In vitro induction culture system of early gonad-like cells and application thereof

Abstract

The present application relates to the technical field of stem cell directional differentiation and reproductive development, in order to solve the problems of limited source of human early gonad-like cells in the prior art, difficulty in stably obtaining early gonad-like cells by the existing induction system, and insufficient stability of differentiation direction, etc., the present application discloses an in-vitro induction culture system of early gonad-like cells and application, which comprises bone morphogenetic protein 7, CHIR99021 and soluble recombinant human neural cell adhesion molecule 1. The present application also provides an in-vitro induction and differentiation method of early gonad-like cells, which places intermediate mesoderm-like cells in the in-vitro induction culture system for induction, and obtains early gonad-like cells. The in-vitro induction culture system in the present application can effectively promote the transformation of intermediate mesoderm-like cells to early gonad-like state, the cells obtained by induction express early gonad-related markers, and present the characteristics of early gonad-related transcription program. The present application can be used to construct an in-vitro early gonad development model.

Description

Technical Field

[0001] This invention belongs to the field of stem cell directed differentiation and reproductive development engineering, specifically relating to an in vitro induction culture system for early gonadal-like cells and its application. Background Technology

[0002] Normal development of the reproductive system is fundamental to maintaining an individual's fertility and gonadal endocrine function. As the core organ of the reproductive system, the gonads begin to develop in the early embryonic period with the formation of gonadal primordia and the differentiation of early gonadal cells. These early gonadal cells further differentiate into various gonadal cell lineages, including supporting cells and interstitial cells, and participate in the subsequent establishment of gonadal cords, the formation of the germ cell microenvironment, and the regulation of sexual differentiation. Therefore, the normality of early gonadal development directly affects the formation of the reproductive system structure, the maintenance of the gamete production environment, and the integrity of reproductive function in adulthood. Abnormalities in these processes can lead to gonadal hypoplasia, sexual development disorders, congenital reproductive system abnormalities, and impaired fertility.

[0003] Reproductive dysfunction and infertility have become significant issues affecting human health and social development. A 2023 report by the World Health Organization indicated that approximately 17.5% of adults worldwide will experience infertility at some point in their lives, suggesting a high incidence of reproductive system-related diseases and a substantial public health burden. Traditional research has largely focused on mature gonadal function, hormone levels, germ cell quality, and clinical phenotype analysis, while research on how abnormalities in early embryonic gonadal formation lead to subsequent gonadal dysfunction remains relatively insufficient. Therefore, establishing in vitro models that can simulate the early human gonadal development process is crucial for elucidating the developmental origins of reproductive-related diseases, constructing disease models, and screening candidate drugs.

[0004] Due to the difficulty in obtaining early human embryonic gonadal tissue, the short developmental window, and ethical restrictions, directly using human embryonic or fetal gonadal tissue to conduct research on early gonadal development has significant limitations. Meanwhile, while animal models can be used to study the basic principles of gonadal development, they are not entirely equivalent to the human gonadal development process. In recent years, advancements in single-cell sequencing, spatial transcriptomics, and in vitro stem cell induction techniques have propelled research on human gonadal development.

[0005] For example, in 2022, Garcia-Alonso et al. constructed single-cell and spatial transcriptome atlases of human and mouse gonadal development (Garcia-Alonso L, Lorenzi V, Mazzeo CI, et al. Single-cell roadmap of human gonadal development. Nature. 2022 Jul 28;607(7919):540-547. doi:10.1038 / s41586-022-04918-4), providing an important reference for understanding the human gonadal somatic cell lineage, supporting cell differentiation, and mesenchymal cell formation, and suggesting that such atlases can guide the construction of in vitro gonadal development models.

[0006] Currently, some studies have attempted to establish in vitro models related to human gonads. For example, cell suspensions derived from human embryonic or fetal gonadal tissue are used for in vitro recombinant culture. Such systems can support the reorganization of gonadal cells to a certain extent and form some testicular or ovarian-like structural features (Oliver E, Katanayeva N, Morotti M, et al. Self-organising human gonads generated by a Matrigel-based gradient system. BMC Biology. 2021 Sep 23;19(1):212. doi:10.1186 / s12915-021-01149-3). However, such methods rely on human embryonic or fetal gonadal tissue, resulting in limited sample sources, ethical restrictions on acquisition, and significant individual differences between different samples, making it difficult to establish a standardized, reproducible, and scalable pluripotent stem cell induction system.

[0007] Another study attempted to establish early testis-like cell or testis-like organoid models using human induced pluripotent stem cells (Knarston IM, Pachernegg S, et al. An In Vitro Differentiation Protocol for Human Embryonic Bipotential Gonad and Testis Cell Development. Stem Cell Reports. 2020 Dec 8;15(6):1377-1391. doi:10.1016 / j.stemcr.2020.10.009). This type of research obtains cells expressing bipotent gonadal markers or early supporting cell-related markers through staged induction, and these cells can form three-dimensional structures under certain conditions, providing a preliminary in vitro research basis for human gonadal cell differentiation and testis-like tissue formation (Pachernegg, Svenja et al. The emergence of multiple testicular celllineages in human stem cell-derived testis-like organoids. Development (Cambridge). 2026 Mar 1;153(5):dev204772. doi:10.1242 / dev.204772). However, existing pluripotent stem cell-derived models still cannot fully reproduce the human embryonic gonadal development process, and mostly focus on testis-like differentiation, supporting cell-like cell formation, or multi-lineage testis-like structure characterization.

