A method for culture of small molecule compounds to directionally induce differentiation of human pluripotent stem cells into ocular surface ectodermal cells.

Abstract

This invention belongs to the field of stem cell biology and cell engineering technology, and discloses a method for the directed induction of human pluripotent stem cells into ocular surface ectodermal cells using a combination of small molecule compounds. This invention employs a serum-free culture medium with well-defined chemical compositions, using Essential 6 medium as the basal medium, and achieves directed induction of ocular surface ectodermal cells through a two-stage small molecule compound induction process. The entire induction process takes a maximum of approximately three weeks, allowing for the efficient extraction of PAX6 cells from human pluripotent stem cells. + / TP63 + High-purity ocular surface ectodermal cells. This method boasts advantages such as a short induction period, high differentiation efficiency, high cell purity, system stability, and good batch-to-batch consistency, filling the technological gap in current directed differentiation systems for ocular surface ectodermal cells. The obtained cells possess the potential to further differentiate into limbal stem cells, providing promising seed cells for the clinical treatment of corneal blindness and holding significant application value in the field of ocular surface regenerative medicine.

Description

Technical Field

[0001] This invention belongs to the field of stem cell biology and cell engineering technology, specifically relating to a method for culture of human pluripotent stem cells that is directed to differentiate into ocular surface ectoderm cells by a combination of small molecule compounds. Background Technology

[0002] Corneal diseases are among the leading causes of blindness, and restoring and reconstructing the structure and function of the cornea is crucial for their treatment. Clinically, corneal tissue transplantation is the primary treatment for corneal blindness, but it faces significant challenges and bottlenecks. Limbic stem cells, located at the limbus, are the sole source of corneal epithelial self-renewal and play a vital role in corneal damage repair and homeostasis maintenance. With the development and optimization of in vitro expansion and culture techniques for limbic stem cells, transplantation of physiologically functional limbic stem cells has become a definitive and effective treatment for corneal blindness. Although autologous limbic stem cell transplantation has become a treatment strategy for corneal regeneration, this method has the following problems: 1) Patients with bilateral limbic stem cell deficiency or genetic defects lack a source of functional stem cells; 2) For unilateral patients, harvesting stem cells from the only healthy eye carries a high risk of iatrogenic blindness. Therefore, finding alternative "seed cells" to achieve functional corneal reconstruction is an inevitable trend in stem cell therapy for corneal blindness.

[0003] Pluripotent stem cells (ESCs and Induced Pluripotent Stem Cells, iPSCs) possess high self-renewal capacity and multi-directional differentiation potential, enabling them to mimic the in vivo development of corneal epithelium and serving as promising "seed cells" for obtaining cells with corneal tissue characteristics. In corneal epithelial development, the ocular surface ectoderm is its developmental origin and a key node. Ocular surface ectoderm derived from iPSCs has been proven to further differentiate into corneal epithelial cells and has been successfully applied in clinical transplantation trials in patients with corneal blindness, achieving long-term safety and significant efficacy. Therefore, establishing a pluripotent stem cell-based directed differentiation system for ocular surface ectoderm is a crucial prerequisite for achieving directed differentiation of limbal stem cells. However, current methods for inducing ocular surface ectoderm have the following major drawbacks: 1) non-directed differentiation, resulting in low spontaneous differentiation efficiency; 2) mixed cell types; 3) long differentiation cycle; and 4) poor batch-to-batch variability. These problems severely hinder the directed regeneration of pluripotent stem cells into limbal stem cells, greatly limiting the widespread application of pluripotent stem cell-derived limbal stem cells in clinical research and treatment. Therefore, establishing a highly efficient, stable, and well-defined system for the directed differentiation and induction of ocular surface ectoderm based on pluripotent stem cells is a critical technical issue that urgently needs to be addressed in the treatment of corneal blindness. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for the culture of human pluripotent stem cells to differentiate into ocular surface ectodermal cells by a combination of small molecule compounds. The method employs a phased, serum-free induction system with clearly defined chemical components to achieve efficient, stable, and high-purity directed differentiation of human pluripotent stem cells into ocular surface ectodermal cells.

