Method for establishing human embryo stem cell line

Abstract

The present application relates to a method of deriving a human embryonic stem cell (hESC) line under feeder-free and heterologous conditions.

Description

Technical Field

[0001] This application relates to the field of stem cells, and more specifically to a standardized method that supports the efficient derivation of clinical-grade human embryonic stem cells under conditions of no feeder layer and no allogeneic origin. Background Technology

[0002] Human pluripotent stem cells (hPSCs) can self-renew indefinitely and differentiate into any cell type in the body, making them highly pluripotent for developing therapies to treat a wide range of diseases. Human embryonic stem cells (hESCs) are a type of hPSC derived directly from early embryos. These embryos have not yet undergone extensive cell division or been exposed to various environmental factors, resulting in minimal accumulation of somatic mutations and the absence of aging-related phenotypic characteristics. Furthermore, as naturally occurring pluripotent cells, hESCs do not require complex reprogramming to achieve pluripotency. Therefore, they do not pose any risk of genetic or epigenetic abnormalities associated with somatic cell reprogramming. Consequently, hESCs are considered a promising cell source for generating a wide variety of human cells for clinical use.

[0003] Previous studies have commonly used human feeder layers to establish clinical-grade human embryonic stem cell (hESC) lines. However, feeder cell-based systems exhibit batch-to-batch variability due to differences in feeder cell preparation methods and sources. Feeder cell-free and xenogeneic component-free culture conditions provide a more consistent and standardized culture environment, crucial for reliability and reproducibility—key requirements for translating stem cell therapy into clinical applications. Recent studies have utilized well-defined extracellular matrix to achieve xenogeneic cell-free differentiation of human embryonic stem cells (hESCs). However, hESC differentiation efficiency is low. Furthermore, there are no reports of differentiating hESCs from low-quality embryos under feeder cell-free and xenogeneic conditions. Summary of the Invention

[0004] This application provides an advanced method for establishing human embryonic stem cell lines efficiently and in a standardized manner under feeder-free and xenogeneic conditions. As pluripotent stem cells naturally occurring during embryonic development, human embryonic stem cells remain an irreplaceable cell source for basic research and clinical applications. The efficient isolation, culture, and storage of human embryonic stem cell lines from frozen-thawed or fresh normal and diseased embryos will provide a valuable cell source for regenerative medicine, disease modeling, and therapeutic development.

[0005] On one hand, this application provides a method for deriving a human embryonic stem cell (hESC) line, comprising the following steps: a) providing a human blastocyst; b) mechanically isolating the inner cell mass (ICM) from the blastocyst in step a); c) seeding the isolated ICM in step b) onto a culture dish coated with a suitable extracellular matrix substrate, wherein the suitable extracellular matrix is ​​capable of supporting the adhesion, survival, and growth of the ICM under feeder-free conditions; d) culturing the seeded ICM in step c) in a heterologous, chemically defined, feeder-free cell culture medium until it forms an initial hESC clone; e) proliferating the initial hESC clone formed in step d) to obtain an hESC line.

[0006] In some implementations, the blastocyst is a clinically surplus or discarded blastocyst with a morphologically distinguishable ICM that is graded A, B, or C according to the Gardner grading system.

[0007] In some implementations, the blastocyst is a blastocyst that has developed for no more than 14 days after in vitro fertilization (IVF).

[0008] In some implementations, the blastocyst is a blastocyst that has developed for 5-8 days after in vitro fertilization (IVF).

[0009] In some implementations, the mechanical separation in step b) does not employ any immunosurgical method that relies on antibodies or complement of non-human animal origin.

[0010] In some implementations, the mechanical separation in step b) is performed using laser-assisted micromanipulation techniques.

[0011] In some implementations, step b) includes using a laser to cut trophoblast cells near the ICM to separate them from the ICM.

[0012] In some implementations, the laser described in step b) is a non-contact infrared laser with a pulse duration of 30 ms to 40 ms.

[0013] In some implementations, when the inner cell mass is tightly connected to the trophoblast cells and difficult to separate, the duration of the laser pulse in step b) is extended to no more than 60 ms.

[0014] In some implementations, the laser drilling operation is performed in real time under a microscope, with the objective lens of the microscope switched to a dedicated laser drilling lens to ensure that the focal plane is aligned with the trophoblast cell junction.

[0015] In some implementations, the laser is applied only to the trophoblast cell junction region surrounding the inner cell mass, and the inner cell mass itself is avoided from being irradiated.

[0016] In some implementations, the laser-assisted separation operation is performed entirely within microdroplets containing fertilization culture medium.

[0017] In some embodiments, the droplets containing the fertilization culture medium are maintained at 37°C and 5% CO2 throughout the culture process.

[0018] In some embodiments, the fertilization culture medium contains the ROCK inhibitor Y27632.

[0019] In some embodiments, the concentration of the ROCK inhibitor Y27632 in the fertilization culture medium is 1 μM-10 μM.

[0020] In some implementations, the laser operation in step b) includes the use of a laser film-breaking device.

[0021] In some implementations, step b) is performed entirely using clinical-grade reagents and consumables, and the entire process is carried out in a heterologous blastocyst culture medium.

[0022] In some implementations, the culture dish described in step c) is coated with laminin.

[0023] In some embodiments, the culture dish described in step c) is coated with laminin-521 (LN521).

[0024] In some embodiments, the coating concentration of said LN521 is 0.25 μg / cm³. 2 Up to 0.75 μg / cm 2 .

[0025] In some embodiments, the preferred coating concentration of said LN521 is 0.50 μg / cm³. 2 .

[0026] In some implementations, step d) includes adding the ROCK inhibitor Y27632 to the cell culture medium from the start of ICM culture and culturing the ICM therein for 4 to 14 days.

[0027] In some implementations, step d) includes not changing the cell culture medium for the first 4 days from the start of ICM culture, and changing the cell culture medium regularly starting from day 5.

[0028] In some implementations, step d) includes changing the cell culture medium every 2 days, starting from day 5, from the start of ICM culturing.

[0029] In some implementations, step d) includes adding the ROCK inhibitor Y27632 to the cell culture medium from the start of ICM culture, culturing the ICM therein for 7-10 days, and removing the Y27632 after 7-10 days.

[0030] In some embodiments, the concentration of the ROCK inhibitor Y27632 is from 1 μM to 15 μM.

[0031] In some embodiments, the preferred concentration of the ROCK inhibitor Y27632 is 5 μM-10 μM.

[0032] In some implementations, the cell culture medium in step d) is TeSR™-AOF medium supplemented with human serum albumin.

[0033] In some implementations, step e) includes passage of the initial hESC clone using a heterologous, GMP-grade enzymatic digestion solution.

[0034] In some implementations, step e) is performed on days 12-16 after the ICM vaccination described in step c).

[0035] In some implementations, the hESC line obtained in step e) undergoes at least three passages.

[0036] In some implementations, the hESC line is cryopreserved as a seed bank after three generations.

[0037] In some embodiments, the hESC system is cryopreserved in cryovials, with each cryovial containing an order of magnitude of 10. 5 Up to 10 7 . cells.

[0038] In some embodiments, the enzymatic hydrolysate is a GMP-grade enzymatic hydrolysate, ReleSR™.

[0039] In some implementations, the efficiency of establishing the hESC lineage from the human blastocysts provided in step a), which are Gardner grade A or B, through steps b) to e) is not less than 60%.

[0040] In some implementations, the efficiency of establishing the hESC lineage from the Gardner grade C human blastocysts provided in step a) through steps b) to e) is not less than 20%.

[0041] In some implementations, the established hESC line has at least one characteristic selected from the group consisting of: a) normal karyotype, b) ability to express pluripotency markers, and c) ability to differentiate into three germ layers.

[0042] In some implementations, the hESC line established is a clinical-grade cell line.

[0043] On the other hand, this application provides initial human embryonic stem cell (hESC) colonies obtained according to the method described in this application, wherein the initial hESC colonies are derived under conditions of no feeder layer and no allogeneic source, have proliferative capacity and exhibit pluripotency.

[0044] On the other hand, this application provides a stable human embryonic stem cell (hESC) line obtained according to the method described in this application, wherein the stable hESC line is derived under conditions of no feeder layer and no allogeneic source, and has a normal karyotype, the ability to express pluripotency markers and / or the ability to differentiate into three germ layers.

[0045] On the other hand, this application provides a cell culture composition comprising the initial hESC colonies described in this application and / or the stable hESC line described in this application, a laminin-521 (LN521) coated surface, and a heterologous, chemically defined culture medium supplemented with ROCK inhibitors.

[0046] On the other hand, this application provides the use of the initial hESC colonies and / or the stable hESC lines described in this application in the preparation of cell products for cell therapy, disease modeling, or drug screening.

[0047] On the other hand, this application provides the use of the initial hESC colonies and / or the stable hESC lines described in this application for directed differentiation into endoderm, mesoderm, or ectoderm lineage cells.

[0048] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0049] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0050] Figure 1A-1B The images show crystal violet staining (A) and quantitative analysis (B) of human pluripotent stem cell (hPSC) clones under different concentrations of substrate LN521 and VN conditions in AOF and E8 media. Figure 1B Results are expressed as mean ± standard deviation (SD); *, p < 0.05; ns, no significant difference.

[0051] Figure 1C The diagram shown illustrates laser-assisted separation of the intracellular matrix (ICM) in the blastocyst, where the dashed lines represent the laser cutting trajectory.