[0008] In addition, some in vitro induction systems mainly focus on obtaining primordial germ cell-like cells, germ cell-like cells, or mesodermal-related cells (Sasaki, Kotaro et al. Robust In Vitro Induction of Human Germ Cell Fate from Pluripotent Stem Cells. Cell Stem Cell. 2015 Aug 6;17(2):178-194. doi:10.1016 / j.stem.2015.06.014 and Mae, Shin-Ichi et al.“Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells.” Nature Communications. 2013 Jan 29;4:1367.doi:10.1038 / ncomms2378). These systems are valuable for studying germ cell development or mesodermal lineage differentiation, but their induction target is not early gonadal cells, nor can they fully simulate the establishment of the somatic cell microenvironment during the formation of gonadal primordia. Therefore, obtaining germ cell-like cells or general mesodermal-related cells alone is insufficient to replace the early gonadal cell induction system.

[0009] In summary, existing technologies have at least the following shortcomings: First, the source of human embryonic or fetal gonadal tissue is limited and subject to ethical restrictions, making it difficult to use as a stable and scalable modeling source; second, primary gonadal cell recombinant systems depend on tissue source and sample state, making them difficult to apply in a standardized and large-scale manner; third, existing pluripotent stem cell-derived early testicular-like cell or testicular organoid models mainly focus on testicular-like differentiation, early supporting cell-like cell formation, or multi-lineage testicular-like structure characterization, which cannot fully meet the need for stable acquisition of early gonadal primordium-like cells or early gonadal somatic cell states; fourth, primordial germ cell-like cells, germ cell-like cells, or generalized mesoderm induction systems cannot replace early gonadal somatic cell induction systems; fifth, it is still necessary to establish an in vitro induction differentiation method that uses pluripotent stem cells as starting cells, proceeds through an intermediate mesoderm-like stage, has a clear induction pathway, controllable culture conditions, a relatively simple operation procedure, and can stably obtain early gonadal-like cells.

[0010] Therefore, there is an urgent need to establish a new in vitro differentiation induction system that enables pluripotent stem cells to differentiate progressively through protostome-like and intermediate mesoderm-like stages, and further obtain cell populations with early gonadal characteristics. This system should possess characteristics such as a clearly defined induction pathway, controllable culture conditions, high reproducibility, and clearly detectable early gonadal markers, thereby providing a reproducible in vitro model for studying the mechanisms of early human gonadal development, modeling gonadal developmental abnormalities, and screening candidate drugs. Summary of the Invention

[0011] To address the shortcomings of existing technologies, this invention provides an in vitro induction and culture system for early gonadal-like cells and its application, thereby solving problems such as the limited source of human early gonadal-like cells, the difficulty in stably obtaining early gonadal-like cells using existing induction systems, insufficient stability in differentiation direction, and the lack of in vitro models of early gonadal development in existing technologies.

[0012] The technical solution adopted in this invention is as follows: In a first aspect, the present invention protects an in vitro induction culture system for early gonadal-like cells, said in vitro induction culture system comprising bone morphogenetic protein 7 (BMP7), CHIR99021, and soluble recombinant human neural cell adhesion molecule 1 (NCAM1). Through the combined induction of these three factors, the transformation of intermediate mesoderm-like cells into an early gonadal-like state can be promoted in vitro.

[0013] CHIR99021 is a WNT signaling pathway agonist that activates the WNT / β-catenin signaling pathway by inhibiting glycogen synthase kinase-3 (GSK-3) activity. BMP7 belongs to the TGF-β superfamily and can activate the BMP signaling pathway. Soluble recombinant human neural cell adhesion molecule 1 (NCAM1) belongs to the immunoglobulin superfamily of cell adhesion molecules; this invention directly adds it to the culture system in a soluble form as a functional inducing factor.

[0014] In some embodiments, the concentration of the soluble recombinant human neural cell adhesion molecule 1 is 5-20 ng / mL, the concentration of the bone morphogenetic protein 7 is 10-100 ng / mL, and the concentration of CHIR99021 is 3 μM.

[0015] In a preferred embodiment, the concentration of the soluble recombinant human neural cell adhesion molecule 1 is 5 ng / mL, and the concentration of the bone morphogenetic protein 7 is 50 ng / mL. The combination with 3 μM CHIR99021 is designated as the N5B50C3 induction system.

[0016] In some embodiments, the in vitro induction culture system further includes a basal culture medium, which is STEMdiff APEL2 medium.

[0017] In some embodiments, the soluble recombinant human neural cell adhesion molecule 1, bone morphogenetic protein 7, and CHIR99021 are simultaneously added to STEMdiff APEL2 medium.

[0018] Secondly, the present invention protects a method for in vitro induction of differentiation of early gonadal-like cells, comprising the following steps: Intermediate mesodermal-like cells were induced and cultured in the in vitro induction culture system described above to obtain early gonadal-like cells.

[0019] In some embodiments, the intermediate mesodermal-like cells are obtained through the following steps: Provide human embryonic stem cells; The human embryonic stem cells were cultured in a first induction culture system to obtain original strip-like cells; The original strip-like cells were cultured in a second induction culture system to obtain intermediate mesoderm-like cells.

[0020] The human embryonic stem cells can be H1 human embryonic stem cells or H9 human embryonic stem cells. The human embryonic stem cells can be maintained in a Matrigel-coated culture dish in mTeSR Plus medium under two-dimensional adherent culture conditions without a feeder layer, and subsequent induction can begin when the cells are in good condition, have regular clonal morphology, have not shown obvious differentiation, and are in the logarithmic growth phase.

[0021] In some embodiments, the first induction culture system uses STEMdiff APEL2 medium as the basal medium and contains activin A, bone morphogenetic protein 4 (BMP4), CHIR99021, fibroblast growth factor 2 (FGF2), and PIK90. In a specific embodiment, the concentration of activin A is 30 ng / mL, the concentration of bone morphogenetic protein 4 is 40 ng / mL, the concentration of CHIR99021 is 6 μM, the concentration of fibroblast growth factor 2 is 20 ng / mL, and the concentration of PIK90 is 100 nM. Human embryonic stem cells are cultured in the first induction culture system for 24 hours to obtain original strip-like cells.