[0005] This invention is achieved through the following technical solution: A method for culture of human pluripotent stem cells that is directionally induced to differentiate into ocular surface ectodermal cells by a combination of small molecule compounds, using a serum-free culture medium with clearly defined chemical composition, wherein the culture medium comprises a basal culture medium and a combination of small molecule compounds added in stages; The basal culture medium is Essential 6 medium; The addition of the small molecule compound consists of two induction phases: Phase 1 induction: The culture medium contained 0.1–5 μM SP600125, 0.1–5 μM JNK-IN-8, 0.5–50 ng / mL TGFβ1, 0.1–5 ng / mL BMP4, and 1×10 -9 ~1×10 -13 M-type retinoic acid (RA); Second stage induction: The culture medium contains 1-30 ng / mL FGF2, 20-300 ng / mL WNT5A, and 1×10 -4 ~1×10 -8 M's RA.

[0006] More preferably, the concentrations of small molecules induced in the first stage are: SP600125 0.5~2 μM, JNK-IN-8 0.1~2 μM, TGFβ1 1~10 ng / mL, BMP4 0.1~2 ng / mL, and RA 1×10 -10 ~1×10 -13 M; The concentrations of small molecules induced in the second stage are: FGF2 5~20 ng / mL, WNT5A 50~200 ng / mL, RA 1×10 -6 ~1×10 -8 M; The basal culture medium is Essential 6 medium.

[0007] More preferably, the induction culture medium is prepared by volume as follows: The first stage of induction medium consists of: 12.5–100 μL of 10 mM SP600125 stock solution, 5–100 μL of 10 mM JNK-IN-8 stock solution, 5–50 μL of 100 μg / mL TGFβ1 stock solution, 0.5–10 μL of 100 μg / mL BMP4 stock solution, and 5–5 mL of 10 nM RA working solution per 500 mL of medium; the remainder is Essential 6 medium. The second stage of induction medium consists of: 25–100 μL of 100 μg / mL FGF2 stock solution, 250–1000 μL of 100 μg / mL WNT5A stock solution, and 0.5–50 μL of 10 mM RA stock solution per 500 mL of medium; the remainder is Essential 6 medium.

[0008] The culture medium of this invention is an induction system with a defined chemical composition, free of serum and animal-derived components. Through the synergistic effect of two-stage small molecule compounds, it induces human pluripotent stem cells to differentiate into ocular surface ectodermal cells. This system has a well-defined composition, stable quality, and strong batch-to-batch consistency, making it easy to standardize and industrialize. It is an ideal solution for the efficient in vitro induction of ocular surface ectodermal cells.

[0009] Furthermore, the method for inducing ocular surface ectodermal cells includes the following steps: S1. Pretreatment for cell differentiation The cell culture medium used for human pluripotent stem cells is pluripotency maintenance medium mTeSR1; Predifferentiation passaging: Clonal clusters of human pluripotent stem cells were digested into single cells using Accutase digestive enzyme, counted, and seeded into cell culture plates pre-coated with hESC-qualified Matrigel at a density of 1000-4000 cells / cm². 2 The passage medium was mTeSR1 medium containing 10 μM of the ROCK inhibitor Y-27632. Single cells were cultured in this medium for 48 h, and then cultured in mTeSR1 medium without Y-27632 for another 24 h to allow the cells to form uniformly sized and evenly distributed clonal clumps. S2. Induction Phase One: 72 hours after passage of human pluripotent stem cells, the culture medium was replaced with the first-stage induction medium, and the cells were induced in this medium for 5 to 10 days. The culture medium was replaced with fresh medium every day and the cell status was observed. After 5 to 10 days of induction, human pluripotent stem cells initially differentiate into intermediate-state cells; S3. Induction Phase Two: Next, the culture medium was replaced with the second-stage induction medium, and the cells were induced in this medium for 5 to 10 days. Fresh medium was replaced every day and the cell status was observed. After 5 to 10 days of induction, the intermediate cells further differentiated into mature, high-purity ocular surface ectoderm cells.

[0010] More preferably, the human pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.

[0011] More preferably, the obtained ocular surface ectodermal cells are PAX6. + TP63 + Double-positive cells.

[0012] The application of the culture method described herein in the preparation of ocular surface ectoderm cells, limbal stem cells, or corneal epithelial repair cells.