[0052] Figure 1D The figure shows the initial clones formed on LN521 medium with or without Y27632 treatment in AOF medium at different days after ICM inoculation (Dpp) (scale bar in 50 µm).

[0053] Figure 1E The image shows the morphology of the initial hESC clone (P0) and the hESC clone after one passage (P1) 12 days after ICM inoculation, indicating that the initial clone formation can be passed down mechanically (scale bar unit is 100 µm).

[0054] Figure 2 This shows the isolation and identification process for clinical-grade hESC lineages.

[0055] Figures 3A-3C The images show the morphology (A), karyotype analysis (B), and alkaline phosphatase staining (C) of HES1 and HES2 (scale bar unit: 100 μm).

[0056] Figure 3D The results show the expression of pluripotency markers as analyzed by immunofluorescence. Transcription factors OCT4 and NANOG, and cell surface markers SSEA4 and TRA-1-60 were detected. Cell nuclei were counterstained with Hoechst 33342 (scale bar unit: 100 μm).

[0057] Figure 3E The figures show the expression levels of pluripotency genes (NANOG, SOX2, REX1, SSEA3, LIN28, GDF3, and FGF4) as assessed by qRT-PCR. Human embryonic stem cell line H9 (positive control) and differentiated cell lines K562 (human leukemia cell line) and A549 (human lung adenocarcinoma cell line) were included to validate experimental specificity (n = 3; **, p < 0.005; ***, p < 0.0005).

[0058] Figure 3FThe result shows the purity as assessed by flow cytometry.

[0059] Figure 3G The display shows the cell doubling time analysis for HES1 and HES2.

[0060] Figure 3H The image shows the formation of the three germ layers in a teratoma section stained with H&E (scale bar unit: 50 μm).

[0061] Figure 4A The figures show flow cytometry results of endoderm precursor cell markers (FOXA2 and SOX17) and pancreatic β-cell markers (NKX6.1 and C-peptide) in stages 1 (top 4 figures) and 6 (bottom 4 figures) of pancreatic β-cell differentiation during HES1 and HES2. DE1 represents definitive endoderm cells derived from HES1 in stage 1, and DE2 represents definitive endoderm cells derived from HES2 in stage 1. β-cell 1 represents pancreatic β-cells derived from HES1 in stage 6, and β-cell 2 represents pancreatic β-cells derived from HES2 in stage 6.

[0062] Figure 4B The image shows the morphology of pancreatic islets during stage 6 differentiation (scale bar unit: 300 μm).

[0063] Figure 4C The results shown are immunofluorescence staining results for β-cell markers. Cell nuclei were stained with Hoechst 33342 (scale bar unit: 50 μm).

[0064] Figure 4D This displays the results of a glucose-stimulated insulin secretion (GSIS) assay. Insulin secretion levels from HES1 and HES2-derived β-cells were measured at low (2.8 mM) and high (16.7 mM) glucose concentrations. The glucose stimulation index values ​​are indicated above the bars.

[0065] Figure 4E The results show immunofluorescence staining of cardiomyocyte markers (cTNT and ACTN) in HES1-derived cardiomyocytes (CM1) and HES2-derived cardiomyocytes (CM2). Cell nuclei were stained with Hoechst 33342 (scale bar unit: 100 μm).

[0066] Figure 4F The figure shows the expression of cardiomyocyte marker genes detected by qRT-PCR (n = 3; **, p < 0.005; ***, p < 0.0005).

[0067] Figure 4GThe image shows the immunofluorescence staining results of corneal endothelial cell markers (ZO1 and ATP1A1) from HES1-derived corneal endothelial cells (CEC1) and HES2-derived corneal endothelial cells (CEC2). Cell nuclei were stained with Hoechst 33342 (scale bar unit: 100 μm).

[0068] Figure 4H The figure shows the expression of corneal endothelial cell marker genes detected by qRT-PCR (n = 3; **, p < 0.005, ***, p < 0.0005).

[0069] Figure 5 The diagram shows the short tandem repeats (STRs) of HES1 (A) and HES2 (B). Detailed Implementation

[0070] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification. Invention Details

[0072] 1. Methods for deriving human embryonic stem cell (hESC) lines

[0073] On one hand, this application provides a method for deriving a human embryonic stem cell (hESC) line, comprising the following steps: a) providing a human blastocyst; b) mechanically isolating the inner cell mass (ICM) from the blastocyst in step a); c) seeding the isolated ICM in step b) onto a culture dish coated with a suitable extracellular matrix substrate, wherein the suitable extracellular matrix is ​​capable of supporting the adhesion, survival, and growth of the ICM under feeder-free conditions; d) culturing the seeded ICM in step c) in a heterologous, chemically defined, feeder-free cell culture medium until it forms an initial hESC clone; e) proliferating the initial hESC clone formed in step d) to obtain an hESC line.

[0074] As used in this application and as understood in the art, “embryonic stem cells (ES cells)” refers to pluripotent cells derived from the inner cell mass (ICM) of mammalian embryos at the blastocyst stage. This includes freshly isolated primary cells as well as cells that have been serially cultured to establish stable cell lines. ES cells may be derived from sperm fertilization of an egg or generated through somatic cell nuclear transfer (SCNT), parthenogenesis, androgenesis, or other techniques designed to produce HLA-homogeneous ES cells. Furthermore, ES cells can be derived from zygotes, blastomeres, or blastocysts resulting from the fusion of sperm and egg, nuclear reprogramming (including chromatin reprogramming followed by integration into the plasma membrane), and other advanced cell engineering techniques. Regardless of their origin or specific method of generation, embryonic stem cells are generally identified based on the following key characteristics: a) the ability to differentiate into cell types representing all three germ layers (endoderm, mesoderm, and ectoderm); b) the expression of key pluripotency markers such as OCT4 and alkaline phosphatase; and c) the ability to form teratomas when transplanted into immunodeficient animals.

[0075] As used herein, the term "inner cell mass (ICM)" generally refers to a cluster of cells within a blastocyst embryo that will develop into fetal tissue. In the context of this application, these cells serve as a sustainable source of pluripotent cells under in vitro culture conditions. Furthermore, the term ICM as used herein also encompasses the internal cell population of an embryo produced through a uniparental process, such as parthenogenesis or androgenesis. That is, the ICM may originate from an embryo formed by the activation of cells containing entirely maternal or paternal DNA. This DNA may be human, for example, derived from human oocytes or sperm, and may be genetically modified or unmodified.

[0076] As used herein, the term "mechanical dissociation" generally refers to a methodology for dissociating cell masses, such as separating the inner cell mass (ICM) from the trophoblast of a blastocyst, using primarily physical forces and tools, including but not limited to micromanipulation needles, pipettes, or laser ablation. This approach relies on physical means to achieve precise and controlled dissection, thereby avoiding the introduction of non-human biological or chemical reagents. Therefore, the term explicitly excludes methods that primarily rely on enzymatic digestion (e.g., using streptoprotein or other animal-derived proteases) or immunological techniques (e.g., immunosurgery, which utilizes antibodies and complement proteins derived from non-human animals to lyse trophoblast cells). Mechanical dissociation supports the generation of clinical-grade pluripotent stem cell lines under heterologous conditions by minimizing exposure to exogenous contaminants.

[0077] As used herein, the term "extracellular matrix (ECM) substrate" generally refers to the surface or underlying matrix that supports optimal cell attachment, survival, and growth under in vitro culture conditions. This includes defined, recombinant, or purified protein coatings; such as laminins (e.g., laminin-521), fibronectin, collagen, gelatin, fibronectin, and fragments thereof, as well as complex biopharmaceuticals like Matrigel®. In some cases, suitable ECM substrates may also contain other ECM molecules, such as collagen IV, nestin, heparan sulfate proteoglycans, and may contain a variety of growth factors essential for cell proliferation and pluripotency maintenance, including but not limited to bFGF, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), nerve growth factor (NGF), and TGF-β1. These substrates provide a biomimetic microenvironment that is crucial for culturing pluripotent stem cells under defined, heterologous, or feeder-free conditions.

[0078] As used herein, the term "feeder-free conditions" generally refers to cell culture systems in which cell expansion and maintenance (particularly pluripotent stem cells) do not require co-culture with a layer of supporting feeder cells (such as mouse or human fibroblasts). Instead, these conditions rely on the addition of defined cytokines and growth factors (e.g., TGFβ, bFGF, LIF) to replace the support functions traditionally provided by feeder cells. Feeder-free systems may use animal-derived substrates (e.g., Matrigel®) or defined extracellular matrix components (e.g., fibronectin, collagen, or tessellation) to promote cell attachment and expansion. Under these conditions, human stem cells can be maintained in a substantially undifferentiated and proliferating state without direct contact with feeder cells. As used herein, the term "feeder layer" refers to a confluence of supporting cells (e.g., irradiated or mitotically inactivated fibroblasts) laid on the surface of a culture dish. These cells release nutrients and other factors into the culture medium and provide a physical substrate for other cells (such as pluripotent stem cells) to attach to, thereby promoting their survival and inhibiting differentiation.

[0079] As used herein, the term "culture medium" generally refers to a cell culture environment. Culture media are typically isotonic solutions and may be liquid, gel, or semi-solid, for example, matrices used to provide cell adhesion or support. As used herein, culture media may include components necessary for the nutritional, chemical, and structural support required for cell culture.