[0022] In some embodiments, the second induction culture system uses STEMdiff APEL2 medium as the basal medium and contains bone morphogenetic protein 4, fibroblast growth factor 2, and A-83-01. In a specific embodiment, the concentration of bone morphogenetic protein 4 is 3 ng / mL, the concentration of fibroblast growth factor 2 is 20 ng / mL, and the concentration of A-83-01 is 1 μM. The original strip-like cells are cultured in the second induction culture system for 24 hours to obtain intermediate mesoderm-like cells.

[0023] In some implementations, intermediate mesodermal-like cells are induced and cultured in an in vitro induction culture system for 24-72 hours. In specific implementations, cell samples are collected at 24, 48, and 72 hours of induction to evaluate the induction effect.

[0024] Thirdly, the present invention protects an early gonadal-like cell population, which is prepared by the in vitro induced differentiation method described above.

[0025] In some embodiments, the early gonad-like cell population expresses one or more early gonad-related marker genes selected from GATA4, WT1, NR5A1 / SF1, LHX9, EMX2, and GADD45G.

[0026] In some embodiments, expression of GATA4 and / or WT1 proteins can be detected in the early gonadal-like cell population.

[0027] In some embodiments, the early gonad-like cell population is derived from H1 human embryonic stem cells, and SOX9 and / or AMH gene expression is also detected therein.

[0028] In some implementations, the early gonadal-like cell population is derived from H9 human embryonic stem cells, and FOXL2 gene expression is also detected therein.

[0029] Fourthly, the application of the aforementioned in vitro induction culture system or early gonad-like cell population in constructing an in vitro early gonadal development model.

[0030] Beneficial effects: 1. This invention employs a combined induction culture system of soluble recombinant human NCAM1, BMP7, and CHIR99021. Soluble recombinant human NCAM1 serves as a functional inducing component, rather than merely as a cell surface marker or cell screening indicator. The induction culture system formed by using NCAM1 simultaneously with BMP7 and CHIR99021 exhibits cross-cell lineage applicability and reproducibility, and can promote the expression of marker genes from different sex-related glandular cell lineages, thereby obtaining early gonadal-like cells.

[0031] 2. The present invention uses intermediate mesodermal-like cells as precursor cells for early gonadal-like cell induction, which is more in line with the developmental logic related to early gonadal development and helps to reduce the problem of unstable differentiation direction caused by direct induction.

[0032] 3. This invention screens the concentrations of soluble recombinant human NCAM1 and BMP7 to determine the preferred induction combination as N5B50C3 (i.e., 5 ng / mL soluble recombinant human NCAM1, 50 ng / mL BMP7, and 3 μM CHIR99021), thus providing the induction culture system with a clear parameter basis and operability.

[0033] 4. This invention sets up a factor-free control system and can be combined with evaluation methods such as real-time quantitative PCR, immunofluorescence staining, and RNA sequencing, which helps to clarify the early gonadal-like cell induction effect and make the results true and reliable.

[0034] 5. The early gonad-like cells obtained by this invention can be used as an in vitro model of early gonadal development, and can also be further extended to serve as a cell model for studying the mechanisms of reproductive developmental abnormalities, constructing disease models, and screening candidate drugs, showing good application potential.

[0035] 6. The induction culture system described in this invention can promote the expression of early gonad-related marker genes in different human embryonic stem cell lines such as H1 and H9, and can promote the expression of male or female gonadal cell lineage-related marker genes in different cell line backgrounds, indicating that the system has good cell line applicability. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a schematic diagram of the in vitro differentiation process of early gonadal-like cells according to the present invention.

[0038] Figure 2 This is a morphological observation of cells during the early gonadal-like cell induction process of the present invention. The scale bar is 500 μm.

[0039] Figure 3 The image shows the RT-qPCR detection results for single-factor concentration screening of NCAM1 and BMP7.

[0040] Figure 4The figure shows the comparison results of RT-qPCR between the N5B50C3 three-factor induction combination and the two-factor control combination.

[0041] Figure 5 The image shows the RT-qPCR results of promoting the expression of early gonadal-related marker genes at different induction time points using H9 human embryonic stem cells as the starting cells and the N5B50C3 induction system.

[0042] Figure 6 The image shows the RT-qPCR results of H1 human embryonic stem cells induced at different times using the N5B50C3 induction system.

[0043] Figure 7 The image shows the immunofluorescence staining results of early gonad-related marker proteins after induction in the N5B50C3 group. The scale bar is 100 μm.

[0044] Figure 8 This figure shows the changes in cellular transcriptional characteristics and the enrichment of early gonadal-related pathways during the N5B50C3 group induction process.

[0045] Figure 9 This figure shows the results of a comparison of transcriptional characteristics and functional enrichment analysis between in vivo developmental reference cells and in vitro induced cells.

[0046] Figure 10 The image shows the RT-qPCR results of gonadal lineage-related marker genes after induction in the N5B50C3 group. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0048] In this embodiment, the procedures involved in human pluripotent stem cell culture, passage, induction, medium change, sampling, RNA extraction, reverse transcription, real-time quantitative PCR, immunofluorescence staining, RNA sequencing library construction, and sequencing can all be performed under aseptic conditions. Cell seeding, medium change, passage, addition of inducing factors, and sample collection can be completed in a laminar flow hood or biosafety cabinet; culture dishes, pipette tips, centrifuge tubes, and culture-related consumables are preferably sterile; cell culture conditions can be 37°C, 5% CO2, or a humidified incubator.