[0013] The present invention has the following advantages over the prior art: Compared to other existing differentiation systems, the method for inducing the directed differentiation of human pluripotent stem cells into ocular surface ectoderm cells by staged combination of small molecule compounds described in this invention has the following advantages: High differentiation efficiency and short cycle: It only takes a maximum of three weeks to transform human pluripotent stem cells into mature and stable ocular surface ectoderm cells, which is significantly shorter than existing methods; High cell purity and high specificity: PAX6 was obtained + TP63 + The double-positive high-purity ocular surface ectodermal cells avoid the mixing of other cell types; the differentiated cells specifically express markers such as PAX6 and TP63, and can further differentiate into functional limbal stem cells; The system is stable and highly reproducible: it is applicable to various human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, with strong batch-to-batch consistency; Clearly defined components, serum-free, and easy to industrialize: The culture medium is defined by chemical components, avoiding interference from unknown components and facilitating quality control and clinical translation.

[0014] This invention provides a stable cell source for basic research on ocular surface ectoderm cells and also provides novel seed cells for cell transplantation therapy for corneal blindness caused by limbal stem cell deficiency. It has important research value and broad clinical application prospects in the field of ocular surface regenerative medicine. Attached Figure Description

[0015] Figure 1 This describes the process by which human embryonic stem cells gradually differentiate into ocular surface ectoderm cells from the first day (Day 1, D1) to the tenth day (D10) in a directed induction system based on this combination of small molecule compounds. Figure 2The ocular surface ectoderm markers PAX6 and TP63 were obtained by induction of human embryonic stem cells; Figure 3 To determine the proportion of PAX6 and TP63 double-positive ocular epiectoderm cells obtained by human embryonic stem cell induction (statistical data were obtained from five independent experiments). Figure 4 To demonstrate the morphology of human induced pluripotent stem cells differentiated into intermediate cells and ocular surface ectoderm cells after five days (Day 5, D5) and ten days (D10) of treatment in this small molecule compound combination directed induction system; Figure 5 The ocular surface ectoderm markers PAX6 and TP63 were specifically expressed by human induced pluripotent stem cells after induction. Figure 6 To determine the proportion of PAX6 and TP63 double-positive ocular epiectodermal cells obtained from human induced pluripotent stem cell differentiation (statistical data were obtained from five independent experiments). Detailed Implementation

[0016] To further explain the present invention, the following specific embodiments are described.

[0017] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0018] Unless otherwise specified, the reagents, consumables, and instruments used in the following experimental examples can be obtained through conventional commercial channels.

[0019] Essential 6 medium was purchased from Life Technologies (catalog number A1516401); mTeSR1 medium was purchased from STEMCELL Technologies (catalog number 85850); ROCK inhibitor Y-27632 was purchased from Tocris (catalog number 1254); JNK inhibitor SP600125 was purchased from MedChemExpress (catalog number HY-12041); JNK inhibitor JNK-IN-8 was purchased from Selleck (catalog number S4901); RA was purchased from R&D Systems (catalog number 0695); BMP4 was purchased from R&D Systems (catalog number 314-BP); TGFβ1 was purchased from MedChemExpress (catalog number HY-P7118); FGF2 was purchased from MedChemExpress (catalog number HY-P7004); WNT5A was purchased from R&D Systems (catalog number 645-WN-010).

[0020] Example 1 Differentiation of human embryonic stem cells into ocular surface ectoderm cells 1. Culture medium formulation 1.1. Preparation of small molecule compounds: SP600125: Dilute the SP600125 powder with DMSO solvent to a final concentration of 10 mM.

[0021] JNK-IN-8: Dilute the JNK-IN-8 powder with DMSO solvent to a final concentration of 10 mM.

[0022] RA: Dilute the RA powder with DMSO solvent to a final concentration of 10 mM; then dilute the stock solution with Essential 6 medium to a working solution of 10 nM.

[0023] BMP4: Dilute the BMP4 powder with ddH2O to obtain a final concentration of 100 μg / mL.

[0024] TGFβ1: Dilute the TGFβ1 powder with ddH2O to obtain a final concentration of 100 μg / mL.

[0025] FGF2: Dilute the FGF2 powder with PBS to a final concentration of 100 μg / mL.

[0026] WNT5A: Dilute the WNT5A powder with PBS to obtain a final stock solution concentration of 100 μg / mL.

[0027] 1.2. Preparation of the first-stage induction medium: Each 500 mL of culture medium contains 12.5–100 μL of 10 mM SP600125 stock solution, 5–100 μL of 10 mM JNK-IN-8 stock solution, 50–500 μL of 10 μg / mL TGFβ1 stock solution, 0.5–10 μL of 100 μg / mL BMP4 stock solution, and 5–5 mL of 10 nM RA working solution; the remainder is Essential 6 culture medium.

