity in the bottom culture medium was measured at different time points. A functional barrier should effectively prevent tracer penetration, manifested by extremely low fluorescence signals in the bottom culture medium. Statistical results are as follows: Figure 6 As shown, statistical analysis revealed that the 3D skin model constructed using the miKCs group achieved a blocking rate of over 60% for sodium fluorescein. The fluorescence intensity detected in the bottom pores of this model was significantly different from that of the mADSC group and approached that of the primary KC (mpKCs) group. These results functionally confirm that the resulting epidermal model not only morphologically mimics skin cells but also establishes a dense epidermal structure with a permeability barrier function.

[0056] The meanings of the relevant legends in the attached figures are as follows: BCEFRSVY: refers to the composition induction system used in Example 1 of this invention.

[0057] Control group: primitive mouse adipose-derived mesenchymal stem cells (mADSCs) that have not been induced by the BCEFRSVY system.

[0058] mKCs / mpKCs: refers to primary mouse keratinocytes, which served as the positive control group in this experiment.

[0059] miKCs: refers to keratinocyte-like cells induced by chemical reprogramming of mADSCs.

[0060] Meaning of molecular markers: TP63 (Trp63): A key transcription factor for keratinocyte development and a characteristic marker of epidermal basal cells.

[0061] CDH1 (E-cadherin): a cadherin that mediates tight junctions between cells and reflects the epithelial properties of cells.

[0062] KRT5 / KRT14: Keratins specific to basal cells of the epidermis, marking the entry of cells into the epithelial lineage.

[0063] KRT1 / KRT10: Markers of terminal differentiation of keratinocytes, reflecting cell maturity.

[0064] GAPDH: Used as an internal control protein to correct the consistency of protein loading.

[0065] Although this invention uses mouse ADSCs as an example, its core mechanism of action targets key developmental signaling pathways (Wnt, TGF-β, BMP) that are highly conserved across species. These pathways play similar roles in mouse and human skin development. Therefore, based on the above experimental results, there is good reason to expect that the methods and compositions described in this invention, after appropriate optimization, can also be applied to human ADSCs, thereby providing a novel and safe cell source for clinical regenerative medicine, which greatly expands the application prospects and value of this invention.

[0066] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A composition for mesenchymal stem cell differentiation into keratinocytes, characterized by, The composition comprises: a histone deacetylase inhibitor, a Wnt signaling pathway activator, a TGF-beta signaling pathway inhibitor, a bone morphogenetic protein pathway activator, and at least one of retinoic acid and a retinoic acid agonist.

2. The composition for differentiation of mesenchymal stem cells into keratinocytes according to claim 1, characterized by, The histone deacetylase inhibitor is VPA, and the Wnt pathway activator is CHIR99021.

3. The composition for differentiation of mesenchymal stem cells into keratinocytes according to claim 1, characterized by, The TGF-beta signaling pathway inhibitor is SB431542.

4. The composition for differentiation of mesenchymal stem cells into keratinocytes according to claim 1, characterized by, The bone morphogenetic protein pathway activator is recombinant human BMP4.

5. The composition for differentiation of mesenchymal stem cells into keratinocytes according to claim 1, characterized by, The retinoic acid agonist is all-trans retinoic acid.

6. The composition for differentiation of mesenchymal stem cells into keratinocytes according to claim 1, characterized by, At least one of a cAMP signaling pathway activator and a ROCK inhibitor is further included.

7. The composition for differentiation of mesenchymal stem cells into keratinocytes according to claim 6, characterized by, The cAMP signaling pathway activator is Forskolin, and the ROCK inhibitor is Y-27632.

8. The composition for differentiation of mesenchymal stem cells into keratinocytes according to claim 7, characterized by, Wherein: The concentration of the histone deacetylase inhibitor ranges from 200 µM to 800 µM; The concentration of the Wnt pathway activator ranges from 0.1 µM to 15 µM; The concentration of the TGF-beta signaling pathway inhibitor ranges from 1 µM to 50 µM; The concentration of the bone morphogenetic protein pathway activator ranges from 2.5 ng / mL to 25 ng / mL; The concentration of the retinoic acid agonist ranges from 1 µM to 10 µM; The concentration of the cAMP signaling pathway activator ranges from 0.5-5 µM; The concentration of the ROCK inhibitor ranges from 5 µM to 20 µM.

9. Use of the composition of any one of claims 1-8 in the production of keratinocytes.

10. A method of producing a population of keratinocyte-like cells, characterized in that, The method comprises contacting a population of mesenchymal stem cells with the composition of any one of claims 1-8 for a sufficient time to induce expression of one or more keratinocyte markers and a change in cell morphology.

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