[0080] As used herein, the term "heterogeneous-free (XF)" refers to cell culture conditions, reagents, culture media, or surfaces that are completely free of non-human animal material. When applied to culture media, extracellular matrix, or culture systems, the term indicates a composition that is substantially free of any animal-derived components. For human cell culture, any protein or biological material derived from non-human animals (such as mice) is considered a heterologous component. Heterogeneous-free conditions explicitly exclude the use of animal-derived substances, such as mouse feeder cells or Matrigel® (a soluble basement membrane formulation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma containing extracellular matrix proteins, including laminin (as a major component), collagen IV, heparan sulfate proteoglycans, and nestin). By using fully defined, recombinant, or synthetic alternatives, heterologous-free systems ensure the absence of non-human biological material, supporting the development of clinically appropriate cell therapies and reducing the risk of zoonotic contamination or immune responses.

[0081] As used herein, the term "chemically defined (cell) culture medium" generally refers to a cell culture medium in which every component is a known chemical entity with a defined structure and precise concentration. This medium is completely free of indeterminate, complex, or biologically variable components, including tissue extracts, hydrolysates, serum (such as fetal bovine serum), and serum-derived proteins (such as bovine or human serum albumin). Instead, it typically consists of a base formulation (e.g., DMEM, DF12, or RPMI 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy) supplemented with recombinant proteins (e.g., recombinant albumin), chemically synthesized lipids, recombinant insulin, and other fully characterized additives. An exemplary chemically defined culture medium is Essential 8™, which supports robust and reproducible culture of pluripotent stem cells under biologically variable conditions, making it suitable for regulatory-compliant and clinical-grade cell production.

[0082] As used herein, the terms “initial hESC colony” or “initial hESC clone” generally refer to the initial cluster of cells that emerges and proliferates directly from the inner cell mass (ICM) of the plating before the first dissociation and passage event (i.e., passage 0). These cells exhibit typical hESC morphology and express pluripotency markers, but may not yet be genomically stable or expanded into a continuous cell line.

[0083] In some implementations, the blastocyst is a clinically surplus or discarded blastocyst with a morphologically distinguishable ICM that is graded A, B, or C according to the Gardner grading system.

[0084] As used herein, the term "clinically surplus or discarded blastocysts" generally refers to human blastocysts created for reproductive purposes during in vitro fertilization (IVF) treatment, but no longer needed for such purposes, donated for research with informed consent, and originally intended to be discarded. This term explicitly excludes blastocysts created specifically for research purposes.

[0085] As used herein, the term "Gardner grading system" refers to a standardized embryo quality assessment system that evaluates blastocysts based on their blastocyst expansion (stages 1-6), inner cell mass (ICM) quality (grade AC), and trophoblast cell quality (grade AC). Within the scope of this application, an ICM graded "Grade A" is considered excellent and well-defined, with a large number of cells that are clustered and tightly packed; "Grade B" is good and well-defined, with a small number of cells that are loosely clustered, and some cells may be scattered; "Grade C" is poor quality, unclear, or poorly developed, with very few or no cells.

[0086] In some embodiments, the blastocyst is a blastocyst that has developed for no more than 14 days after in vitro fertilization (IVF). For example, the blastocyst is a blastocyst that has developed for 5, 6, 7, 8, 9, or 10 days after in vitro fertilization (IVF).

[0087] In some implementations, the blastocyst is a blastocyst that has developed for 5 or 6 days after in vitro fertilization (IVF).

[0088] In some implementations, the mechanical separation in step b) does not employ any immunosurgical method that relies on antibodies or complement of non-human animal origin.

[0089] As used herein, the term "immunosurgery" generally refers to a specific technique for isolating the inner cell mass (ICM) from a blastocyst-stage embryo. This method involves sequentially exposing the embryo to antibodies—typically produced by immunizing a non-human animal with human cells—and complement proteins (typically derived from non-human animal serum, such as guinea pig or rabbit serum). The antibodies bind to antigens on the trophoblast layer, followed by complement activation leading to selective cleavage of the outer layer, while the ICM remains intact due to its relatively low antigenicity. While this method is effective for obtaining ICM, it introduces non-human biological materials, such as allogeneic antibodies and animal serum components, thus limiting its applicability in the derivation of clinical-grade or allogeneic-free human pluripotent stem cells. Therefore, immunosurgery is explicitly excluded from mechanical or allogeneic-free isolation protocols for therapeutic applications.

[0090] In some implementations, the mechanical separation in step b) is performed using laser-assisted micromanipulation techniques.

[0091] As used herein, the term “laser-assisted micromanipulation” generally refers to a mechanical separation method that uses a non-contact laser system integrated with a micromanipulation device to precisely ablate, cut, or perforate trophoblast cells adjacent to the embryonic micromolecular mass (ICM) to facilitate separation, without direct physical contact between the tool and the embryo.

[0092] In some implementations, step b) includes using a laser to cut trophoblast cells near the ICM to separate them from the ICM.

[0093] As used herein, the term "trophoblast" generally refers to the outer cell layer of a blastocyst embryo, responsible for forming extraembryonic tissues, including the placenta and other supporting structures during early development. This tissue facilitates embryo implantation and the establishment of initial attachment to the maternal endometrium. As used herein, the term also encompasses the trophoblast derived from embryos produced through uniparental mechanisms, such as parthenogenesis (completely maternal) or androgenesis (completely paternal) activation of cells containing entirely maternal or paternal DNA via artificial activation of human oocytes or sperm. Regardless of its origin, the trophoblast plays a crucial role in embryonic development and is distinct from the inner cell mass (ICM), which forms the embryonic body.

[0094] In some implementations, the laser described in step b) is a non-contact infrared laser with a pulse duration of 30 ms to 40 ms.

[0095] In some implementations, when the inner cell mass is tightly connected to the trophoblast cells and difficult to separate, the duration of the laser pulse in step b) is extended to no more than 60 ms.

[0096] In some implementations, the laser drilling operation is performed in real time under a microscope, with the objective lens of the microscope switched to a dedicated laser drilling lens to ensure that the focal plane is aligned with the trophoblast cell junction.

[0097] In some implementations, the laser is applied only to the trophoblast cell junction region surrounding the inner cell mass, and the inner cell mass itself is avoided from being irradiated.

[0098] In some implementations, the laser-assisted separation operation is performed entirely within microdroplets containing fertilization culture medium.

[0099] In some embodiments, the droplets containing the fertilization culture medium are maintained at 37°C and 5% CO2 throughout the culture process.

[0100] In some embodiments, the fertilization culture medium contains the ROCK inhibitor Y27632.

[0101] In some embodiments, the concentration of the ROCK inhibitor Y27632 in the fertilization culture medium is 1 μM-10 μM.

[0102] In some implementations, the laser operation in step b) includes the use of a laser film-breaking device.

[0103] In some implementations, step b) is performed entirely using clinical-grade reagents and consumables, and the entire process is carried out in a heterologous blastocyst culture medium.

[0104] Inner cell mass (ICM) separation is performed using a non-contact infrared laser. The basic pulse duration is controlled at 30–40 ms to ensure sufficient severing of the connections between trophectoderm cells. Depending on the developmental stage or structural density of the blastocyst (e.g., Gardner C grade), the pulse duration can be appropriately extended to no more than 60 ms to improve the separation success rate. This ensures effective severing while avoiding thermal damage, maintaining ICM pluripotency and subsequent cell line establishment capabilities. Laser manipulation is performed in real-time under a microscope, using a dedicated laser perforation lens to precisely focus on the trophectoderm junction region, strictly avoiding the ICM itself. This technique protects ICM integrity, preventing DNA breakage, mitochondrial damage, or apoptosis; it also improves hESC cell line establishment efficiency because intact and healthy ICMs are more likely to adhere, proliferate, and form typical pluripotent clones. All these procedures also comply with clinical safety standards, avoiding genomic instability introduced by physical / thermal damage. The entire process is carried out in microdroplets containing fertilization culture medium, maintained at 37°C and 5% CO2. The addition of 1–10 μM of the ROCK inhibitor Y27632 to the culture medium significantly reduces stress and apoptosis of ICMs during the procedure, enhancing their adhesion and subsequent colony formation abilities. Combined with the use of a clinical-grade laser perforation device, this protocol not only improves the efficiency and quality of hESC line establishment but also demonstrates excellent reproducibility and GMP compliance, providing a safe and efficient technical guarantee for the stable establishment of clinical-grade human embryonic stem cell lines.

[0105] In some implementations, the culture dish described in step c) is coated with laminin.

[0106] In some embodiments, the culture dish described in step c) is coated with laminin-521 (LN521).

[0107] In some embodiments, the coating concentration of said LN521 is 0.25 μg / cm³. 2 Up to 0.75 μg / cm 2 For example, the coating concentration of the LN521 is approximately 0.25 μg / cm³. 2 Approximately 0.30 μg / cm 2 Approximately 0.35 μg / cm2 Approximately 0.40 μg / cm 2 Approximately 0.45 μg / cm 2 Approximately 0.50 μg / cm 2 Approximately 0.55 μg / cm 2 Approximately 0.60 μg / cm 2 Approximately 0.65 μg / cm 2 Approximately 0.70 μg / cm 2 or approximately 0.75 μg / cm 2.

[0108] In some embodiments, the preferred coating concentration of said LN521 is 0.50 μg / cm³. 2 .