[0028] 1.3. Preparation of the second-stage induction medium: Each 500 mL of culture medium contains 25–100 μL of 100 μg / mL FGF2 stock solution, 250–1000 μL of 100 μg / mL WNT5A stock solution, and 0.5–50 μL of 10 mM RA stock solution; the remainder is Essential 6 culture medium.

[0029] 2. Steps for inducing differentiation of ocular surface ectodermal cells S1. Pretreatment for cell differentiation The cell culture medium used for human embryonic stem cells was pluripotency maintenance medium mTeSR1. Predifferentiation passage: Clonal clusters of human embryonic stem cells were digested into single cells using Accutase, counted, and then seeded into cell culture plates pre-coated with hESC-qualified Matrigel at a density of 1000–4000 cells / cm². 2 The passage medium was mTeSR1 medium containing 10 μM ROCK inhibitor Y-27632. Single cells were cultured in this medium for 48 h, and then cultured in mTeSR1 medium without Y-27632 for another 24 h to allow the cells to form uniformly sized and evenly distributed clonal clumps.

[0030] S2. Induction Phase One 72 hours after passage of human embryonic stem cells, the culture medium was replaced with the first-stage induction medium, and induction was performed in this medium for 5-10 days, with fresh medium changed daily and cell status observed. After 5-10 days of induction, human embryonic stem cells began to differentiate into intermediate cells.

[0031] S3. Induction Phase Two Next, the culture medium was replaced with the second-stage induction medium, and the cells were induced in this medium for 5-10 days, with fresh medium being used daily and the cell status observed. After 5-10 days of induction, the intermediate cells further differentiated into mature, high-purity ocular surface ectoderm cells.

[0032] 3. Analysis of Experimental Results: (1) Cell morphology observation: like Figure 1 As shown, during the directed differentiation of ocular surface ectoderm, cells gradually transform from the dense clonal clusters of embryonic stem cells into a uniform ocular surface ectoderm epithelial morphology.

[0033] (2) Detection of cell gene expression: like Figure 2 As shown, in the ocular surface ectoderm directed differentiation system of the present invention, ocular surface ectoderm cells obtained by small molecule combination induction specifically highly express the marker genes PAX6 and TP63; (3) Differentiation efficiency detection: like Figure 3 As shown, this ocular surface ectoderm directed differentiation system can induce human embryonic stem cells into PAX6 and TP63 double-positive ocular surface ectoderm cells in a proportion as high as 95.5%.

[0034] Example 2 Human induced pluripotent stem cells differentiate into ocular surface ectodermal cells 1. Culture medium formulation 1.1. Preparation of small molecule compounds: SP600125: Dilute the SP600125 powder with DMSO solvent to a final concentration of 10 mM.

[0035] JNK-IN-8: Dilute the JNK-IN-8 powder with DMSO solvent to a final concentration of 10 mM.

[0036] RA: Dilute the RA powder with DMSO solvent to a final concentration of 10 mM; then dilute the stock solution with Essential 6 medium to a working solution of 10 nM.

[0037] BMP4: Dilute the BMP4 powder with ddH2O to obtain a final concentration of 100 μg / mL.

[0038] TGFβ1: Dilute the TGFβ1 powder with ddH2O to obtain a final concentration of 100 μg / mL.

[0039] FGF2: Dilute the FGF2 powder with PBS to a final concentration of 100 μg / mL.

[0040] WNT5A: Dilute the WNT5A powder with PBS to obtain a final stock solution concentration of 100 μg / mL.

[0041] 1.2. Preparation of the first-stage induction medium: Each 500 mL of culture medium contains 12.5–100 μL of 10 mM SP600125 stock solution, 5–100 μL of 10 mM JNK-IN-8 stock solution, 50–500 μL of 10 μg / mL TGFβ1 stock solution, 0.5–10 μL of 100 μg / mL BMP4 stock solution, and 5–5 mL of 10 nM RA working solution; the remainder is Essential 6 culture medium.

[0042] 1.3. Preparation of the second-stage induction medium: Each 500 mL of culture medium contains 25–100 μL of 100 μg / mL FGF2 stock solution, 250–1000 μL of 100 μg / mL WNT5A stock solution, and 0.5–50 μL of 10 mM RA stock solution; the remainder is Essential 6 culture medium.