[0049] like Figure 1As shown, this embodiment uses human embryonic stem cells as the starting cells, which are sequentially induced by proto-stripe cells and intermediate mesoderm-like cells. The optimal concentrations of NCAM1 and BMP7 are then determined through single-factor screening and combined with CHIR99021 to form the N5B50C3 induction system. Subsequently, the induced cells are subjected to RT-qPCR, RNA sequencing, and immunofluorescence staining to evaluate their early gonadal-like cell characteristics.

[0050] Example 1: In vitro induction method for early gonadal-like cell differentiation This embodiment discloses an in vitro method for inducing differentiation of early gonadal-like cells, comprising the following steps: Intermediate mesodermal-like cells were induced and cultured in an in vitro induction culture system to obtain early gonadal-like cells.

[0051] The in vitro induction culture system (i.e., the third induction culture system) in this embodiment uses STEMdiff APEL2 medium as the basal medium and contains soluble recombinant human neural cell adhesion molecule 1 (catalog number: HY-P75932, MedChemExpress), bone morphogenetic protein 7 (catalog number: 120-03P, PeproTech), and CHIR99021 (catalog number: HY-10182, MedChemExpress). Intermediate mesodermal-like cells are placed in the third induction culture system for induction culture for approximately 24-72 hours. In a specific implementation, cell samples are collected at 24, 48, and 72 hours of induction culture to evaluate the induction effect.

[0052] In some embodiments, the final concentration of soluble recombinant human neural cell adhesion molecule 1 in the third induction culture system is 5-20 ng / mL, for example, 5 ng / mL, 10 ng / mL, or 20 ng / mL. The final concentration of bone morphogenetic protein 7 in the third induction culture system is 10-100 ng / mL, for example, 10 ng / mL, 50 ng / mL, or 100 ng / mL. The final concentration of CHIR99021 in the third induction culture system is preferably 3 μM.

[0053] In a preferred embodiment, the in vitro induction culture system comprises 5 ng / mL human neural cell adhesion molecule 1, 50 ng / mL bone morphogenetic protein 7, and 3 μM CHIR99021. This third induction culture system may be referred to as the N5B50C3 induction system.

[0054] In a preferred embodiment, the soluble recombinant human neural cell adhesion molecule 1, bone morphogenetic protein 7, and CHIR99021 are simultaneously added to the STEMdiff APEL2 medium; wherein, the soluble recombinant human neural cell adhesion molecule 1 (NCAM1) is added directly to the STEMdiff APEL2 medium in free form.

[0055] After treating intermediate mesodermal-like cells with the above-mentioned in vitro induction culture system, early gonad-like cells with an early gonad-like state can be obtained.

[0056] Example 2: Obtaining intermediate mesodermal-like cells This embodiment mainly describes the acquisition of intermediate mesoderm-like cells in Example 1, including the following steps: Step 1: Provide human embryonic stem cells (including maintenance culture, passage, and quality control of human embryonic stem cells) This embodiment provides a method for the maintenance culture and quality control of human embryonic stem cells for subsequent induction. In this embodiment, the human embryonic stem cells can be H9 human embryonic stem cells or H1 human embryonic stem cells. Specifically, H9 human embryonic stem cells: Wuhan Pronosai Life Technology Co., Ltd. #CL-0890. H1 human embryonic stem cells: Wuhan Pronosai Life Technology Co., Ltd. #CL-0889.

[0057] The main materials and reagents used in this embodiment include: Matrigel (catalog number: 354277, Corning), mTeSR Plus medium (catalog number: 100-0274, STEMCELL Technologies), Y-27632 (catalog number: HY-10071, MedChemExpress), ReLeSR (catalog number: 100-0483, STEMCELL Technologies), phosphate buffer (catalog number: 14190144, Gibco), sterile culture dishes, sterile centrifuge tubes, sterile pipetting consumables, and routine cell culture equipment.

[0058] An exemplary culture and passaging process includes the following steps: (1) Thaw and dilute Matrigel 354277 according to the manufacturer’s recommended conditions, add it to a 35mm culture dish, and cover the bottom of the culture dish to form a coating layer suitable for the growth of human embryonic stem cells; (2) Remove excess coating solution, seed H9 or H1 human embryonic stem cells into a culture dish coated with Matrigel 354277, and add mTeSR Plus medium for culture; (3) After cell seeding, add 10 μM Y-27632 to the culture medium and incubate overnight; (4) The next day, remove the culture medium containing Y-27632 and replace it with fresh mTeSR Plus culture medium; (5) Change the culture medium according to the cell state during the culture process, and observe the cell morphology, clonal edges and whether there are obvious differentiation areas under a microscope; (6) After the cells enter the logarithmic growth phase, the subsequent induction experiment begins; (7) Remove the old culture medium and gently wash the cells with phosphate buffer; (8) Add ReLeSR and treat the cells according to the manufacturer's instructions; (9) After the cell clone edges loosen, remove ReLeSR and add fresh mTeSR Plus medium; (10) Gently blow or tap the culture dish to allow the cells to detach in cell clusters of appropriate size; (11) Seed the cells into new Matrigel 354277 coated culture dishes and add 10 μM Y-27632 for overnight culture.

[0059] In some implementations, the human embryonic stem cells used for induction experiments are passaged less than 20 times. In some implementations, the cells undergo routine mycoplasma testing before induction to confirm the absence of mycoplasma contamination before being used in subsequent experiments.

[0060] Step 2: Obtain intermediate mesodermal-like cells; This step involves a method for obtaining intermediate mesoderm-like cells from human embryonic stem cells. These intermediate mesoderm-like cells can serve as the starting cells for subsequent early gonadal-like cell induction.

[0061] In this embodiment, the induction culture of intermediate mesodermal-like cells includes two consecutive stages: induction of protostyloid cells and induction of intermediate mesodermal-like cells.