[0109] In some embodiments, step d) includes adding the ROCK inhibitor Y27632 to the cell culture medium from the start of ICM culture and culturing the ICM therein for 4 to 14 days. For example, step d) includes adding the ROCK inhibitor Y27632 to the cell culture medium from the start of ICM culture and culturing the ICM therein for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

[0110] In some implementations, step d) includes not changing the cell culture medium for the first 4 days from the start of ICM culture, and changing the cell culture medium regularly starting from day 5.

[0111] In some implementations, step d) includes changing the cell culture medium every 2 days, starting from day 5, from the start of ICM culturing.

[0112] In some implementations, step d) includes adding the ROCK inhibitor Y27632 to the cell culture medium from the start of ICM culture, culturing the ICM therein for 7 days, and removing the Y27632 after 7 days.

[0113] In some embodiments, the concentration of the ROCK inhibitor Y27632 is from 1 μM to 15 μM. For example, the concentration of the ROCK inhibitor Y27632 is about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, or about 15 μM.

[0114] In some embodiments, the preferred concentration of the ROCK inhibitor Y27632 is 5 μM to 10 μM.

[0115] In some implementations, the cell culture medium in step d) is TeSR™-AOF medium supplemented with human serum albumin.

[0116] In some implementations, step e) includes passage of the initial hESC clone using a heterologous, GMP-grade enzymatic digestion solution.

[0117] In some implementations, step e) is performed on days 12-21 after the ICM vaccination described in step c). For example, step e) is performed on days 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 after the ICM vaccination described in step c).

[0118] In some implementations, the hESC line obtained in step e) undergoes at least three passages.

[0119] In some implementations, the hESC line is cryopreserved as a seed bank after three generations.

[0120] In some embodiments, the hESC system is cryopreserved in cryovials, with each cryovial containing an order of magnitude of 10. 5 Up to 10 7 . cells.

[0121] In some embodiments, the enzymatic hydrolysate is a GMP-grade enzymatic hydrolysate, ReleSR™.

[0122] In some implementations, the efficiency of establishing the hESC lineage from the human blastocysts provided in step a), which are Gardner grade A or B, through steps b) to e) is not less than 60%.

[0123] In some implementations, the efficiency of establishing the hESC lineage from the Gardner grade C human blastocysts provided in step a) through steps b) to e) is not less than 20%.

[0124] In some implementations, the established hESC line has at least one characteristic selected from the group consisting of: a) normal karyotype, b) ability to express pluripotency markers, and c) ability to differentiate into three germ layers.

[0125] a) “Normal karyotype,” as used in this application and as understood in the art, refers to a cytogenetic map that shows the correct number and structure (46,XY or 46,XX) of chromosomes characteristic of the human species, without numerical abnormalities such as aneuploidy or structural aberrations such as translocations, deletions, or duplications. Karyotype stability is crucial for the safe clinical application of hESCs because chromosomal abnormalities can impair genomic integrity and lead to functional defects or tumorigenicity.

[0126] b) "Ability to express pluripotency markers" refers to the ability of hESCs to express key molecular markers related to pluripotency at the gene and protein levels. These include transcription factors such as OCT4, NANOG, and SOX2, as well as surface antigens such as SSEA-4, TRA-1-60, and TRA-1-81. Sustained expression of these markers under undifferentiated culture conditions indicates the cell's self-renewal capacity and pluripotent status.

[0127] c) “Capacity to differentiate into all three germ layers,” as used herein and as understood in the art, refers to the potential of hESC to differentiate into derivatives of all three embryonic germ layers—ectoderm, mesoderm, and endoderm—under appropriate in vitro or in vivo conditions. This capacity is typically verified by: in vitro directed differentiation protocols producing specific cell types (e.g., neurons (ectoderm), cardiomyocytes (mesoderm), or pancreatic β-cells (endoderm)); or in vivo teratoma formation assays in immunodeficient mice, wherein injection of hESC results in the production of tumors containing tissues representing all three germ layers.

[0128] In some implementations, the hESC line established is a clinical-grade cell line.

[0129] As used herein, the term "clinical grade" generally refers to cells, reagents, or processes manufactured, processed, and tested in accordance with current Good Manufacturing Practice (GMP) regulations and guidelines to ensure their suitability for potential human therapeutic applications. This includes derivatization and amplification using GMP-grade reagents in controlled environments (e.g., Class A / B cleanrooms) and comprehensive quality control testing for sterility, mycoplasma, exogenous viruses, and endotoxins.

[0130] In the specific implementation of this application, the entire experimental process for establishing human embryonic stem cell (hESC) lines is conducted in a GMP-compliant Class A environment within a Class B cleanroom. All reagents used for hESC derivatization, amplification, and cryopreservation are commercially available GMP-grade reagents and have been validated through specific tests detailed in their respective Certificates of Analysis (COA). The derivatization process strictly adheres to GMP-compliant Standard Operating Procedures (SOPs) and record-keeping forms.

[0131] 2. Initial human embryonic stem cell (hESC) colonies

[0132] On the other hand, this application provides initial human embryonic stem cell (hESC) colonies obtained according to the method described in this application, wherein the initial hESC colonies are derived under conditions of no feeder layer and no allogeneic source, have proliferative capacity and exhibit pluripotency.

[0133] In a specific embodiment of this application, the initial hESC colonies are obtained by seeding isolated inner cell masses (ICMs) onto laminin-521 (LN521)-coated culture dishes and culturing them in TeSR™-AOF medium supplemented with Y27632 (preferably at a concentration of 10 μM). For blastocysts with invisible ICMs, all cell material obtained by biopsy is collected and seeded under the same conditions. To promote initial adhesion of ICMs or cell masses, the medium is not changed for the first 4 days after seeding. From day 5, the medium (TeSR™-AOF containing 10 μM Y27632) is changed every 2 days. From day 7, Y27632 is removed, and the medium is changed daily. After 12-16 days of culture, the initial hESC clones are ready for mechanical passage. From passage 1 onwards, hESCs are amplified using GMP-grade enzymatic digestion solution ReleSR™ according to the manufacturer's instructions. By the third generation, the hESC line was harvested and cryopreserved using GMP-grade cryopreservation medium CryoStar® CS10 as the master seed bank.

[0134] 3. Human embryonic stem cell (hESC) line

[0135] On the other hand, this application provides a stable human embryonic stem cell (hESC) line obtained according to the method described in this application, wherein the stable hESC line is derived under conditions of no feeder layer and no allogeneic source, and has a normal karyotype, the ability to express pluripotency markers and / or the ability to differentiate into three germ layers.

[0136] As used in this article, the term "stable hESC lineage" generally refers to a population of human embryonic stem cells that has been successfully expanded through multiple passages (e.g., beyond the 3rd generation), exhibits stable growth, maintains a normal karyotype, expresses pluripotency markers, and retains the potential for differentiation into the three germ layers under appropriate stimulation.

[0137] 4. Cell culture composition

[0138] On the other hand, this application provides a cell culture composition comprising the initial hESC colonies described in this application and / or the stable hESC line described in this application, a laminin-521 (LN521) coated surface, and a heterologous, chemically defined culture medium supplemented with ROCK inhibitors.

[0139] In a specific embodiment of this application, the cell culture system employs a defined, heterologous culture medium (such as TeSR™-AOF) supplemented with the ROCK inhibitor Y27632 to support clonal expansion and pluripotency maintenance in colony formation analysis. For directed differentiation, stage-specific culture medium formulations are further utilized, containing essential components such as B27 supplementation, ascorbic acid, recombinant growth factors, and small molecule inhibitors to promote lineage-specific directed differentiation. For example, AggreWell™ microplates are used to promote pancreatic β-cell differentiation during the aggregation stage (Stage 3). Subsequently, cell clumps are cultured in ultra-low adsorption 6-well plates under dynamic conditions (e.g., 90 rpm, 37 °C, 5% CO2, 85% humidity) to enhance their maturity and functionality. For mesodermal and ectodermal differentiation, such as cardiomyocyte and corneal endothelial cell induction, the culture surface is pre-coated with Matrigel® or other defined extracellular matrix to support cell attachment, proliferation, and differentiation. This composable and scalable culture system enables robust derivation, expansion, and trilaminar differentiation of hESCs under standardized and clinically compliant conditions.

[0140] 5. Applications of initial hESC colonies and stable hESC systems

[0141] On the other hand, this application provides the use of the initial hESC colonies and / or the stable hESC lines described in this application in the preparation of cell products for cell therapy, disease modeling, or drug screening.

[0142] On the other hand, this application provides the use of the initial hESC colonies and / or the stable hESC lines described in this application for directed differentiation into endoderm, mesoderm, or ectoderm lineage cells.

[0143] The initial hESC colonies and stable hESC lines (such as HES1 and HES2 provided in the embodiments of this application) can be directed to differentiate into functional cell types representing three germ layers, including pancreatic β cells derived from the endoderm, cardiomyocytes derived from the mesoderm, and corneal endothelial cells derived from the ectoderm.

[0144] The differentiated pancreatic β cells exhibited typical islet-like morphology at stage 6, with approximately 50% of the cells identified as insulin-positive β cells. These cells demonstrated glucose-responsive insulin secretion, reflecting their physiological activity. Differentiated cardiomyocytes expressed key structural and functional markers such as ACTN1 and cTNT, and showed significant upregulation of core cardiac transcription factors such as MEF2C, TBX5, GATA4, and NKX2.5. Corneal endothelial cells exhibited a characteristic hexagonal morphology and expressed specific markers such as the tight junction protein ZO1 and the sodium-potassium ATPase ATP1A1, while the expression of other corneal endothelial-related genes was significantly upregulated.

