Compositions and methods for expanding keratinocytes

Abstract

The present disclosure relates to medium compositions and / or supplements added to medium and to methods for culturing and / or expanding keratinocytes or keratinocyte-like cells. In another aspect, the present disclosure relates to medium compositions and / or supplements added to medium and to methods that provide enrichment or selection of a population of keratinocytes or keratinocyte-like cells. Keratinocytes or keratinocyte-like cells may be derived from pluripotent stem cells, more particularly from a differentiated population of pluripotent stem cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 290,480, filed December 16, 2021, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to cell culture applications, more particularly to the culture and / or proliferation of keratinocytes, and even more particularly to the culture and / or proliferation of epidermal keratinocytes. [Background technology]

[0003] In addition to their use as a research tool, the ability to grow keratinocytes, such as epidermal keratinocytes, would be important for medical applications: a growable and potentially unlimited source of keratinocytes would be of great importance to burn victims and others requiring skin grafts.

[0004] In addition, a proliferative and potentially unlimited source of keratinocytes, such as epidermal keratinocytes, would be a valuable resource for industries, such as the cosmetics industry, to test the effects of products and product ingredients in in vitro model systems.

[0005] Furthermore, a proliferative and potentially limitless source of keratinocytes, such as epidermal keratinocytes, would enable scientific research studying the developmental biology of the skin and diseases related to the skin, such as melanoma and other skin cancers.

[0006] However, primary skin samples are scarce and primary human keratinocyte cultures have a limited life span. Therefore, the development of media and methods for deriving keratinocytes from pluripotent stem cells (PSCs) would provide research and industrial laboratories, and potentially the medical field, with an unlimited source of experimental or therapeutic material. Indeed, PSC-derived keratinocytes would enable the modeling of skin diseases, whether hereditary or spontaneous, bringing hope for patient-specific cell therapy in the clinic.

[0007] Thus, there is a need for robust and efficient media, kits, methods and systems for generating and expanding populations of PSC-derived keratinocytes, such as PSC-derived epidermal keratinocytes. Summary of the Invention

[0008] In one aspect of the disclosure, a method is provided for generating and / or expanding and / or enriching keratinocytes, such as epidermal keratinocytes. The method may include contacting a differentiated population of pluripotent stem cells (PSCs) with a growth medium comprising a basal medium and one or more of an inhibitor of transforming growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure, and culturing the differentiated population of PSCs in the growth medium to generate expanded keratinocytes.

[0009] In one embodiment, the growth medium is supplemented with two or more of an inhibitor of TGF signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure.

[0010] In one embodiment, the growth medium does not contain serum and / or bovine pituitary extract.

[0011] In one embodiment, the contacting and culturing steps are performed in feeder cell-free conditions.

[0012] In one embodiment, the contacting and culturing steps are carried out on or within a support that comprises one or more extracellular matrix proteins.

[0013] In one embodiment, the method can further comprise dissociating the PSC-derived skin organoid to obtain a differentiated population of PSCs.In one embodiment, the PSC-derived skin organoid is formed under serum- and / or feeder cell-free conditions.

[0014] In one embodiment, the PSC-derived skin organoids are PSC-derived hair-containing skin organoids.

[0015] In one embodiment, the method may further include producing more epidermal keratinocytes than contaminating cell types when the contacting and culturing steps are performed in a growth medium, as compared to a basal medium not supplemented with one or more of an inhibitor of TGFB signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure.

[0016] In one embodiment, the inhibitor of TGF signaling is an inhibitor of TGFβ signaling. In one embodiment, the inhibitor of TGFβ signaling is one or more of A83-01, A77-01, and SB431542.

[0017] In one embodiment, the gamma secretase inhibitor is DAPT.

[0018] In one embodiment, the agent that disrupts the cytoskeleton structure is a Rho / Rock kinase inhibitor. In one embodiment, the Rho / Rock kinase inhibitor is Y-27632.

[0019] In another aspect of the present disclosure, a medium for generating and / or proliferating and / or enriching keratinocytes, such as epidermal keratinocytes, is provided. The medium may comprise a basal medium (e.g., a growth medium) supplemented with one or more of an inhibitor of transforming growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure.

[0020] In one embodiment, the inhibitor of TGF signaling is an inhibitor of TGFβ signaling. In one embodiment, the inhibitor of TGFβ signaling is one or more of A83-01, A77-01, and SB431542.

[0021] In one embodiment, the gamma secretase inhibitor is DAPT.

[0022] In one embodiment, the agent that disrupts the cytoskeleton structure is a Rho / Rock kinase inhibitor. In one embodiment, the Rho / Rock kinase inhibitor is Y-27632.

[0023] In one embodiment, the medium allows the (epidermal) keratinocytes to grow without contact with feeder cells.

[0024] In one embodiment, the (epidermal) keratinocytes are derived from a differentiated population of PSCs.

[0025] In one embodiment, the medium is serum and / or BPE free.

[0026] In one embodiment, the medium does not contain exogenously added inhibitors of TGF signaling.

[0027] In one embodiment, the medium does not contain an exogenously added gamma secretase inhibitor.

[0028] In one embodiment, the medium supports the growth of epithelial cells. [Brief description of the drawings]