[0062] The main reagents included: STEMdiff APEL2 medium (catalog number: 05275, STEMCELL Technologies); activin A (catalog number: 10429-HNAH, Sino Biological); bone morphogenetic protein 4 (catalog number: CR93, Novoprotein); CHIR99021 (catalog number: HY-10182, MedChemExpress); fibroblast growth factor 2 (catalog number: 100-18B, PeproTech); PIK90 (catalog number: S1187, Selleck); A-83-01 (catalog number: SML0788, Sigma); and phosphate-buffered saline (catalog number: 14190144, Gibco). All reagents were purchased commercially and stored and used according to the product instructions.

[0063] For example, the induction process of proto-stripe cells and intermediate mesodermal-like cells includes the following steps: a. Select human embryonic stem cells that are in good condition and in the logarithmic growth phase from step 1; b. Aspirate the mTeSR Plus culture medium and gently wash the human embryonic stem cells with phosphate buffer; c. Add the first induction culture system, which is based on STEMdiff APEL2 medium and contains 30 ng / mL activin A, 40 ng / mL bone morphogenetic protein 4, 6 μM CHIR99021, 20 ng / mL fibroblast growth factor 2 and 100 nM PIK90; d. Human embryonic stem cells and the first induction culture system were placed in an incubator and cultured for 24 hours to obtain original strip-like cells; e. After the original strip-like cells have been induced, remove the first induction culture system; f. Add the second induction culture system, which is based on STEMdiff APEL2 medium and contains 3 ng / mL bone morphogenetic protein 4, 20 ng / mL fibroblast growth factor 2 and 1 μM A-83-01; g. Continue culturing the cells in an incubator for 24 hours to obtain intermediate mesodermal-like cells.

[0064] Example 3: An in vitro induction and culture system for early gonadal-like cells This embodiment includes screening the concentrations of early gonadal-inducing factors (soluble recombinant human neural cell adhesion molecule-1, bone morphogenetic protein 7, and CHIR99021) to obtain an optimal induction combination. The screening can be performed on intermediate mesoderm-like cells, testing different concentrations of soluble recombinant human neural cell adhesion molecule-1 and bone morphogenetic protein 7 separately, and further establishing a combined induction group of soluble recombinant human neural cell adhesion molecule-1, bone morphogenetic protein 7, and CHIR99021. The optimal induction combination is determined by comparing the expression of early gonadal-related markers under different induction conditions.

[0065] The cells used in this example were intermediate mesodermal-like cells obtained according to Example 2. The main reagents used included: STEMdiff APEL2 medium, soluble recombinant human neural cell adhesion molecule 1, bone morphogenetic protein 7, CHIR99021, and reagents required for subsequent detection.

[0066] The main reagents used in the embodiments of this invention and their sources are as follows: STEMdiff APEL2 medium (Catalog No.: 05275, STEMCELL Technologies); Soluble recombinant human neural cell adhesion molecule 1 (Catalog No.: HY-P75932, MedChemExpress). Bone morphogenetic protein 7 (Catalog No.: 120-03P, PeproTech).

[0067] All reagents were purchased through commercial channels and stored and used in accordance with the product instructions.

[0068] An example grouping method is as follows: a. Factor-free control group: No soluble recombinant human neural cell adhesion molecule 1, bone morphogenetic protein 7 and CHIR99021 were added, and only the basic culture conditions (STEMdiff APEL2 medium) were maintained. b. NCAM1 single-factor concentration screening group: 5 ng / mL, 10 ng / mL or 20 ng / mL soluble recombinant human neural cell adhesion molecule 1 were added to STEMdiff APEL2 medium, respectively, without the addition of bone morphogenetic protein 7 and CHIR99021; c. BMP7 single-factor concentration screening group: 10 ng / mL, 50 ng / mL or 100 ng / mL bone morphogenetic protein 7 were added to STEMdiff APEL2 medium, respectively, without adding soluble recombinant human neural cell adhesion molecule 1 and CHIR99021. d. N5B50C3 three-factor induction group (i.e. N5B50C3 group): Based on the results of single-factor concentration screening, 5 ng / mL soluble recombinant human neural cell adhesion molecule 1, 50 ng / mL bone morphogenetic protein 7 and 3 μM CHIR99021 were added simultaneously. e. The two-factor control group (i.e., the B50C3 group) with NCAM1 removed: 50 ng / mL bone morphogenetic protein 7 and 3 μM CHIR99021 were added to STEMdiff APEL2 medium, without the addition of soluble recombinant human neural cell adhesion molecule 1. f. BMP7-removed factor 2 control group (i.e., N5C3 group): 5 ng / mL soluble recombinant human neural cell adhesion molecule 1 and 3 μM CHIR99021 were added to STEMdiff APEL2 medium, without the addition of bone morphogenetic protein 7.

[0069] An exemplary screening and verification process includes the following steps: g. Obtain intermediate mesodermal-like cells according to Example 2; h. The intermediate mesodermal-like cells obtained in the same batch were first divided into a factor-free control group, an NCAM1 single-factor concentration screening group, and a BMP7 single-factor concentration screening group; i. Each single-factor concentration screening group was given the corresponding concentration of soluble recombinant human neural cell adhesion molecule 1 or bone morphogenetic protein 7, and cultured for a longer period of time. j. Collect cell samples at 24, 48 and / or 72 hours after induction to detect the expression levels of GATA4, WT1, NR5A1 / SF1, LHX9 and / or other early gonad-associated marker genes; k. Based on the results of single-factor concentration screening, 5 ng / mL of soluble recombinant human neural cell adhesion molecule 1 and 50 ng / mL of bone morphogenetic protein 7 were determined as the preferred concentrations; l. 5 ng / mL soluble recombinant human neural cell adhesion molecule 1, 50 ng / mL bone morphogenetic protein 7 and 3 μM CHIR99021 were combined for the induction of intermediate mesodermal-like cells to form the N5B50C3 three-factor optimal induction combination; m. Based on the optimal induction combination of N5B50C3 three factors, two-factor control groups with the removal of soluble recombinant human neural cell adhesion molecule 1 and two-factor control groups with the removal of bone morphogenetic protein 7 were set up respectively. n. Compare the N5B50C3 three-factor optimal induction combination with the above two-factor control combination to evaluate the expression levels of early gonad-related marker genes and / or marker proteins under different treatment conditions.