[0145] These functionally validated cells are suitable for a variety of applications, including disease modeling (such as diabetes, heart disease, and corneal diseases), high-throughput drug screening and toxicity assessment, and the development of cell therapies in regenerative medicine. Specifically, they can be used to build in vitro disease models, discover new drug targets, and produce clinical-grade cell products for transplantation.

[0146] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the cell line derivation methods and uses of this application, and are not intended to limit the scope of the invention.

[0147] Example

[0148] Example 1: Method for Derivation of Human Embryonic Stem Cell (hESC) Lines

[0149] 1. Experimental Methods

[0150] Embryo thawing

[0151] This study was approved by the Medical Ethics Committee of the Affiliated Hospital of Yunnan University (Ethics No.: 2023224). All embryos used in this study were donated by couples who provided informed ethical consent. Embryo thawing was performed using a commercial thawing medium kit (KITAZATO, VT102). The specific procedure was as follows: Thawing culture dishes were prepared 24 hours in advance, with 1 mL of blastocyst medium covered with sterile mineral oil added, and equilibrated in an incubator for more than 6 hours. On the day of thawing, the thawing solution was preheated at 37 °C for 60 minutes, while the dilution solution, wash solution 1, and wash solution 2 were equilibrated at room temperature for ≥45 minutes. During thawing, 500 μL of the preheated thawing solution was transferred to a 60 mm culture dish. The vitrified carrier was quickly removed from liquid nitrogen using pre-cooled forceps and immersed in the thawing solution, releasing the embryo by gentle agitation within 60 seconds. Subsequently, the embryos were sequentially transferred to dilution solution and incubated for 3 minutes, followed by incubation for 5 minutes with 100 μL of wash solution 1, and then incubated for 5 minutes with 100 μL of wash solution 2. Finally, after washing with pre-equilibrated medium, they were transferred to pre-equilibrated microdroplets using a Pasteur pipette. Assisted hatching was performed by creating perforations in the zona pellucida using a non-contact laser, and the embryos were cultured until the inner cell mass separated.

[0152] Inner cell mass separation

[0153] Microdroplets (each containing 30 µL of fertilization culture medium and coated with mineral oil) were prepared one day in advance and equilibrated in an incubator for more than 6 hours. Embryos were transferred into microdroplets 1 hour before inner cell mass separation. The holder and biopsy needle were rinsed with 30 µL of polyvinylpyrrolidone solution, followed by washing with 30 µL of fertilization culture medium to remove excess polyvinylpyrrolidone. The holder was used to locate the ectoderm cells at the distal end of the inner cell mass, and the embryo was rotated to observe the inner cell mass boundary. Trophoblast cells near the inner cell mass were aspirated using the biopsy needle. A 30–40 ms laser pulse was applied to penetrate the trophoblast cells near the inner cell mass, and then the inner cell mass was gently separated from the trophoblast cells. The separated inner cell mass was then used for the derivation of human embryonic stem cells.

[0154] Isolation of human embryonic stem cells

[0155] The isolated inner cell mass was seeded with laminin 521 (0.50 μg / cm). 2In coated culture dishes, the medium was TeSR™-AOF containing 10 μM Y27632, and cultured at 37 °C and 5% CO2. For blastocysts where the inner cell mass was not observable, all biopsy-derived cell material was collected and used for the isolation of human embryonic stem cells. The medium was not changed for the first 4 days post-inoculation to promote inner cell mass adhesion. From day 5 post-inoculation, the medium (TeSR™-AOF containing 10 μM Y27632) was changed every 2 days. From day 7 post-inoculation, Y27632 was removed and the medium was changed daily. Initial human embryonic stem cell clones could be mechanically passaged after day 12 post-inoculation. After one passage, human embryonic stem cells could be passaged using commercially available GMP-grade enzymatic digestion solution ReleSR™ according to instructions. Human embryonic stem cell lines passaged to passage 3 could be harvested in batches and cryopreserved, with 10 cryovials per batch serving as a seed bank, each cryovial containing approximately 5 × 10⁶ cells. 5 Up to 2 × 10 7 The viable cells were cryopreserved using GMP-grade cryopreservation solution CryoStar® CS10. Clinical-grade human embryonic stem cell lines were established in a Grade A environment within a Grade B laboratory. The GMP-grade reagents used for establishing human embryonic stem cells are listed in the table below.

[0156] Table 1 GMP-grade reagents for the isolation, culture, and storage of human embryonic stem cells.

[0157]

[0158] Colony formation experiment

[0159] Human embryonic stem cells in the logarithmic growth phase were digested into single cells using Accutase and suspended in TeSR™-AOF or E8 medium, both supplemented with 10 µM Y27632. Cells were adjusted to a density of 300 cells / well and seeded onto wells pre-coated with laminin 521 (0.75, 0.50, and 0.25 μg / cm³). 2 ) or vitamin protein (3, 2 and 1 μg / cm 2 Cells were cultured in 12-well plates. After 7 days, cells were fixed with 4% formaldehyde and stained with 0.1% crystal violet. Images were acquired and cell population area was quantified using ImageJ software.

[0160] alkaline phosphatase staining

[0161] Alkaline phosphatase staining was performed using an alkaline phosphatase chromogenic kit according to the manufacturer's instructions. After washing with DPBS, cells were stained with alkaline phosphatase for 20 minutes, followed by washing three times with pure water and photographing.

[0162] Flow cytometry

[0163] Single cells were fixed with 4% paraformaldehyde for 15 minutes, treated with permeation buffer for 15 minutes, and then blocked with blocking buffer for 30 minutes. Cell samples were incubated with primary antibody at 4 °C for 1 hour, followed by incubation with fluorescein-labeled secondary antibody at room temperature for 30 minutes. 10,000 single cells were collected from each sample using flow cytometry, and sorting was based on negative controls and the unique gene expression patterns of each cell population. The antibodies used are shown in the table below.

[0164] Table 2 Antibody List

[0165]

[0166]

[0167] Teratoma formation

[0168] All animal experiments were approved by the Animal Ethics Committee of the Hangzhou Institute of Medical Sciences, Chinese Academy of Sciences, and conducted in accordance with relevant guidelines (ethics number: AP2024-10-0307). SCID-Beige mice (6-8 weeks old, male) were purchased from the Charles River Laboratories and housed in a constant temperature environment of 23 ± 2 ℃, a 12:12 light-dark cycle, free access to water and food, and a specific pathogen-free (SPF) environment. To induce teratoma formation, approximately 2 × 10⁻⁶ mice were used. 6 Personal embryonic stem cells were digested with Accutase and then suspended in a 1:1 mixture of cryogel and culture medium. The cell suspension was then subcutaneously injected into SCID-Beige mice anesthetized with isoflurane. When the teratoma reached a diameter of 10 mm, the animals were euthanized using carbon dioxide exposure. The teratomas were subsequently collected, fixed, and processed by Wuhan CyberBio Technology Co., Ltd. for sectioning and hematoxylin-eosin (H&E) staining.

[0169] Doubling time

[0170] Cell proliferation was analyzed using Cell Counting Kit-8 (CCK8). hESCs were seeded at a density of 5,000 cells per well into matrix-coated 96-well plates and cultured at 37 °C with 5% CO2. On days 2, 3, and 4, 10% (v / v) of CCK8 reagent was added to each well. After 2 hours of incubation, absorbance was measured at 450 nm using a microplate reader. Five replicate wells were analyzed at each time point, with two additional replicate wells serving as blank controls. Cell doubling time was calculated using the following formula: Td = Δt × Lg² / (LgN) t - LgN0), where Δt is the culture time, N0 is the initial cell number, and N tLet t be the number of cells at time t.

[0171] STR analysis

[0172] Short tandem repeat (STR) mapping analysis was performed by Shanghai Biyuntian Biotechnology Co., Ltd. Genomic DNA was extracted from cells, and STR loci were amplified by PCR to generate specific fragments. The amplification products were separated by capillary electrophoresis, and the number of repeat units at each STR locus was detected to generate a unique STR map. The STR maps were analyzed and compared with standard databases (such as ATCC) to confirm genetic identity, purity, and absence of interspecies contamination.

[0173] Karyotype analysis

[0174] For karyotype analysis, well-dispersed metaphase chromosomes are obtained through cell culture and metaphase arrest techniques. The chromosomes are stained to produce banded structures, and high-resolution images are taken using a microscope. Karyotype diagrams are arranged according to chromosome size and banding patterns and compared with standard reference karyotypes to identify numerical abnormalities or structural aberrations.

[0175] RNA extraction and quantitative PCR

[0176] Cell lysis was achieved by adding 1 mL of RNAiso Plus solution to each sample and then vigorously vortexing for 30 seconds to ensure complete lysis. Samples were then incubated on ice for 10 minutes. To achieve phase separation, 200 μL of chloroform was added to the lysis buffer. The tubes were vigorously shaken for 30 seconds and then incubated on ice for 5 minutes to promote complete phase separation. The mixture was centrifuged at 12,000 g for 10 minutes at 4 °C, and the aqueous phase was carefully transferred to a new tube. RNA was precipitated by adding 500 μL of isopropanol, incubating on ice for 10 minutes, and then centrifuging at 12,000 g for 10 minutes at 4 °C. The resulting RNA precipitate was washed with 1 mL of ice-cold 75% ethanol, centrifuged at 7,500 g for 5 minutes at 4 °C, dried, and resuspended in DEPC-treated water. RNA concentration and purity were determined using a Nanodrop spectrophotometer. Reverse transcription was performed using the PrimeScript RT kit (containing gDNA remover), followed by qPCR using TB Green® premixed Taq according to the manufacturer's instructions. The primer list is as follows.