[0029] For a better understanding of the various embodiments described herein, and to more clearly show how they may be effectively put into practice, reference is made to the accompanying drawings, which illustrate at least one exemplary embodiment, and which will now be described, and which are not intended to limit the scope of the teachings described herein. [Figure 1] Representative photographs of human cells in culture are shown. (A) Human primary neonatal keratinocytes (HEKn3) were cultured as monolayers in DermaCult™ (STEMCELL Technologies) medium and imaged at day 25 of the third passage. (B) Human PSCs were cultured as monolayers in Defined Keratinocyte Serum Free Medium (ThermoFisher) using published protocols (Kajiwara K, et al, 2017). Scale bar is 200 μm. [Diagram 2] Representative images corresponding to different stages of the disclosed keratinocyte differentiation protocol using WLS-1C iPS cells are shown. Compared to the results shown in Figure 1, the efficiency of keratinocyte induction from PSCs may be improved when cells of hair-containing skin organoids are used as starting points. Panel A) shows PSC-derived hair-containing skin organoids at days 70-120. Images in panels B)-D) correspond to days 4, 7, and 10 after plating dissociated cells of PSC-derived hair-containing skin organoids in a monolayer in the disclosed culture medium. Scale bar is 200 μm. [Diagram 3] 1 shows a line graph of keratinocyte population doublings, comparing the population doublings of H9 ES cells, H1 ES cells, and STiPS-F022 iPS cells with primary neonatal and adult keratinocytes during extended culture in culture medium of the present disclosure. [Figure 4] Figure 1 shows the effect of extracellular matrix coating on keratinocyte proliferation. Differentiated STiPS-F022 iPS cells were plated as a monolayer either directly on plastic dishes or on collagen coatings, and contacted with culture medium of the present disclosure to grow the cells for at least six passages (p6). (A) Images of each condition were taken 5 days after plating and 5 days into the sixth passage. (B) Proliferation of STiPS-F022 iPS-derived keratinocytes was assessed among cells plated either directly on plastic dishes or on collagen coatings. Scale bar is 200 μm. [Diagram 5] Immunocytochemical staining for expression of keratinocyte-specific markers among keratinocytes derived / expanded from PSCs. (A) WLS-1C iPS cells were differentiated into keratinocyte-like cells according to the present disclosure and assessed for expression of keratin 14 (K14), keratin 5 (K5), and TP63 (p63) at passage 1. (B) WLS-1C iPS cells were differentiated into keratinocyte-like cells according to the present disclosure and assessed for expression of keratin 14 (K14), keratin 5 (K5), and TP63 (p63) at passage 9. (C) H9 ES cells were differentiated into keratinocyte-like cells according to the present disclosure and assessed for expression of K14, K5, and p63 at passage 4. (D) Expression levels of K14, K5, and p63 among PSC-derived keratinocytes were assessed relative to control cultures of human primary keratinocytes. The scale bar is 200 μm. [Figure 6] Shows the morphology of keratinocyte colonies seeded at a certain clonal cell density. Both primary neonatal keratinocytes (HEKn3) and dissociated PSC-derived skin organoids (STiPS-F022) cells were seeded at 200 cells / well in 6-well plates and cultured in DermaCult™ in the absence of coating or feeder cell support. In the image, grey indicates K14+ cells / colonies. [Figure 7] Morphology of PSC-derived cells after culturing in various commercially available media is shown. After STiPS-F022 iPS cells were formed into hair-containing skin organoids as described herein and dissociated, cells were plated in (A) EpiLife™ EDGS medium (ThermoFisher), (B) EpiLife™ HKGS medium (ThermoFisher), or (C) CnT-57 medium (CELLnTEC). Images of cells grown in each media condition were taken at the indicated time points. Scale bar is 200 μm. [Figure 8]1 shows a line graph of population doublings of PSC-derived cells grown in various culture media. H9 ES cells were grown in either the keratinocyte differentiation medium of the present disclosure or in EpiLife HKGS medium, and population doublings were evaluated over several weeks. STiPS-F022 iPS cells were grown in either the keratinocyte differentiation medium of the present disclosure or in EpiLife EDGS medium, and population doublings were evaluated over several weeks. H1 ES cells were grown in the keratinocyte differentiation medium of the present disclosure, and population doublings were evaluated over several weeks. [Figure 9] 1 shows expression of keratin 14 among PSC-derived cells cultured in different media formulations. (A) Representative immunocytochemistry images of keratin 14 staining among cells derived from H1 and H9 ES cells, and WLS-1C and STiPS-F022 iPS cells using either the media of the present disclosure, EpiLife HKGS, EpiLife EDGS, or CnT57 culture medium. (B) Plots show the percentage of keratin 14 positive cells differentiated in the indicated culture medium from the indicated PSC lines. Scale bar is 200 μm. [Figure 10] Figure 1 shows the expression of TP63 among PSC-derived cells cultured in different media formulations. (A) Representative immunocytochemistry images of TP63 staining among cells derived from H1 and H9 ES cells, and WLS-1C and STiPS-F022 iPS cells using either the media of the present disclosure, EpiLife HKGS, EpiLife EDGS, or CnT57 culture media. (B) Plots show the percentage of TP63-positive cells among keratinocytes differentiated in the indicated culture media from the indicated PSC lines. Scale bar is 200 μm. [Figure 11]Figure 2 shows the morphology of PSC-derived cells after culturing in various media formulations. H9 ES cells and WLS-1C iPS cells were formed into hair-containing skin organoids as described herein, and after dissociation, the cells were plated in DermaCult, EpiLife HKGS, or EpiLife HKGS supplemented with different small molecules. Images of cells grown in each media condition were taken at the indicated time points. Scale bar is 200 μm. [Figure 12] Figure 1 shows the FACS analysis of TP63+ cells generated in different media formulations.WLS-1C iPS cells are formed into hair-containing skin organoids as described herein, and then dissociated, and cells are plated on EpiLife HKGS or EpiLife HKGS supplemented with the indicated small molecules.Cells are stained for TP63 expression and analyzed by FACS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present disclosure relates to medium compositions and / or supplements added to medium and to methods for culturing human keratinocytes. More specifically, the present disclosure relates to the growth of human keratinocytes or conditions for the growth of human keratinocytes, but not the growth (conditions) of off-target or contaminating cell types present in the starting cell population. In one embodiment, the present disclosure relates to the preferential or selective growth of keratinocytes (e.g., epidermal keratinocytes) in comparison to other cell types found in the skin, such as fibroblasts.

[0031] As used in this disclosure, the term "keratinocyte" refers to a cell type found in various animals, particularly in their skin, but also, for example, in the esophagus. In humans and other animals, keratinocytes constitute the majority of the outer layer of the skin (i.e., the epidermis). Thus, in embodiments of the present disclosure, the term "keratinocyte" refers to epidermal keratinocytes, such as human epidermal keratinocytes. As such, the terms "keratinocyte" and "epidermal keratinocyte" are used interchangeably herein. In the context of skin, keratinocytes differentiate from the underlying basal layer and migrate upward toward the surface of the epidermis. In humans, keratinocytes may constitute about 90% or more of the skin cells of the epidermis. During the development of keratinocytes from epidermal stem cells, different stages of keratinocyte development are characterized by the expression of stage-specific markers. Keratinocytes may be characterized by expression of TP63, keratin 5 ("K5"), and keratin 14 ("K14"). In vivo, keratinocytes have at least some proliferative capacity before they differentiate and / or senesce and / or are sloughed off.

[0032] As used in this disclosure, the term "pluripotent stem cells" or "PSCs" or its plural variants refer to a population of cells that can self-renew and differentiate into all three germ layers and beyond. Conditions for culturing PSCs to maintain them in an undifferentiated state are known, but improving these conditions is the subject of ongoing research. PSCs is a broad term that describes both embryonic stem cells ("ESCs") and induced pluripotent stem cells (iPSCs), among others. There can be ethical challenges regarding the procurement of ESCs, but to date, several ESC lines are well established in the art. iPSCs, on the other hand, are less associated with ethical constraints because they can be induced from a rich variety of readily available cells, including adult somatic cells. Thus, new iPSC lines constantly emerge. PSCs are important models to study differentiation mechanisms, model disease, and provide important medical opportunities. PSCs can be harvested, derived, or induced from any source species, but in this disclosure, PSCs are preferably human-derived.