[0070] The induction effect was evaluated by comparing the expression levels of one or more marker genes among GATA4, WT1, NR5A1 / SF1, and LHX9 in different treatment groups.

[0071] The results of this embodiment show that, as Figure 3 As shown, in the single-factor concentration screening, 5 ng / mL soluble recombinant human neural cell adhesion molecule-1 and 50 ng / mL bone morphogenetic protein 7 exhibited superior induction effects. Further, 5 ng / mL soluble recombinant human neural cell adhesion molecule-1, 50 ng / mL bone morphogenetic protein 7, and 3 μM CHIR99021 were combined to form the N5B50C3 induction system. Compared with the two-factor control combination that removed soluble recombinant human neural cell adhesion molecule-1 or bone morphogenetic protein 7, the N5B50C3 induction system more effectively promoted the expression of early gonadal-related markers. Therefore, this combination was identified as the preferred induction system and used in subsequent early gonadal-like cell induction experiments. Figure 3 As shown, the single-factor concentration screening results of NCAM1 and BMP7 revealed changes in the expression of early gonadal-related marker genes after different concentration treatments, with 5 ng / mL NCAM1 and 50 ng / mL BMP7 showing the best concentrations. Figure 4 As shown, compared with the factorless control group and the two-factor control group, the N5B50C3 combination can significantly increase the expression of early gonad-related marker genes.

[0072] This example is sufficient to demonstrate that NCAM1 is not a non-functional additive, but rather makes a real contribution to the induction effect of the complete culture system.

[0073] Example 4: Early gonadal-like cells induced by N5B50C3 and their comparison with the factor-free control system This embodiment provides a method for obtaining early gonadal-like cells using the N5B50C3 induction system, and sets up a factor-free control system to compare the induction effect.

[0074] In this embodiment, the intermediate mesodermal-like cells obtained according to Example 2 are divided into the following two groups: FF group: Factor-free control group, using STEMdiff APEL2 medium as the basic culture system, without the addition of soluble recombinant human neural cell adhesion molecule 1, bone morphogenetic protein 7 and CHIR99021.

[0075] N5B50C3 group: 5 ng / mL soluble recombinant human neural cell adhesion molecule 1, 50 ng / mL bone morphogenetic protein 7 and 3 μM CHIR99021 were added to STEMdiff APEL2 medium.

[0076] An exemplary induction process includes the following steps: a. Obtain intermediate mesodermal-like cells according to Example 2; b. Aspirate the induction medium for intermediate mesodermal-like cells and gently wash the cells with phosphate-buffered saline. c. Add the corresponding culture systems to the FF group and the N5B50C3 group respectively; d. Continue culturing the cells in the incubator; e. Cell samples were collected at 24, 48, and 72 hours after induction. f. The collected cell samples are used for real-time quantitative PCR and RNA sequencing analysis; in some embodiments, a portion of the sample may also be used for protein level detection.

[0077] Each sample represents an independent biological replicate, which can be defined as an independent differentiation experiment initiated from an independently resuscitated batch of human embryonic stem cells. In some embodiments, at least three biological replicates are set up per group; in real-time quantitative PCR detection, three technical replicates are set up for each biological replicate.

[0078] The results of this embodiment show that, as Figure 5 As shown, compared with the FF group, the expression levels of early gonad-related markers were increased in the N5B50C3 group, indicating that the N5B50C3 induction system can promote the transformation of intermediate mesoderm-like cells into early gonad-like cells. Figure 2 The images shown are representative bright-field images of human embryonic stem cells, protostomatal cells, intermediate mesoderm-like cells, and early gonadal-like cells from day 1 to day 6 of induction. During induction, cell morphology gradually changes, exhibiting denser sheet-like and locally aggregated states. Figure 5 As shown, when H9 human embryonic stem cells were used as the starting cells, compared with H9 human embryonic stem cells and the corresponding factor-free control group, the N5B50C3 group showed increased expression of early gonadal-related marker genes such as GADD45G, GATA4, WT1, NR5A1 / SF1, LHX9, and EMX2 at different induction time points. Figure 6 As shown, similar expression changes were obtained when H1 human embryonic stem cells were used as the starting cells, indicating that the N5B50C3 induction system can be applied to different human embryonic stem cell lines. Furthermore, compared with H1 human embryonic stem cells and the corresponding factor-free control group, the N5B50C3 group increased the expression of early gonadal-related marker genes such as GADD45G, GATA4, WT1, NR5A1 / SF1, LHX9, and EMX2 on days 1, 2, and / or 3 of induction, suggesting that the N5B50C3 induction system is reproducible in different human embryonic stem cell lines.

[0079] Example 5: Detection of early gonadal-like cell characteristics by RT-qPCR, immunofluorescence, and RNA sequencing This embodiment provides a method for detecting and identifying early gonadal-like cells. The detection method can be used to evaluate the characteristics of early gonadal-like cells under different induction conditions in Examples 3 and 4.

[0080] 5.1 RNA extraction, reverse transcription, and real-time quantitative PCR Real-time quantitative PCR can be used to detect the expression of early gonadal-related marker genes in cells after induction. Preferred marker genes for detection include one or more of GATA4, WT1, NR5A1 / SF1, and LHX9.