[0177] Table 3 Primer List (F - forward primer, R - reverse primer)

[0178]

[0179]

[0180] Immunofluorescence staining

[0181] Cell cultures or tissue sections were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. Samples were washed twice with PBS (5 minutes each time), then permeabilized with PBS containing 0.2% Triton X-100 at room temperature for 30 minutes, and then blocked with PBS containing 5% bovine serum albumin (BSA) at room temperature for 1 hour. Primary antibodies were incubated overnight at 4 °C, and secondary antibodies (containing Hoechst 33342) were incubated at 37 °C for 1 hour. Samples were washed three times with PBS (5 minutes each time), and then observed and photographed under a microscope after adding PBS. A list of antibodies used is provided above.

[0182] Aseptic operation

[0183] Sterility testing was performed according to the Pharmacopoeia of the People's Republic of China, using a culture method. The sterility test was conducted using BACT / ALERT® (BioMérieux). After 24 hours of cell culture, the culture supernatant was collected and placed in a culture flask for further incubation. Aerobic and anaerobic tests were performed simultaneously. Contamination was confirmed after 14 days.

[0184] Mycoplasma testing

[0185] Mycoplasma was detected by polymerase chain reaction (PCR). Before passage, the supernatant of cell culture was collected, heated at 95 °C for 10 minutes, and then centrifuged at 12,000 g for 1 minute. The supernatant was used as a template for PCR amplification using specific primers. The amplified products were photographed after gel electrophoresis.

[0186] Virus testing

[0187] Viral detection was performed using quantitative real-time PCR (TaqMan probes). Primers and probes were designed to target conserved regions of the virus. The effectiveness of the primers and probes was tested by serially diluting positive fragments to obtain the corresponding equations. Subsequently, cDNA and DNA from cell lines were used as templates to detect viral contamination.

[0188] Endotoxin testing

[0189] Endotoxins were detected by colorimetric assay at Hangzhou ChainMedical Laboratory. During the coagulation enzyme hydrolysis reaction, the chromogenic substrate caused changes in absorbance. By dynamically detecting the rate of change in absorbance of the solution, the concentration of lipopolysaccharide from Gram-negative bacteria was quantitatively determined.

[0190] 2. Experimental Results

[0191] Establishment of hESC preparation method

[0192] To establish a clinically compliant hESC preparation method under feeder-free and xenogeneic conditions, extracellular matrix (ECM) components and human pluripotent stem cell (hPSC) culture media were evaluated according to clinical-grade specifications. Evaluation criteria included chemical composition certainty, xenogeneicity, GMP compliance, and commercial availability. Laminin-521 (LN521) and vilinkin (VN), meeting all requirements, were selected for testing. Colony-forming ability was assessed using early-passaged hESC lines. LN521 demonstrated excellent support for cell adhesion and growth in a concentration-dependent manner. The coating density of LN521 was increased from 0.25 µg / cm³. 2 Increased to 0.50 µg / cm 2 This leads to a 2-fold increase in cell number. Figure 1A-1B Evaluation of the culture medium showed that, on LN521 and VN-coated plates, albumin-supplemented TeSR™-AOF (AOF) medium significantly enhanced colony formation and cell growth while maintaining typical hESC morphology. Figure 1A-1B Therefore, 0.5 µg / cm was chosen. 2 LN521-coated density-binding AOF medium supplemented with albumin was used for subsequent hESC establishment.

[0193] All embryos used were frozen, clinically discarded embryos that were unsuitable for clinical use due to exceeding demand or developmental deficiencies. In accordance with ethical guidelines, all donor couples underwent a fully traceable informed consent process. Donor candidates underwent multi-stage medical evaluations, including infectious disease combinations (HIV-1 / 2, HBV, HCV, TP) and detailed family history to identify potential neoplastic, hereditary, and congenital diseases. Eligible embryos were recruited for hESC establishment.

[0194] Inner cell mass biopsy

[0195] To avoid the inherent risk of heterologous contamination in immunosurgical procedures, a laser-assisted ICM separation method was developed for the precise ablation of trophoblast cells. Figure 1CThis mechanical separation method was optimized using a standardized preimplantation genetic diagnosis (PGD) biopsy workflow and a micromanipulation system equipped with a biopsy needle. All separation procedures used clinical-grade reagents and consumables, including in vitro fertilization (IVF) validated culture media and embryo-tested mineral oil, ensuring compliance with GMP standards for therapeutic-grade hESC derivatization processes. For blastocysts with morphologically visible ICM (grade ≥ B), the ICM was selectively removed via minimally invasive surgery, along with the removal of most of the trophectoderm cell mass. For malformed blastocysts with no visible ICM (grade C), all biopsy-obtained cellular material was collected and cultured for hESC line derivation.

[0196] Materials required for the experiment included: fertilization culture medium (ORIGIO, 10315060F); mineral oil (RBC-1016); 7% PVP handling solution (Irvine Scientific, 90121); 60mm culture dishes (BD Falcon, 353652); micromanipulation oocyte holding needles (Sunlight Medical, SHP-100-30); micromanipulation biopsy needles (Sunlight Medical, SBB-20Z-30); stereomicroscope (NIKON, Japan); inverted microscope (NIKON, Japan); laser membrane breaking device (MTG, Germany); micromanipulation system (RI, UK); 1000µl micropipette and tip; sterile Pasteurized pipettes (Darwin, D1230-5P).

[0197] One day in advance, prepare the following: Using the lid of a 60mm culture dish, prepare biopsy culture dishes (place 3 rows of 30µl microdrops of fertilization culture medium in the center of the dish, 5 drops per row, and 3 30µl PVP drops on the far right), cover with mineral oil, and place in an incubator for equilibration, ensuring equilibration time exceeds 6 hours. Place 5 embryos in each biopsy dish; prepare the appropriate number of biopsy culture dishes based on the number of embryos to be biopsied. Draw the Pasteur tubes used for embryo transfer, and heat the ends near the 300µm diameter.

[0198] One hour before the biopsy, the thawed blastocysts, awaiting biopsy, are transferred from the blastocyst culture dish to the second column of the biopsy dish using a prepared Pasteur tube. The embryo number and other relevant information are labeled next to the microdroplet before placing the dish in an incubator to await biopsy. The micromanipulation oocyte holding needle and micromanipulation biopsy needle are then attached to the two manipulator arms of the micromanipulation system, respectively.

[0199] After completing the above preparations, perform an inner cell mass biopsy. Remove the biopsy culture dish and place it on the microsurgical stage. Under low magnification, place the oocyte-holding needle and biopsy needle into the rightmost PVP droplet, aspirating a small amount of PVP solution to rinse the tips of the needles. Transfer the oocyte-holding needle and biopsy needle to the third column of fertilization culture droplets to wash away excess PVP solution. After rinsing, transfer the oocyte-holding needle and biopsy needle to the second column of droplets containing the embryo to be biopsied. Use the oocyte-holding needle to aspirate the trophoblast cells furthest from the inner cell mass, rotating the embryo to make the boundary of the inner cell mass clearly visible. Then, use the biopsy needle to aspirate cells on one side of the inner cell mass. Adjust the objective lens to the laser-drilling lens and adjust the focus to make the trophoblast cells clearly visible. Adjust the laser target center and use a laser beam of 30-40ms to drill holes starting from the junction of the trophoblast cells next to the inner cell mass, while simultaneously using the biopsy needle to pull the inner cell mass until it is completely separated from the trophoblast cells. For embryos that are difficult to separate, the laser emission time can be appropriately increased. When drilling with the laser, try to remove the trophoblast layer as completely as possible, but avoid damaging the inner cell mass. Use an oocyte-holding needle to drag the separated trophoblast cells into the first column of droplets, leaving the inner cell mass in the second column of droplets for inoculation. Under a stereomicroscope, use a pre-prepared Pasteur tube (first rinsed in PVP handling solution and then washed with fertilization culture medium to remove excess PVP) to inoculate the inner cell mass into a pre-prepared 12-well plate and label it.

[0200] hESC System

[0201] After mechanical separation from the blastocysts, the ICM was inoculated into AOF medium coated with LN521 and supplemented with the ROCK inhibitor Y27632. Clinical-grade hESC lineage was established using GMP-grade Y27632. Morphological monitoring showed that the initial ICM clumps effectively adhered to LN521 within 24 hours. Figure 1D However, gradual cell death was observed after Y27632 was removed starting on day 2. Therefore, Y27632 treatment was extended to the end of the first week. The results showed that continuous Y27632 treatment significantly enhanced the survival and proliferative capacity of naïve pluripotent stem cells derived from ICM. Figure 1D After 5-7 days of culture, small clusters of cells exhibiting typical hESC morphology appeared. Figure 1D Subsequently, Y27632 was no longer required for the expansion of hESC clones. These findings indicate that ROCK inhibition is crucial for the adhesion, survival, and expansion of primary human embryonic stem cells by naïve hESCs under feeder-free conditions. Naïve hESC clones were mechanically passaged 12–16 days post-inoculation. Figure 1EFrom generation 2 onwards, hESCs were maintained on LN521-coated plates using AOF medium and amplified in small clumps using GMP-grade heterologous enzyme digestion solution. By generation 3, more than 20 million hESCs were obtained, and the hESC seed bank was cryopreserved at or after this stage.