[0033] As used in this disclosure, the term "differentiated population of PSCs" refers to a population of cells that emerges from one or more undifferentiated PSCs at a certain point in time and is no longer in an undifferentiated state as a result of exposure to various internal or external cues. Differentiated populations of PSCs still maintain some capacity for self-renewal, but the ability of cells of such populations to differentiate into any lineage may be constrained by the developmental trajectory they follow (as well as being influenced by the internal or external cue(s) they are exposed to). In the context of this disclosure, differentiated populations of PSCs are populations of cells that are driven toward the ectodermal lineage. More specifically, differentiated populations of PSCs are populations of cells that are directed toward the epidermal lineage or further downstream of the early ectoderm to the epidermal lineage. Starting from PSCs, differentiated populations of PSCs may differentiate directly into the cell type of interest or may pass through one or more intermediate stages. Thus, differentiated populations of PSCs that can give rise to epidermal stem cells may have passed through one or more early stages, including the ectodermal progenitor stage. In one embodiment, the differentiation population of PSCs corresponds to the cells of dissociated PSC-derived skin organoids. PSC-derived skin organoids may be generated by any method known in the art, including the method published by Lee et al. (Nature, 2020). In one embodiment, the differentiation population of PSCs corresponds to the cells of hair-containing PSC-derived skin organoids. In one embodiment, the differentiation population of PSCs corresponds to the cells of dissociated hair-containing PSC-derived skin organoids. The differentiation population of PSCs may correspond to any source species, but in the present disclosure, the differentiation population of PSCs is preferably derived from humans.

[0034] Media and supplements In one aspect of the present disclosure, a medium for growing keratinocytes, such as epidermal keratinocytes, is provided. In one embodiment, the (epidermal) keratinocytes are of primary origin, i.e., taken directly from a subject or patient. In one embodiment, the (epidermal) keratinocytes are derived from PSCs. In one embodiment, the (epidermal) keratinocytes are derived from a differentiation population of PSCs. Thus, the differentiation population of PSCs may be intermediate between PSCs and (epidermal) keratinocytes.

[0035] In one embodiment, the differentiated population of PSCs (e.g., from skin organoids or hair-containing skin organoids) can comprise one or more epidermal stem cells. In one embodiment, the differentiated population of PSCs (e.g., from skin organoids or hair-containing skin organoids) can comprise one or more progenitor cells of keratinocytes or keratinocyte-like cells. In one embodiment, the differentiated population of PSCs (e.g., from skin organoids or hair-containing skin organoids) can comprise both one or more epidermal stem cells and one or more progenitor cells of keratinocytes or keratinocyte-like cells.

[0036] In one embodiment, the (epidermal) keratinocytes or keratinocyte-like cells are of mammalian origin. In one embodiment, the (epidermal) keratinocytes or keratinocyte-like cells are of human origin. In one embodiment, the keratinocytes are of PSC origin. In one embodiment, the keratinocytes or keratinocyte-like cells are proliferative and / or self-renewing. Thus, in one embodiment, the keratinocytes may be or may comprise stem cells and / or keratinocyte progenitor cells.

[0037] In one embodiment, a differentiation population of PSCs (e.g., from skin organoids or hair-containing skin organoids) may comprise one or more holoclones. In one embodiment, a differentiation population of PSCs (e.g., from skin organoids or hair-containing skin organoids) may comprise one or more meroclones. In one embodiment, a differentiation population of PSCs (e.g., from skin organoids or hair-containing skin organoids) may comprise one or more paraclones. In one embodiment, a differentiation population of PSCs (e.g., from skin organoids or hair-containing skin organoids) may comprise two or more holoclones and two or more meroclones. In one embodiment, a differentiation population of PSCs (e.g., from skin organoids or hair-containing skin organoids) may comprise more holoclones and / or meroclones than paraclones.

[0038] The keratinocyte medium of the present disclosure may be used to grow keratinocytes, such as epidermal keratinocytes and / or keratinocyte-like cells, and may therefore be referred to as keratinocyte growth medium or epidermal keratinocyte growth medium. The foregoing terms may be used interchangeably herein.

[0039] The keratinocyte medium of the present disclosure includes a basal medium. In one embodiment, the basal medium may be any medium capable of supporting the growth of keratinocytes, such as epidermal keratinocytes or keratinocyte-like cells. In one embodiment, the basal medium may be any medium capable of supporting the growth of PSC-derived keratinocytes, such as PSC-derived epidermal keratinocytes. Basal media are known in the art and may include RPMI, DMEM, F-12, MCDB153, DMEM / F-12, Adv DMEM, Adv DMEM / F-12. Basal media typically include carbohydrates, amino acids, trace elements, lipids, buffers, salts, and the like. In some embodiments, the basal medium may not include one or more of the aforementioned types of components, which may be included in a supplement. In some embodiments, the basal medium may be further supplemented with one or more of the aforementioned types of components.

[0040] The basal medium used to formulate the (epidermal) keratinocyte growth medium of the present disclosure may also be supplemented with additional components to make a complete medium. Thus, the keratinocyte medium of the present disclosure (e.g., epidermal keratinocyte growth medium) may be formulated as a complete medium or as a basal medium provided (whether in a kit or otherwise) with one or more supplements that are added to the basal medium before being used to make the complete medium.

[0041] To formulate a complete (epidermal) keratinocyte growth medium, it may be beneficial and / or necessary to include (or further supplement) one or more of the following: one or more growth factors or other proteins, one or more hormones, salts, vitamins, an albumin source, one or more small molecules, etc.

[0042] In one embodiment, an albumin source is included in the keratinocyte growth medium of the present disclosure. In one embodiment, the albumin source can be serum, such as serum collected from a mammal. In one embodiment, the albumin source is a more defined component than serum. For example, the albumin source can be isolated albumin. In one embodiment, the isolated albumin is recombinant albumin. In one embodiment, the isolated albumin is bovine serum albumin. In one embodiment, the isolated albumin is human albumin.

[0043] In one embodiment, the keratinocyte medium (eg, keratinocyte growth medium or epidermal keratinocyte growth medium) of the present disclosure is serum-free.

[0044] In one embodiment, the keratinocyte growth medium benefits from and / or requires supplementation with one or more small molecules. In one embodiment, the one or more small molecules include one or more small molecule inhibitors. In one embodiment, the one or more small molecules (e.g., small molecule inhibitors) are included in the supplement.