[0081] An exemplary detection process includes the following steps: a. Collect human embryonic stem cells, intermediate mesoderm-like cells, FF group cells, and N5B50C3 group cells at different time points after induction; b. Process cells according to the RNA extraction kit instructions, performing cell lysis, DNA removal, RNA adsorption, protein removal, and washing sequentially to obtain total RNA; c. RNA integrity was detected by agarose gel electrophoresis; d. Reverse transcribe the extracted RNA into cDNA according to the instructions of the reverse transcription kit; e. Prepare a 20 μL real-time quantitative PCR reaction system, which includes 2×Fast Real qPCRPremix, nuclease-free water, cDNA template and specific primers; f. Add the reaction mixture to a 96-well plate and detect it using a real-time quantitative PCR system; g. Based on the obtained Ct value, the relative expression level of the target gene is calculated using the ΔΔCt method; h. Statistical analysis was performed on the differences in target gene expression among different experimental groups.

[0082] In some implementations, each experimental group includes three biological replicates and three technical replicates. In some implementations, multiple group comparisons may be performed using one-way ANOVA combined with Tukey's multiple comparison test to assess statistical differences. All data are expressed as mean ± standard deviation (Mean ± SD). Multiple group comparisons were performed using one-way ANOVA combined with Tukey's multiple comparison test, and a p-value < 0.05 was considered statistically significant.

[0083] In addition, to evaluate whether the induced early gonad-like cells have the ability to further initiate gonadal sex differentiation-related transcriptional programs, RT-qPCR was used to detect gonadal lineage-related marker genes in the cells on day 3 of induction. Specifically, the male gonadal lineage-related marker genes SOX9 and AMH were detected in the H1 human embryonic stem cell-derived induction system, while the female gonadal lineage-related marker gene FOXL2 was detected in the H9 human embryonic stem cell-derived induction system.

[0084] like Figure 10 As shown, compared with the corresponding human embryonic stem cell group and the FF group on day 3 of induction, the N5B50C3 group derived from H1 showed increased expression of male gonadal cell lineage-related marker genes, SOX9, and AMH on day 3 of induction, while FOXL2 expression was increased in the N5B50C3 group derived from H9. These results indicate that, compared with the corresponding factor-free control group, the N5B50C3 group derived from H9 showed increased expression of the female gonadal cell lineage-related marker gene FOXL2 on day 3 of induction. This suggests that the N5B50C3 induction system, in addition to initiating early gonadal-related transcriptional programs, can also promote the expression of male and female gonadal cell lineage-related marker genes, thus the induced early gonad-like cells have the potential to further differentiate into different sex-related gonadal lineages.

[0085] In some implementations, one or more of SOX9, AMH, and FOXL2 may be used as supplementary indicators for evaluating the further differentiation status of early gonadal-like cells, but this does not limit the number of cells obtained by induction to mature supporting cell-like cells or granular cell-like cells.

[0086] The sequences of specific primers for genes such as GATA4, WT1, NR5A1 / SF1, and LHX9 in this embodiment are shown in Table 1: Table 1. Specific primer sequences for early gonadaloid cell evaluation indicators: ; 5.2 Immunofluorescence detection This embodiment further employs immunofluorescence staining to detect the expression of early gonadal-associated marker proteins in cells after induction. GATA4 and WT1 proteins are preferably detected.

[0087] An exemplary immunofluorescence staining procedure includes the following steps: a. Cells of the N5B50C3 group induced on day 3 were cultured in culture dishes suitable for microscopic imaging. After induction, they were fixed and stained. After the culture medium was removed, the cells were washed with phosphate buffer. b. Add 4% paraformaldehyde to fix for 15 minutes, wash the cells with phosphate buffer after fixation, and then add 0.3% Triton X-100 to permeate for 10 minutes; c. After permeabilization, block with 5% BSA for 30 minutes, then add GATA4 primary antibody and / or WT1 primary antibody, and incubate overnight at 4°C; d. After washing the cells the next day, add the corresponding Alexa Fluor-labeled secondary antibody and incubate for 1 hour, then add DAPI to counterstain the cell nuclei; e. After washing with PBS or PBST, observe and record cell morphology and fluorescence signals under a confocal microscope.

[0088] The antibody information for GATA4 primary antibody and WT1 primary antibody in this embodiment is shown in Table 2 below: Table 2. Antibody information for GATA4 primary antibody and WT1 primary antibody: ; This embodiment quantifies the induction effect by statistically analyzing the proportions of GATA4-positive cells, WT1-positive cells, or GATA4 / WT1 double-positive cells. For example... Figure 7 As shown, GATA4 and WT1 positive signals can be detected in the cells induced by the N5B50C3 group. DAPI was used to label the cell nucleus. The composite figure shows that the GATA4 or WT1 signal corresponds to the distribution of the cell nucleus, suggesting that the induced cells have the protein expression characteristics of early gonadal-like cells.

[0089] 5.3 RNA sequencing validation RNA sequencing can be used to evaluate changes in cell state during induction at the overall transcriptional level and to determine whether early gonadal-associated transcriptional programs are activated.

[0090] a. Collect human embryonic stem cell samples, intermediate mesoderm-like cell samples, FF group cell samples, and N5B50C3 group cell samples on day 1, day 2 and day 3 after induction, and extract total RNA from them respectively; b. After quality testing of the extracted total RNA, eukaryotic mRNA was enriched using Oligo(dT) magnetic beads, and fragmentation buffer was added to fragment the mRNA; c. Using fragmented mRNA as a template, first-strand cDNA is synthesized using random primers, and second-strand cDNA is further synthesized to obtain double-stranded cDNA; d. The double-stranded cDNA is purified, its ends are repaired, A-tails are added, adapters are ligated, fragments are screened, and PCR amplification is performed to construct a sequencing library; in some implementations, approximately 350 bp fragments are recovered for subsequent library amplification. e. Quality control of sequencing libraries, including using Qubit 3.0 to detect library concentration, using Agilent 2100 to detect library fragment distribution, and using a real-time quantitative PCR system to detect the effective concentration of the library; f. Mix the qualified libraries according to the effective concentration and target data volume, and perform high-throughput sequencing; in some implementations, the sequencing method is 150 bp paired-end sequencing; g. Further analysis of the sequencing data was conducted to evaluate the initiation of early gonad-related transcriptional programs during induction by comparing the transcriptional characteristics of human embryonic stem cells, intermediate mesoderm-like cells, FF group cells, and cells at different time points after N5B50C3 induction.