[0202] After optimizing culture conditions and ICM separation protocols, a standardized hESC derivatization process was established. Figure 2 All reagents used were GMP-grade and commercially available as described above. Using this protocol, 16 hESC lines were successfully established from 35 eligible blastocysts, achieving an overall efficiency of approximately 45.7% (Table 4). Establishment efficiency was positively correlated with ICM quality (Tables 4-6). Blastocysts at 5 days post-fertilization (dpf) showed higher derivatization rates than those at 6 days post-fertilization (dpf) (Tables 5 and 6). For 5-dpf blastocysts with visible ICM (Grades A and B), derivatization efficiency exceeded 70% (Table 5). Even for blastocysts with barely visible ICM (Grade C), derivatization efficiency reached approximately 30% (Table 4). Overall, these findings demonstrate that this feeder-free and heterologous derivatization protocol effectively supports efficient differentiation of hESCs from blastocysts.

[0203] Table 4 Overall Derivation Efficiency of hESC Series

[0204]

[0205] Table 5. hESC line derivation efficiency of blastocysts at 5 days post-fertilization (5-dpf).

[0206]

[0207] Table 6. hESC line derivation efficiency of blastocysts at 6 days post-fertilization (6-dpf).

[0208]

[0209] Establishment and Characterization of Clinical-Grade hESC Lines

[0210] according to Figure 2 The workflow shown established two clinical-grade hESC lines (HES1 and HES2). The process included informed consent, donor eligibility assessment, detailed cell line establishment procedures, process monitoring, and quality control of the derived hESCs. The clinical-grade hESCs were produced in a Grade A environment within a Grade B facility. All reagents used for hESC establishment, amplification, and cryopreservation were heterologous and GMP-grade, and the hESC quality was validated through specific tests detailed in the Certificate of Analysis (COA). Standard operating procedures (SOPs) and record forms for the establishment process were performed in accordance with GMP requirements.

[0211] HES1 and HES2 were fully characterized. Both cell lines exhibited typical human pluripotent stem cell morphology. Figure 3A Biosafety testing conducted at generation 5, including sterility, mycoplasma, viral (HIV-1 / 2, HBV, HCV, TP, HTLV-1, HCMV, EBV, HHV6, and HHV7) and endotoxin testing, confirmed the safety of these cell lines (Table 7). Short tandem repeat (STR) mapping analysis confirmed the absence of cell cross-contamination between hESC lines. Figure 5 Pluripotency characterization performed around generation 10 showed that HES1 and HES2 exhibited normal karyotype, were alkaline phosphatase positive, and expressed typical hPSC markers (). Figure 3B-3E Flow cytometry analysis using OCT4 and NANOG showed that HES1 and HES2 had high purity (>97%). Figure 3F The proliferation rates of HES1 and HES2 were comparable to those of the H9 control line. Figure 3G After transplantation into immunodeficient mice, both cell lines formed teratomas containing all three germ layers. Figure 3H In summary, these results indicate that HES1 and HES2 represent high-quality hESC seeds suitable for establishing clinical-grade master cell banks for future therapeutic applications.

[0212] Table 7 Biosafety Tests

[0213]

[0214] Example 2: Directed Differentiation of Clinical-Grade Human Embryonic Stem Cells (hESCs)

[0215] 1. Experimental Methods

[0216] Pancreatic β-cell differentiation

[0217] The differentiation of human embryonic stem cells into β cells was performed using a six-stage protocol (Reference: Du Y, et al., Human pluripotent stem-cell-derived islets ameliorate diabetes in non-human primates. Nature Medicine 2022, 28:272-282). In brief, human embryonic stem cells were digested into single cells using Accutase and suspended in mTESR1 medium supplemented with 10 µM Y27632. The cells were then cultured at 1.5 × 10⁻⁶ cells / cells. 5 cells / cm 2The inoculum was seeded at a density on the surface coated with Matrigel. Differentiation began 24 hours after inoculation, using the corresponding specialized culture medium formulation for each stage.

[0218] Phase 1 (4 days): On day 1, cells were maintained in MCDB 131 medium supplemented with 1×Glutamax, 4.5 mM glucose, 1% B27, 0.25 mM ascorbic acid, 100 ng / mL Activin A, and 6 μM CHIR99021. From day 2 to day 4, the medium was replaced with MCDB 131 medium containing 1×Glutamax, 4.5 mM glucose, 1% B27, and 100 ng / mL Activin A.

[0219] Phase 2 (2 days): Cells were treated with MCDB 131 medium supplemented with 1×Glutamax, 4.5 mM glucose, 1% B27, 0.25 mM ascorbic acid, 50 ng / mL KGF, 100 nM Wnt-C59 and 5 μM SB431542.

[0220] Phase 3 (4 days): Cells were cultured in DMEM containing 1% B27, 0.25 mM ascorbic acid, 0.25 mM SANT-1, 2 μM retinoic acid, and 100 nM LDN193189. At the end of Phase 3, cells were digested with Accutase at 37 °C for 5–8 min, washed with DMEM, and then cultured at 6 × 10⁻⁶ cells / year. 6 Cells were seeded at a density of 10 μM Y27632 in six-well AggreWell microplates. After 24 hours, the cell clusters were transferred to ultra-low adhesion six-well plates and cultured at 37°C, 5% CO2, and 85% humidity with shaking at 90 rpm.

[0221] Phase 4 (6 days): Cell aggregates were maintained in DMEM medium containing 1% B27, 0.25 mM ascorbic acid, 100 ng / mL epidermal growth factor (EGF), 200 nM TPB, 0.25 mM SANT-1 and 10 mM nicotinamide.

[0222] Phase 5 (6 days): Cell populations were further enriched with 1% B27, 0.25 mM ascorbic acid, 10 μM ALK5 inhibitor II, 300 nM LDN193189, 1 μM T3, 10 μM ISX9, 10 μg / ml heparin, 100 nM γ-secretase inhibitor XXI, 100 nM Wnt-C59, and 10 μM Y27632. ISX9 was added from day 1 to day 3.

[0223] Phase 6 (2 days): Cells from Phase 5 were treated with DMEM medium containing 1% B27, 0.25 mM ascorbic acid, 10 μM ALK5 inhibitor II, 0.5 μM R428, 1 μM T3, 10 μg / mL heparin, 10 μM zinc sulfate and 2 mM N-acetyl-L-cysteine.

[0224] The reagents used are shown in the table below.

[0225] Table 8 Reagents and Kits

[0226]

[0227]

[0228] Glucose-stimulated insulin secretion

[0229] Human embryonic stem cell-derived islets (10–20 clusters) were collected into 12-well plates and washed twice with Krebs buffer (129 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1 mM Na2HPO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 10 mM HEPES, and 0.1% BSA). Subsequently, these aggregates were incubated sequentially for 1 hour each in Krebs buffer containing 2.8 mM glucose and 16.7 mM glucose. After each incubation, the supernatant was collected, and the aggregates were washed with fresh Krebs buffer when the solution was changed. The supernatant samples were frozen at -80 °C until analysis. After the experiment, the cells were dispersed into single cells using Accutase and counted using a Countess II automated cell counter.

[0230] Cardiac cell differentiation

[0231] Human embryonic stem cell-derived cardiomyocytes were prepared using a two-dimensional monolayer differentiation method (Reference: Wang H, et al, Hyperactivation of platelet-derived growth factor signalling contributions to arrhythmogenesis in Brugada syndrome. Clin Transl Med 2022,12:e715). The simplified steps are as follows: Undifferentiated human embryonic stem cells were dispersed and then... 4 cells / cm 2hESCs were seeded at a density of [missing information - likely a specific density] into 6-well plates coated with matrix gel. After reaching 85% confluence, hESCs were treated for 2 days with RPMI 1640 medium (RPMI + B27-insulin) supplemented with 1% B27 but without insulin, and 6 μM CHIR99021 was added. On day 3, cells were transferred to RPMI + B27-insulin medium without CHIR99021. On days 4-5, cells were treated with 5 μM IWR-1 to inhibit the Wnt signaling pathway. On days 5-6, IWR-1 treatment was stopped, and cells were placed in RPMI + B27-insulin medium. From day 7 onwards, cells were cultured in RPMI 1640 medium without glucose and supplemented with B27 containing insulin until cell pulsation was observed. The reagents used in this experiment are listed in the table above.

[0232] Corneal endothelial cell differentiation

[0233] The corneal endothelial cell differentiation method was a modification of a prior method (Reference: Li Z, et al, Long-term corneal recovery by simultaneous delivery of hPSC-derived cornealendothelial precursors and nicotinamide. J Clin Invest 2022, 132). Briefly, human embryonic stem cells were differentiated at 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 100% in 12-well matrix gel-coated plates and cultured until the cell density reached 30%. Then, they were transferred to neural crest differentiation medium for further culture. This medium consisted of DMEM / F12, 20% knockout serum substitute, 4 ng / mL bFGF, and 1 μM retinoic acid. After 5 days of culture, the medium was replaced with corneal endothelial cell differentiation medium, which consisted of DMEM / F12, 8 ng / mL bFGF, 10 ng / mL PDGF-BB, 10 ng / mL DKK-2, 50 μg / mL ascorbic acid, 10 ng / mL Heregulin β-1, 200 ng / mL IGF-1, 1 × B27, 0.01 mM β-mercaptoethanol, 10 μM Y27632, and 1 μM SB431542. Neural crest cells were cultured for 10 days to induce corneal endothelial cell differentiation. During differentiation, cells were cultured at 37 °C and 5% CO2, with the culture medium changed daily. The reagents used in this experiment are listed in the table above.