[0045] In one embodiment, the keratinocyte medium (e.g., keratinocyte growth medium or epidermal keratinocyte growth medium) comprises a basal medium supplemented with one or more of an inhibitor of transforming growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure.

[0046] The inhibitor of transforming growth factor (TGF) signaling may be any factor that functions to inhibit signaling by TGF. In one embodiment, the inhibitor of transforming growth factor (TGF) signaling is a small molecule, such as a small molecule inhibitor. In one embodiment, the inhibitor of transforming growth factor (TGF) signaling is a protein. In one embodiment, the inhibitor of TGF signaling is an inhibitor of TGFβ signaling. In one embodiment, the inhibitor of TGF signaling is one or more of A83-01, A77-01, and SB431542, galunisertib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947.

[0047] The gamma secretase inhibitor may be any agent that functions to inhibit signal transduction by gamma secretase. In one embodiment, the gamma secretase inhibitor is a small molecule, such as a small molecule inhibitor. In one embodiment, the gamma secretase inhibitor is a protein. In one embodiment, the gamma secretase inhibitor is one or more of DAPT, RO4929097, semagacestat (LY450139), avagacestat (BMS-708163), dibenzazepine (YO-01027), LY411575, L-685,458, crenigacestat (LY3039478).

[0048] The agent that disrupts the cytoskeleton may be any factor that performs the function of disrupting the cytoskeleton. In one embodiment, the agent that disrupts the cytoskeleton is a small molecule, such as a small molecule inhibitor. In one embodiment, the agent that disrupts the cytoskeleton is a protein. In one embodiment, the agent that disrupts the cytoskeleton is a Rho / Rock kinase inhibitor. In one embodiment, the agent that disrupts the cytoskeleton is one or more of Y-27632, thiazovivin, fasudil (HA-1077), GSK429286A, RKI-1447, Y-27632, H-1152 dihydrochloride, and azaindole 1 (TC-S 7001).

[0049] In one embodiment, the keratinocyte culture medium (e.g., keratinocyte growth medium or epidermal keratinocyte growth medium) comprises a basal medium supplemented with two or more of an inhibitor of transforming growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure. In one embodiment, two or more small molecules (e.g., small molecule inhibitors) are included in the supplement.

[0050] In one embodiment, the keratinocyte medium (e.g., keratinocyte growth medium or epidermal keratinocyte growth medium) comprises a basal medium supplemented with each of an inhibitor of transforming growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure. In one embodiment, each of the small molecules (e.g., small molecule inhibitors) is included in the supplement.

[0051] In one embodiment, the epidermal keratinocyte growth medium of the present disclosure may be defined. That is, in one embodiment, the epidermal keratinocyte growth medium of the present disclosure does not contain and / or is not in contact with undefined components. Examples of undefined components that are often included in or in contact with cell culture media include bovine pituitary extract ("BPE"), serum, human platelet lysate, feeder cells, isolated extracellular matrix proteins produced by cells (e.g., Matrigel™), conditioned media, etc.

[0052] In one embodiment, the keratinocyte medium (e.g., keratinocyte growth medium or epidermal keratinocyte growth medium) of the present disclosure does not contain serum and / or BPE. In one embodiment, the medium of the present disclosure does not contact feeder cells or an undefined cell support (e.g., matrix) such as Matrigel™.

[0053] In one embodiment, keratinocytes (e.g., epidermal keratinocytes) growing or grown in the presence of the medium of the present disclosure may be cultured in the presence of an extracellular matrix, preferably in the presence of a defined extracellular matrix coating, such as on a collagen coating.

[0054] Thus, the keratinocyte medium of the present disclosure (e.g., keratinocyte growth medium or epidermal keratinocyte growth medium), whether provided as a complete medium or as a basal medium supplemented as described herein, supports the proliferation of (epidermal) keratinocytes, whether PSC-derived or primary. In one embodiment, the medium of the present disclosure supports 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, or 60 or more keratinocyte population doublings.

[0055] In one embodiment, keratinocytes, such as epidermal keratinocytes, grown using the medium of the present disclosure express characteristic markers. In one embodiment, more than 40% of the grown cells are positive for characteristic markers of (epidermal) keratinocytes. In one embodiment, more than 50% of the grown cells are positive for characteristic markers of (epidermal) keratinocytes. In one embodiment, more than 60% of the grown cells are positive for characteristic markers of (epidermal) keratinocytes. In one embodiment, more than 70% of the grown cells are positive for characteristic markers of (epidermal) keratinocytes. In one embodiment, more than 80% of the grown cells are positive for characteristic markers of (epidermal) keratinocytes. In one embodiment, more than 90% of the grown cells are positive for characteristic markers of (epidermal) keratinocytes. In one embodiment, nearly 100% of the grown cells are positive for characteristic markers of (epidermal) keratinocytes.

[0056] In one embodiment, characteristic markers of keratinocytes, such as epidermal keratinocytes, include one or more of K14 and p63. In one embodiment, more than 40% of the expanded cells express one or both of K14 and p63 at the second, third, fourth, or fifth passage. In one embodiment, more than 50% of the expanded cells express one or both of K14 and p63 at the second, third, fourth, or fifth passage. In one embodiment, more than 60% of the expanded cells express one or both of K14 and p63 at the second, third, fourth, or fifth passage. In one embodiment, more than 70% of the expanded cells express one or both of K14 and p63 at the second, third, fourth, or fifth passage. In one embodiment, greater than 80% of the expanded cells express one or both of K14 and p63 at the second, third, fourth or fifth passage. In one embodiment, greater than 90% of the expanded cells express one or both of K14 and p63 at the second, third, fourth or fifth passage. In one embodiment, nearly 100% of the expanded cells express one or both of K14 and p63 at the second, third, fourth or fifth passage.

[0057] In one embodiment, the keratinocyte medium (e.g., keratinocyte growth medium or epidermal keratinocyte growth medium) of the present disclosure is superior (in terms of the percentage of cells that actually produce keratinocytes) to commercially available media. For example, media designed to grow primary keratinocytes include EpiLife HKGS, EpiLife EDGS, KSFM, DKSFM, KGM2, KGM gold, CnT-07, CnT-57, and CnT-Prime. In addition, various of these media products may have other limitations, such as not being defined.

[0058] Thus, the keratinocyte culture medium of the present disclosure may support more population doublings than the aforementioned medium formulations. Additionally, or alternatively, the epidermal keratinocyte growth medium of the present disclosure may produce a greater proportion of proliferated keratinocytes, such as epidermal keratinocytes, than the aforementioned medium formulations.