[0091] This embodiment evaluates the initiation of early gonad-related transcriptional programs during induction by comparing the transcriptional characteristics of human embryonic stem cells, intermediate mesoderm-like cells, FF group cells, and N5B50C3 group early gonad-like cells. In a further embodiment, functional enrichment analysis can be performed on differentially expressed genes to evaluate whether biological processes related to gonadal development are enhanced. For example... Figure 8 As shown, during the induction process of the N5B50C3 group, the overall transcriptional characteristics of the cells gradually deviated from the state of human embryonic stem cells and intermediate mesoderm-like cells, and showed a high correlation with early gonad-related cells in vivo. At the same time, the enrichment of the "Gonad development" related pathway in the cells gradually increased from day 1 to day 3 of induction. Figure 9 As shown in the figure, the early gonad-related gene module gradually increases during in vitro induction, accompanied by elevated expression of early gonad-related genes such as GATA4, WT1, LHX9, and GADD45G. Functional enrichment results indicate that this gene module is associated with biological processes such as gonadal development, sexual differentiation, and extracellular matrix tissue formation. This sufficiently demonstrates that the present invention can induce the development of intermediate mesoderm-like cells into an in vitro early gonadal development model using an in vitro induction culture system formed by the N5B50C3 combination.

[0092] In summary, Examples 1 to 5 illustrate the in vitro induction differentiation method for early gonadal-like cells of the present invention from aspects such as maintaining the initial cells, obtaining intermediate mesoderm-like cells, optimizing the concentration of inducing factors, induction with preferred combinations and control comparisons, and multi-level detection and verification. The above examples demonstrate that the method of the present invention has the characteristics of a clear induction pathway, well-defined parameter basis, a complete detection system, and good application scalability, and can be used to obtain induced cells with early gonadal-like characteristics.

Claims

1. An in vitro induction and culture system for early gonadal-like cells, characterized in that, The in vitro induction culture system contains bone morphogenetic protein 7, CHIR99021, and soluble recombinant human neural cell adhesion molecule 1.

2. The in vitro induction and culture system for early gonadal-like cells according to claim 1, characterized in that, The concentration of the soluble recombinant human neural cell adhesion molecule 1 is 5-20 ng / mL, the concentration of the bone morphogenetic protein 7 is 10-100 ng / mL, and the concentration of CHIR99021 is 3 μM.

3. The in vitro induction and culture system for early gonadal-like cells according to claim 2, characterized in that, The concentration of the soluble recombinant human neural cell adhesion molecule 1 is 5 ng / mL, and the concentration of the bone morphogenetic protein 7 is 50 ng / mL.

4. The in vitro induction and culture system for early gonadal-like cells according to claim 1, characterized in that, The in vitro induction culture system also includes a basal culture medium, which is STEMdiff APEL2 medium.

5. A method for in vitro induction of early gonadal-like cell differentiation, characterized in that, Includes the following steps: Intermediate mesodermal-like cells are induced and cultured in the in vitro induction culture system described in any one of claims 1-4 to obtain early gonadal-like cells.

6. The method for in vitro induction of early gonadal-like cell differentiation according to claim 5, characterized in that, The intermediate mesodermal-like cells were obtained through the following steps: Provide human embryonic stem cells; The human embryonic stem cells were cultured in a first induction culture system to obtain original strip-like cells; The original strip-like cells were cultured in a second induction culture system to obtain intermediate mesoderm-like cells.

7. The method for in vitro induction of early gonadal-like cell differentiation according to claim 6, characterized in that, The first induction culture system uses STEMdiff APEL2 medium as the basal medium and contains activin A, bone morphogenetic protein 4, CHIR99021, fibroblast growth factor 2 and PIK90.

8. The method for in vitro induction of early gonadal-like cell differentiation according to claim 7, characterized in that, In the first induction culture system, the concentration of activin A is 30 ng / mL, the concentration of bone morphogenetic protein 4 is 40 ng / mL, the concentration of CHIR99021 is 6 μM, the concentration of fibroblast growth factor 2 is 20 ng / mL, and the concentration of PIK90 is 100 nM.

9. The method for in vitro induction of early gonadal-like cell differentiation according to claim 8, characterized in that, The second induction culture system uses STEMdiff APEL2 medium as the base medium and contains bone morphogenetic protein 4, fibroblast growth factor 2 and A-83-01.

10. The method for in vitro induction of early gonadal-like cell differentiation according to claim 9, characterized in that, In the second induction culture system, the concentration of bone morphogenetic protein 4 is 3 ng / mL, the concentration of fibroblast growth factor 2 is 20 ng / mL, and the concentration of A-83-01 is 1 μM.

11. The method for in vitro induction of early gonadal-like cell differentiation according to claim 5, characterized in that, The induction culture time is 24-72 hours.

12. An early population of gonadal-like cells, characterized in that, The early gonadal-like cell population was prepared by the in vitro induced differentiation method according to any one of claims 6-11.

13. The application of the in vitro induction culture system according to any one of claims 1-4 or the early gonad-like cell population according to claim 12 in constructing an in vitro early gonadal development model.

Images