[0234] 2. Experimental Results

[0235] HES1 and HES2 were directed to differentiate into representative cell types of the three germ layers. For endoderm differentiation, both cell lines were differentiated into pancreatic β cells. Stage 1 differentiation produced endoderm progenitor cells in which FOXA2 and SOX17 were co-expressed in over 90% of the cells. Figure 4A By stage 6, cell clusters exhibiting typical islet morphology were observed. Figure 4B Approximately 50% of the differentiated cells were identified as pancreatic β cells, a proportion comparable to that of primary human islets. Figure 4A Immunofluorescence staining confirmed the expression of typical β-cell markers ( Figure 4C ), and differentiated cells exhibit glucose-stimulated insulin secretion function ( Figure 4D For mesodermal differentiation, cardiomyocytes were generated from both cell lines. Immunofluorescence staining confirmed the expression of cardiac markers ACTN1 and cTNT. Figure 4E qRT-PCR analysis showed that after the appearance of pulsatile clusters, the expression of key cardiac transcription factors and functional genes (including MEF2C, TBX5, GATA4, NKX2.5, cTNT, RYR2, and ACTN1) was significantly upregulated. Figure 4F For ectoderm differentiation, corneal endothelial cells were derived through neural crest induction. Three weeks after differentiation, uniform hexagonal cells were observed to express tight junction protein ZO1 and sodium-potassium ATPase ATP1A1. Figure 4G qRT-PCR confirmed the upregulation of corneal endothelial markers. Figure 4H ).

[0236] These results demonstrate the strong trigerm layer differentiation capacity of HES1 and HES2, confirming that they can generate functional cell types from all three germ layers using the derivation methods provided in this application.

Claims

1. A method for deriving a human embryonic stem cell (hESC) line, comprising the following steps: a) Provide human embryonic sacs; b) Mechanically separate the inner cell mass (ICM) from the blastocyst described in step a); c) The ICM isolated in step b) is seeded onto a culture dish coated with a suitable extracellular matrix substrate, wherein the suitable extracellular matrix can support the adhesion, survival and growth of the ICM without a feeder layer; d) Culture the inoculated ICM from step c) in a heterologous, chemically defined, feeder-free cell culture medium until it forms an initial hESC clone; e) Proliferate the initial hESC clone formed in step d) to obtain an hESC line.

2. The method according to claim 1, wherein the blastocyst is a clinical surplus or discarded embryo having a morphologically distinguishable ICM that is rated as Grade A, B, or C according to the Gardner grading system.

3. The method according to claim 1 or 2, wherein the blastocyst is a blastocyst that has developed for no more than 14 days after in vitro fertilization (IVF).

4. The method according to any one of claims 1-3, wherein the blastocyst is a blastocyst that has developed for 5 to 8 days after in vitro fertilization (IVF).

5. The method according to any one of claims 1-4, wherein the mechanical separation in step b) does not employ any immunosurgical method that relies on antibodies or complement of non-human animal origin.

6. The method according to any one of claims 1-5, wherein the mechanical separation in step b) is performed by laser-assisted micromanipulation.

7. The method according to any one of claims 1-6, wherein step b) comprises using a laser to cut trophoblast cells near the ICM to separate them from the ICM.

8. The method according to claim 6 or 7, wherein the laser in step b) is a non-contact infrared laser with a pulse duration of 30 ms to 40 ms.

9. The method according to claim 8, wherein when the inner cell mass is tightly connected to the trophoblast cells and difficult to separate, the duration of the laser pulse in step b) is extended to no more than 60 ms.

10. The method according to any one of claims 6 to 9, wherein the laser drilling operation is performed in real time under a microscope, and the objective lens of the microscope is switched to a dedicated laser drilling lens to ensure that the focal plane is aligned with the trophoblast cell junction.

11. The method according to any one of claims 6-10, wherein the laser acts only on the trophoblast cell junction region surrounding the inner cell mass and avoids irradiating the inner cell mass itself.

12. The method according to any one of claims 6-11, wherein the laser-assisted separation operation is carried out entirely in a microdroplet containing fertilization culture medium.

13. The method according to claim 12, wherein the microdroplets containing the fertilization culture medium are maintained at 37°C and 5% CO2 throughout the culture process.

14. The method of claim 12 or 13, wherein the fertilization culture medium contains the ROCK inhibitor Y27632.

15. The method according to claim 14, wherein the concentration of the ROCK inhibitor Y27632 is 1 μM-10 μM.

16. The method according to any one of claims 6 to 15, wherein the laser operation in step b) comprises using a laser film-breaking device.

17. The method according to any one of claims 1-16, wherein step b) is performed entirely using clinical-grade reagents and consumables and is carried out entirely in a heterologous blastocyst culture medium.

18. The method according to any one of claims 1-17, wherein the culture dish in step c) is coated with laminin.

19. The method of claim 18, wherein the culture dish in step c) is coated with laminin-521 (LN521).

20. The method according to claim 19, wherein the coating concentration of LN521 is 0.25 μg / cm³. 2 Up to 0.75 μg / cm 2 .

21. The method according to claim 19, wherein the preferred coating concentration of LN521 is 0.50 μg / cm³. 2 .

22. The method according to any one of claims 1-21, wherein step d) comprises adding the ROCK inhibitor Y27632 to the cell culture medium from the start of ICM culture and culturing the ICM therein for 4 to 20 days.

23. The method according to any one of claims 1-22, wherein step d) includes not changing the cell culture medium for the first 4 days from the start of ICM culture, and changing the cell culture medium periodically from day 5 onwards.

24. The method of claim 23, wherein step d) comprises changing the cell culture medium every 2 days starting from day 5, beginning from the start of ICM culture.

25. The method according to any one of claims 1-24, wherein step d) comprises adding the ROCK inhibitor Y27632 to the cell culture medium from the start of ICM culture, culturing the ICM therein for 7-10 days, and removing the Y27632 after 7-10 days.

26. The method according to claim 22 or 25, wherein the concentration of the ROCK inhibitor Y27632 is from 1 μM to 15 μM.

27. The method of claim 26, wherein the preferred concentration of the ROCK inhibitor Y27632 is 5 μM-10 μM.

28. The method according to any one of claims 1-27, wherein the cell culture medium in step d) is TeSR™-AOF medium supplemented with human serum albumin.

29. The method according to any one of claims 1-28, wherein step e) comprises passage the initial hESC clone using a heterologous, GMP-grade enzymatic digestion solution.

30. The method according to any one of claims 1-29, wherein step e) is performed on day 12-16 after the inoculation with ICM in step c).

31. The method according to any one of claims 1-30, wherein the hESC line obtained in step e) has undergone at least three subcultures.

32. The method according to claim 31, wherein the hESC line is cryopreserved as a seed bank after three generations.

33. The method according to claim 31 or 32, wherein the hESC system is cryopreserved in cryovials, and each cryovial contains an order of magnitude of 10 5 Up to 10 7 . cells.

34. The method of claim 29, wherein the enzymatic hydrolysis solution is a GMP-grade enzymatic hydrolysis solution ReleSR™.

35. The method according to any one of claims 1-34, wherein the efficiency of establishing the hESC lineage from the human blastocysts provided in step a), which are Gardner grade A or B, through steps b) to e) is not less than 60%.

36. The method according to any one of claims 1-34, wherein the efficiency of establishing the hESC lineage from the human blastocysts of Gardner grade C provided in step a) through steps b) to e) is not less than 20%.

37. The method according to any one of claims 1-36, wherein the established hESC line has at least one characteristic selected from the group consisting of: a) normal karyotype, b) ability to express pluripotency markers, and c) ability to differentiate into three germ layers.

38. The method according to any one of claims 1-37, wherein the established hESC line is a clinical-grade cell line.

39. The initial human embryonic stem cell (hESC) colonies obtained by the method according to any one of claims 1-38, wherein the initial hESC colonies are derived under conditions of no feeder layer and no allogeneic source, have proliferative capacity and exhibit pluripotency.

40. A stable human embryonic stem cell (hESC) line obtained by the method of any one of claims 1-38, wherein the stable hESC line is derived under conditions of no feeder layer and no allogeneic origin and has a normal karyotype, the ability to express pluripotency markers and / or the ability to differentiate into three germ layers.

41. A cell culture composition comprising the initial hESC colony of claim 39 and / or the stable hESC line of claim 40, a laminin-521 (LN521) coated surface, and a heterologous, chemically defined culture medium supplemented with ROCK inhibitors.

42. Use of the initial hESC colony of claim 39 and / or the stable hESC line of claim 40 in the preparation of cell products for cell therapy, disease modeling or drug screening.

43. Use of the initial hESC colony of claim 39 and / or the stable hESC lineage of claim 40 for directed differentiation into endoderm, mesoderm or ectoderm lineage cells.

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