[0059] In one embodiment, the keratinocyte medium of the present disclosure (e.g., keratinocyte growth medium or epidermal keratinocyte growth medium) preferentially or selectively (compared to other media) supports the growth of keratinocytes, such as epidermal keratinocytes, whether derived from PSCs or sourced from a primary tissue sample. In such an embodiment, when cultured in the presence of the keratinocyte medium of the present disclosure (e.g., keratinocyte growth medium or epidermal keratinocyte growth medium), off-target cells may be outcompeted by target cells (e.g., keratinocytes). In contrast, when cultured in the presence of currently available media, such as the commercially available media listed in the preceding paragraph, target cells (e.g., keratinocytes) may be outcompeted by off-target cells (e.g., fibroblasts, epidermal cells other than keratinocytes).

[0060] method In one aspect of the present disclosure, a method for expanding keratinocytes, such as epidermal keratinocytes, is provided. In one embodiment, the (epidermal) keratinocytes are of primary origin, i.e., taken directly from a subject or patient. In one embodiment, the (epidermal) keratinocytes are derived from PSCs. In one embodiment, the (epidermal) keratinocytes are derived from a differentiated population of PSCs. Thus, the differentiated population of PSCs may be intermediate between PSCs and (epidermal) keratinocytes.

[0061] In one embodiment, the (epidermal) keratinocytes or keratinocyte-like cells are of mammalian origin. In one embodiment, the (epidermal) keratinocytes or keratinocyte-like cells are of human origin. In one embodiment, the mammalian keratinocytes are of PSC origin. In one embodiment, the keratinocytes or keratinocyte-like cells are proliferative and / or self-renewing. Thus, in one embodiment, the keratinocytes may be or may comprise stem cells and / or keratinocyte progenitor cells.

[0062] In one embodiment, the differentiation population of PSCs can be derived from or correspond to the cells of skin organoid (e.g., PSC-derived skin organoid).In one embodiment, dissociated skin organoid can correspond to the cells of hair-containing skin organoid.In one embodiment, the differentiation population of PSCs can be a dissociated population of cells derived from skin organoid, such as hair-containing skin organoid.

[0063] The description of keratinocyte medium (eg keratinocyte growth medium or epidermal keratinocyte growth medium) above applies to any reference to such medium in connection with the methods, kits and uses described herein.

[0064] A method for expanding keratinocytes, such as epidermal keratinocytes, may include contacting a differentiated population of pluripotent stem cells (PSCs) with a keratinocyte medium (as described above) and culturing the differentiated population of PSCs in such medium to generate expanded keratinocytes, such as epidermal keratinocytes.

[0065] In another aspect, a method of enriching or selecting a population of keratinocytes, such as epidermal keratinocytes, comprises contacting a differentiated population of pluripotent stem cells (PSCs) with a keratinocyte medium and culturing the differentiated population of PSCs in such medium to generate enriched and / or expanded keratinocytes, such as epidermal keratinocytes. An enriched population of cells refers to a scenario in which the target cells, in this case keratinocytes, predominate over each population, whether alone or in combination with off-target cells (e.g., fibroblasts, epidermal cells other than keratinocytes, etc.).

[0066] In one embodiment, the differentiated population of PSCs (e.g., from skin organoids or hair-containing skin organoids) can comprise one or more epidermal stem cells. In one embodiment, the differentiated population of PSCs (e.g., from skin organoids or hair-containing skin organoids) can comprise one or more progenitor cells of keratinocytes or keratinocyte-like cells. In one embodiment, the differentiated population of PSCs (e.g., from skin organoids or hair-containing skin organoids) can comprise both one or more epidermal stem cells and one or more progenitor cells of keratinocytes or keratinocyte-like cells.

[0067] In one embodiment, a differentiation population of PSCs (e.g., from skin organoids or hair-containing skin organoids) may comprise one or more holoclones. In one embodiment, a differentiation population of PSCs (e.g., from skin organoids or hair-containing skin organoids) may comprise one or more meroclones. In one embodiment, a differentiation population of PSCs (e.g., from skin organoids or hair-containing skin organoids) may comprise one or more paraclones. In one embodiment, a differentiation population of PSCs (e.g., from skin organoids or hair-containing skin organoids) may comprise two or more holoclones and two or more meroclones. In one embodiment, a differentiation population of PSCs (e.g., from skin organoids or hair-containing skin organoids) may comprise more holoclones and / or meroclones than paraclones.

[0068] The contacting and culturing steps may be for any duration that will produce a desired amount of keratinocytes, such as enriched and / or expanded epidermal keratinocytes, hi one embodiment, the contacting and culturing steps are carried out for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

[0069] In one embodiment, the contacting and culturing steps are carried out for more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, or more than 10 passages. Of course, the population of expanded or proliferating (epidermal) keratinocytes may be temporarily excluded from contact with the keratinocyte medium during the period in which the culture is being passaged.

[0070] In one embodiment, the contacting and culturing steps are performed in feeder cell-free conditions. In one embodiment, the contacting and culturing steps are performed on or in a support or coating comprising one or more extracellular matrix proteins. In one embodiment, the support or coating comprising one or more extracellular matrix proteins is undefined, such as Matrigel™. In one embodiment, the support or coating comprising one or more extracellular matrix proteins is defined, such as collagen, vitronectin, laminin. In one embodiment, the one or more extracellular matrix proteins are recombinant. In one embodiment, the contacting and culturing steps are performed in the absence of a support or coating comprising one or more extracellular matrix proteins.

[0071] As previously described, the growth medium of the present disclosure comprises a basal medium supplemented with one or more of an inhibitor of transforming growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure.

[0072] The inhibitor of transforming growth factor (TGF) signaling may be any factor that functions to inhibit signaling by TGF. In one embodiment, the inhibitor of transforming growth factor (TGF) signaling is a small molecule, such as a small molecule inhibitor. In one embodiment, the inhibitor of transforming growth factor (TGF) signaling is a protein. In one embodiment, the inhibitor of TGF signaling is an inhibitor of TGFβ signaling. In one embodiment, the inhibitor of TGF signaling is one or more of A83-01, A77-01, and SB431542, galunisertib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947.

[0073] The gamma secretase inhibitor may be any agent that functions to inhibit signal transduction by gamma secretase. In one embodiment, the gamma secretase inhibitor is a small molecule, such as a small molecule inhibitor. In one embodiment, the gamma secretase inhibitor is a protein. In one embodiment, the gamma secretase inhibitor is one or more of DAPT, RO4929097, semagacestat (LY450139), avagacestat (BMS-708163), dibenzazepine (YO-01027), LY411575, L-685,458, crenigacestat (LY3039478).