[0043] 2. Steps for inducing differentiation of ocular surface ectodermal cells S1. Pretreatment for cell differentiation The cell culture medium used for human induced pluripotent stem cells was mTeSR1, a pluripotency maintenance medium. Predifferentiation passage: Clonal clusters of human embryonic stem cells were digested into single cells using Accutase, counted, and seeded into pre-coated hESC-qualified Matrigel cell culture plates at a density of 1000–4000 cells / cm². 2 The passage medium was mTeSR1 medium containing 10 μM ROCK inhibitor Y-27632. Single cells were cultured in this medium for 48 h, and then cultured in mTeSR1 medium without Y-27632 for another 24 h to allow the cells to form uniformly sized and evenly distributed clonal clumps.

[0044] S2. Induction Phase One 72 hours after passage of human induced pluripotent stem cells, the culture medium was replaced with the first-stage induction medium, and induction continued for 5–10 days, with fresh medium changed daily and cell status observed. After 5–10 days of induction, human induced pluripotent stem cells began to differentiate into intermediate cells.

[0045] S3. Induction Phase Two Next, the culture medium was replaced with the second-stage induction medium, and the cells were induced in this medium for 5-10 days, with fresh medium being used daily and the cell status observed. After 5-10 days of induction, the intermediate cells further differentiated into mature, high-purity ocular surface ectoderm cells.

[0046] 3. Analysis of Experimental Results: (1) Cell morphology observation: like Figure 4 As shown, during the directed differentiation of ocular surface ectoderm, cells begin to differentiate from human induced pluripotent stem cells, and the morphological changes of cells in intermediate and ocular surface ectoderm stages are illustrated.

[0047] (2) Detection of cell gene expression: like Figure 5 As shown, in the ocular surface ectoderm directed differentiation system of the present invention, ocular surface ectoderm cells obtained by human induced pluripotent stem cells through small molecule combination induction specifically highly express the marker genes PAX6 and TP63; (3) Differentiation efficiency detection: like Figure 6 As shown, this ocular ectoderm-directed differentiation system can induce human induced pluripotent stem cells into PAX6 and TP63 double-positive ocular ectoderm cells in a proportion as high as 96.3%.

[0048] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for cultured human pluripotent stem cells to differentiate into ocular surface ectodermal cells by a combination of small molecule compounds, characterized in that, A serum-free culture medium with a clearly defined chemical composition is used, which comprises a basal culture medium and a combination of small molecule compounds added in stages; The basal culture medium is Essential 6 medium; The addition of the small molecule compound consists of two induction phases: Phase 1 induction: The culture medium contained 0.1–5 μM SP600125, 0.1–5 μM JNK-IN-8, 0.5–50 ng / mL TGFβ1, 0.1–5 ng / mL BMP4, and 1×10 -9 ~1×10 -13 M's RA; Second stage induction: The culture medium contains 1-30 ng / mL FGF2, 20-300 ng / mL WNT5A, and 1×10 -4 ~1×10 -8 M's RA.

2. The cultivation method according to claim 1, characterized in that, The concentrations of small molecules induced in the first stage were: SP600125 0.5–2 μM, JNK-IN-8 0.1–2 μM, TGFβ1 1–10 ng / mL, BMP4 0.1–2 ng / mL, and RA 1 × 10⁻⁶. -10 ~1×10 -13 M; The concentrations of small molecules induced in the second stage are: FGF2 5~20 ng / mL, WNT5A 50~200 ng / mL, RA 1×10 -6 ~1×10 -8 M.

3. The cultivation method according to claim 1, characterized in that, Includes the following steps: S1. Pre-differentiation treatment: Human pluripotent stem cells were digested into single cells using Accutase at a concentration of 1000-4000 cells / cm³. 2 The cells were plated and cultured in mTeSR1 containing 10 μM Y-27632 for 48 h, followed by culture in mTeSR1 without Y-27632 for 24 h. S2, First-stage induction: Replace with the first-stage induction medium and induce culture for 5-10 days; S3, Second Stage Induction: Replace with the second stage induction medium and induce culture for 5-10 days to obtain ocular surface ectoderm cells.

4. The cultivation method according to claim 3, characterized in that, The human pluripotent stem cells mentioned are human embryonic stem cells or human induced pluripotent stem cells.

5. The cultivation method according to claim 3, characterized in that, The obtained ocular surface ectoderm cells were PAX6. + TP63 + Double-positive cells.

6. The application of the culture method according to any one of claims 1 to 5 in the preparation of ocular surface ectoderm cells, limbal stem cells or corneal epithelial repair cells.

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