[0074] The agent that disrupts the cytoskeleton may be any factor that performs the function of disrupting the cytoskeleton. In one embodiment, the agent that disrupts the cytoskeleton is a small molecule, such as a small molecule inhibitor. In one embodiment, the agent that disrupts the cytoskeleton is a protein. In one embodiment, the agent that disrupts the cytoskeleton is a Rho / Rock kinase inhibitor. In one embodiment, the agent that disrupts the cytoskeleton is one or more of Y-27632, thiazovivin, fasudil (HA-1077), GSK429286A, RKI-1447, Y-27632, H-1152 dihydrochloride, and azaindole 1 (TC-S 7001).

[0075] In one embodiment, a keratinocyte medium (e.g., keratinocyte growth medium or epidermal keratinocyte growth medium) of the present disclosure comprises a basal medium supplemented with two or more of an inhibitor of transforming growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure.

[0076] In one embodiment, a keratinocyte medium (e.g., keratinocyte growth medium or epidermal keratinocyte growth medium) of the present disclosure comprises a basal medium supplemented with each of an inhibitor of transforming growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure.

[0077] In one embodiment, the basal medium supplemented as described herein (e.g., epidermal keratinocyte growth medium) does not contain serum and / or bovine pituitary extract. In one embodiment, the basal medium supplemented as described herein (e.g., epidermal keratinocyte growth medium) is defined.

[0078] In one embodiment, the method may further comprise dissociating the PSC-derived skin organoid to obtain a differentiated population of PSCs. In one embodiment, the method further comprises providing a dissociated sample of the differentiated population of PSCs. In one embodiment, the dissociated sample is obtained by dissociating the skin organoid. In one embodiment, the skin organoid is a hair-containing skin organoid. In one embodiment, the skin organoid and / or the hair-containing skin organoid is derived from PSCs. In one embodiment, the skin organoid and / or the hair-containing skin organoid is derived from human cells, such as human PSCs.

[0079] In one embodiment, skin organoid and / or hair-containing skin organoid are derived according to published methods.In one embodiment, skin organoid and / or hair-containing skin organoid are derived using STEMdiff™ Skin Organoid Kit (STEMCELL Technologies).

[0080] In one embodiment, skin organoids and / or hair-containing skin organoids are formed under serum- and / or feeder cell-free conditions. In one embodiment, skin organoids and / or hair-containing skin organoids are formed in the presence of Matrigel™. In one embodiment, skin organoids and / or hair-containing skin organoids are formed in the absence of Matrigel™. In one embodiment, skin organoids and / or hair-containing skin organoids are formed using low-attachment, ultra-low-attachment, or non-tissue culture treated plates (e.g., 96-well, 24-well, or 6-well, etc.). In one embodiment, the plate used to form skin organoids and / or hair-containing skin organoids can be coated with a surfactant, such as Anti-adherence Rinse Solution (STEMCELL Technologies), before seeding cells thereon.

[0081] In one embodiment, the skin organoids and / or hair-containing skin organoids are formed in a stepwise manner and take at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 or more days before structures and cell types develop.

[0082] In one embodiment, skin (or hair-containing skin) organoids can be dissociated using any known dissociation means. In one embodiment, the dissociation means can be protease-based. In one embodiment, the dissociation means can be trypsin, dispase, TryplLE™, Accutase™.

[0083] In one embodiment, skin (or hair-containing skin) organoids can be dissociated at various times during their development timeline and still produce expanded keratinocytes when cultured according to the medium and method of the present disclosure. Thus, in one embodiment, skin (or hair-containing skin) organoids (e.g., populations of differentiated PSCs) dissociated at various times can still be used to harvest expanded and / or enriched keratinocytes (e.g., epidermal keratinocytes). Without being bound by theory, suitable progenitor cells of keratinocytes (e.g., epidermal keratinocytes) may exist before skin (or hair-containing skin) organoids are fully developed and may survive to the later stages of such development. In one embodiment, 70-day, 100-day, or 140-day organoids can be used to induce expanded and / or enriched (epidermal) keratinocytes in the medium (or by carrying out the method) of the present disclosure.

[0084] The disclosed method (and use of the medium) produces expanded epidermal keratinocytes. Expanded epidermal keratinocytes should exhibit cobblestone-like and polygonal epithelial cell morphology. In addition, elongated or spindle-shaped cells that may resemble fibroblasts and / or other contaminating, off-target cell types should be few or absent. In one embodiment, the amount of fibroblasts and / or other contaminating or off-target cells decreases during or after passaging, while the amount of (epidermal) keratinocytes or keratinocyte-like cells increases during or after passaging.

[0085] In one embodiment, the expanded keratinocytes grow as colonies. In one embodiment, the expanded keratinocytes grow as clonally derived colonies. In one embodiment, the colonies are derived from holoclones or holoclon-like cells (in the case of PSC-derived cultures). In one embodiment, the colonies are derived from meroclones or meroclones-like cells (in the case of PSC-derived cultures). In one embodiment, the colonies are derived from paraclones or paraclones-like cells (in the case of PSC-derived cultures). In one embodiment, the majority of the colonies are derived from holoclones or holoclones-like cells (in the case of PSC-derived cultures) and / or from meroclones or meroclones-like cells (in the case of PSC-derived cultures).

[0086] In one embodiment, the colony formation efficiency of the differentiation population (among which one or more holoclones and / or meroclones) of PSCs (e.g., among the cells of skin organoids, such as dissociated hair-containing skin organoids) is greater than 1%. In one embodiment, the colony formation efficiency of the differentiation population (among which one or more holoclones and / or meroclones) of PSCs (e.g., among the cells of skin organoids, such as dissociated hair-containing skin organoids) is greater than 2%. In one embodiment, the colony formation efficiency of the differentiation population (among which one or more holoclones and / or meroclones) of PSCs (e.g., among the cells of skin organoids, such as dissociated hair-containing skin organoids) is greater than 3%. In one embodiment, the colony formation efficiency of the differentiation population (among which one or more holoclones and / or meroclones) of PSCs (e.g., among the cells of skin organoids, such as dissociated hair-containing skin organoids) is greater than 4%. In one embodiment, the colony formation efficiency of the differentiation population (among which one or more holoclones and / or meroclones) of PSCs (e.g., among the cells of skin organoids, such as dissociated hair-containing skin organoids) is greater than 5%. In one embodiment, the colony formation efficiency of the differentiation population (among which one or more holoclones and / or meroclones) of PSCs (e.g., among the cells of skin organoids, such as dissociated hair-containing skin organoids) is greater than 6%. In one embodiment, the colony formation efficiency of the differentiation population (among which one or more holoclones and / or meroclones) of PSCs (e.g., among the cells of skin organoids, such as dissociated hair-containing skin organoids) is greater than 7%. In one embodiment, the colony formation efficiency of the differentiation population (among which one or more holoclones and / or meroclones) of PSCs (e.g., among the cells of skin organoids, such as dissociated hair-containing skin organoids) is greater than 8%. In one embodiment, the colony formation efficiency of a differentiated population (among which one or more holoclones and / or meroclones) of PSCs (e.g., among cells of a skin organoid, such as a dissociated hair-containing skin organoid) is greater than 9%.In one embodiment, the colony formation efficiency of a differentiated population (among which one or more holoclones and / or meroclones) of PSCs (e.g., among cells of a skin organoid, such as a dissociated hair-containing skin organoid) is 10% or greater.

[0087] In one embodiment, the method may further comprise producing more (epidermal) keratinocytes than contaminating (e.g., off-target) cell types when the contacting and culturing steps are performed using the medium of the present disclosure, compared to the use of a basal medium that is not supplemented with one or more, two or more, or each of an inhibitor of TGFB signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure. Thus, preferential proliferation / generation / enrichment of keratinocytes, such as epidermal keratinocytes, may be advantageously and surprisingly achieved using the medium and methods as disclosed herein, compared to other commercially available media.

[0088] The efficiency of the media and methods disclosed herein for expanding / generating / enriching keratinocytes, such as epidermal keratinocytes, can be assessed by examining biomarkers. Markers of keratinocytes, such as epidermal keratinocytes, may include basal layer markers, such as keratin 14 (K14), keratin 5 (K5), keratin 15 (K15), TP63, integrin alpha 6 (ITGA6), integrin beta 1 (ITGB1), and collagen XVII (BP180). Efficiency can also be assessed by examining separately or in parallel biomarkers that should not be expressed at measurable levels among (epidermal) keratinocytes, including suprabasal layer markers, such as keratin 10 (K10) and / or involucrin.

[0089] The method may further comprise differentiating the keratinocytes, such as epidermal keratinocytes, generated / enriched / expanded using a medium or according to the method disclosed herein. The keratinocytes, such as epidermal keratinocytes, may be differentiated using any method or medium known to those skilled in the art. In one embodiment, the (epidermal) keratinocytes may be differentiated in the presence of a solution containing a high calcium concentration, such as greater than 1 mM CaCl2. In one embodiment, the (epidermal) keratinocytes may be differentiated under air-liquid interface conditions. When differentiated, whether under ALI conditions or not, primary keratinocytes or PSC-derived keratinocytes should be capable of generating stratified epithelial cells with appropriate cellular architecture and marker expression. In the case of PSC-derived keratinocytes, such appropriate architecture and marker expression should be consistent, similar, or comparable to their in vivo counterparts.

[0090] In one embodiment, keratinocytes, such as epidermal keratinocytes, generated and / or expanded and / or enriched using the media and / or methods of the present disclosure may be used for therapeutic or cosmetic purposes. In one embodiment, such keratinocytes may be combined with a suitable scaffold to generate ex vivo derived skin grafts. In one embodiment, such keratinocytes may be used to study skin development and / or the biology of various skin-related diseases. In one embodiment, such keratinocytes or ex vivo derived skin grafts may be used to test compounds or other substances intended for use on the skin of a subject.

[0091] The following non-limiting examples illustrate the present disclosure. EXAMPLES

[0092] Example 1: Maintenance of pluripotent stem cells Pluripotent stem cells (PSCs) were maintained in PSC maintenance medium (mTeSR1™, TeSR™-E8™, or mTeSR™ Plus) (STEMCELL Technologies) and passaged according to the manufacturer's recommended maintenance culture and passaging protocol. At the start of the keratinocyte induction protocol, 1000-3500 PSCs were aggregated into each microwell of an AggreWell™ 800 (STEMCELL Technologies) microwell device or per well of a low attachment 96-well plate and cultured for 2 days in the same medium used to maintain the PSCs (in this case one of mTeSR1™, TeSR™-E8™, or mTeSR™ Plus (STEMCELL Technologies)) to form aggregates.

[0093] Example 2: Formation of skin organoids The aggregates of Example 1 were plated in AggreWell™ or 96-well plates under non-adherent conditions and cultured in a skin organoid induction medium such as STEMdiff™ Skin Organoid Induction Medium (STEMCELL Technologies) for 12 days. The skin organoids were then transferred to STEMdiff™ Skin Organoids Maturation Medium (STEMCELL Technologies) for at least 8 weeks, with medium addition or replacement every 3-4 days. After the skin organoids mature and preferably appear as hair-containing skin organoids (e.g., hair placodes present skin organoids), they were dissociated overnight in Dispase solution in a 37°C incubator.

[0094] Example 3: Plating of dissociated cells of skin organoids under adherent conditions Dissociated organoids from Example 2 are plated in cell culture medium of the present disclosure (e.g., in DermaCult™, STEMCELL Technologies) at an appropriate density, typically ranging from one dissociated organoid per well of a 6-well plate to a T25 flask. When large colonies appear, they are passaged using Accutase™ or a trypsin-based dissociation reagent at or just before reaching confluence. After transferring the cells to DermaCult™ medium, the cultures are passaged according to the manufacturer's recommended protocol, with cm 2 1.25~5×10 3 Cells are seeded.

[0095] Primary human keratinocytes in culture exhibit a cobblestone-like epithelial cell morphology (Figure 1A). Primary human keratinocytes should also express basal layer markers K14, K5, p63, ITGA6, and ITGB1, but not significant levels of suprabasal layer markers K10 and involucrin. However, when PSCs are directly seeded in monolayer conditions in keratinocyte medium and differentiated using published protocols (Kajiwara K, et al, 2017), the morphology of the resulting cells is not cobblestone-like. Rather, the resulting cells appear elongated (fibroblast-like), spread, and overlapping (Figure 1B). Briefly, the Kajiwara K, et al, 2017 protocol involves culturing PSCs for 4 days on vitronectin-coated plates in Defined Keratinocyte Serum Free Medium ("DKSFM") (Thermo Fisher) supplemented with retinoic acid and BMP4. From days 4 to 14, cells are cultured in DKSFM supplemented with 20 ng / mL EGF. After 14 days, cells are cultured on fibronectin- and type 1 collagen-coated plates in DKSFM supplemented with 20 ng / mL EGF and 10 uM Y-27632.

[0096] Thus, improved means of generating PSC-derived keratinocytes are needed, and the above observations prompted experimentation that led to the unexpected discovery that cells from dissociated skin organoids appear to provide a surprisingly effective starting point for growing relatively pure cultures of PSC-derived keratinocytes.

[0097] Skin organoids can be generated from PSCs using STEMdiff™ Skin Organoids Induction Medium (STEMCELL Technologies). Figure 2A shows a representative image of hair-containing skin organoids formed using STEMdiff™ Skin Organoid Induction Medium (STEMCELL Technologies) formed as described in Examples 1 and 2. Skin organoids were initiated in the aforementioned medium according to the manufacturer's recommended protocol, and after 12 days, the induced skin organoids were transferred to STEMdiff™ Skin Organoid Maturation Medium (STEMCELL Technologies). Skin organoids were allowed to mature under non-adherent conditions for approximately 6 weeks or more, with cell culture medium changes every 3-4 days. Skin organoids can be considered mature when hair follicles are observed on their outer surface toward the cell culture medium.

[0098] Once the (hair-containing) skin organoids are sufficiently mature, they can be incubated overnight at 37°C in Dispase™ (STEMCELL Technologies) to dissociate the structures into a suspension of cells. The dissociated suspension of cells is plated into tissue-culture treated culture plates or flasks and cultured in DermaCult™ (STEMCELL Technologies) under adherent conditions. The medium is changed approximately every 2-3 days, and before the cells reach confluence, they are passaged using Accutase or a trypsin-based dissociation reagent. After the initial seeding of the differentiated population of PSCs, few colonies of keratinocytes are observed on day 3 or 4 (Figure 2B). However, after approximately 7 days of culture, large colonies of keratinocytes appear (Figures 2C and 2D).

[0099] The population doublings of keratinocytes derived from three different human PSC lines (H9 ES cells, H1 ES cells, and STiPS F022 iPS cells) were measured over several weeks according to this Example 3. The proliferation of human PSC-derived keratinocytes was compared to commercially available primary adult and primary neonatal human keratinocytes. Figure 3 shows that over the course of this experiment, the population doublings of PSC-derived keratinocytes were only slightly reduced and therefore comparable to the primary keratinocyte controls.

[0100] Example 4: Proliferation of PSC-derived keratinocytes is increased when cells are plated on collagen-coated plates The effect of extracellular matrix on population doubling of PSC-derived keratinocytes was investigated. Keratinocytes derived from F022 iPS cells were formed and plated essentially as described in Example 3. However, cell suspensions of dissociated skin organoids were plated either directly on plastic dishes or on collagen-coated dishes (STEMCELL Technologies). At both early passage (passage 0) and late passage (passage 6), more PSC-derived keratinocytes appeared when grown on collagen-coated surfaces (Figure 4A). This observation was confirmed when the number of PSC-derived keratinocytes was quantified over several weeks in the aforementioned culture conditions. When cell suspensions of dissociated skin organoids were cultured on collagen-coated plates, the number of population doublings increased by about 10 compared to conditions in which cells were plated directly on plastic (Figure 4B).

[0101] Example 5: PSC-derived keratinocytes exhibit comparable levels of basal cell markers compared to control cultures of primary keratinocytes PSC-derived keratinocytes generated according to Example 3 or 4 were evaluated for the expression of keratinocyte markers by immunocytochemistry. Briefly, samples were fixed in 4% PFA for 1 hour at room temperature or overnight at 4°C. Samples were then exposed to blocking buffer for 1 hour at room temperature. Samples were then incubated with primary antibodies (as shown in the table below) in diluted PBST overnight at 4°C Celsius. After a series of washes, samples were incubated with secondary antibodies diluted in PBST for 1 hour at room temperature in the dark. After a series of washes, samples were incubated with DAPI dye (1:10000 in PBS) for 8 minutes at room temperature. A series of washes with PBS was performed, after which the samples were visualized using fluorescence microscopy. [Table 1]

[0102] Expression of keratin 14 (K14), keratin 5 (K5), and TP63 (p63) was confirmed in WLS-1C iPS cells at passage 1 and passage 9 (Figures 5A and 5B) and in H9 ES cells at passage 4 (Figure 5C). The expression levels of these markers appeared to be comparable to the expression of these markers among cultures of human primary keratinocytes (Figure 5D). These results indicate that PSC-derived keratinocytes are well similar to primary keratinocytes.

[0103] To test the ability of PSC-derived skin organoids to form colonies of dissociated cells, a limited number of primary keratinocytes and PSC-derived cells (total 200) were plated in a well of a 6-well plate in DermaCult™ without any coating or feeder layer (Figure 6). The emerging colonies were stained for K14, revealing that both types of starting cell populations had cells capable of forming K14+ colonies. Apparent colony formation efficiencies of over 5% and probably about 10% were achieved. Considering morphology and colony size, it was revealed that both starting cell populations contained a large number of holoclones and meroclones among 200 seeded cells. These data suggest that the media and methods disclosed herein support the proliferation and clonal induction of keratinocyte stem cells or early keratinocyte progenitor cells.

[0104] Example 6: Analysis of cells grown using commercially available media Various commercially available cell culture media are commercially available for culturing human epidermal keratinocytes: EpiLife™ Defined Growth Supplement for use with EpiLife™ medium; Gibco™ Human Keratinocyte Growth Supplement for use with EpiLife medium or Medium 154; and Cnt-57 Epithelial Proliferation Medium. These media were tested in an experiment to generate a population of expanded PSC-derived epidermal keratinocytes, essentially starting from a cell suspension of dissociated skin organoids harvested as described in Example 3. Images of the resulting cells taken at different time points after culturing in either EpiLife EDGS (Figure 7A), EpiLife HKGS (Figure 7B), and CnT-57 (Figure 6C) show that these media are unable to grow PSC-derived keratinocytes that exhibit a cobblestone-like morphology. Population doublings of keratinocytes grown from various PSC lines in the culture medium of the present disclosure were also evaluated over a period of several weeks in comparison to EpiLife EDGS and EpiLife HKGS. DermaCult™ outperformed EpiLife EDGS and EpiLife HKGS in all cell lines tested (FIG. 8). These results suggest that EpiLife EDGS, EpiLife HKGS, and CnT-57 are unable to generate populations of expanded PSC-derived keratinocytes.

[0105] Further experiments were performed using DermaCult™ and the commercial media previously described, in which expression of keratin 14 and TP63 was assessed among keratinocytes differentiated from different PSC lines.

[0106] Figure 9A shows representative images of keratin 14 expression among cells differentiated from two ES cell lines (H1 and H9) and two iPS cell lines (WLS-1C and STiPS F022) in either EpiLife HKGS, EpiLife EDGS, CnT-57, and DermaCult™. The images suggest that the DermaCult™ formulation is superior to the commercial formulation, and to confirm these observations, the resulting PSC-derived keratinocytes were quantified (Figure 9B). Cells were quantified using Image J, and there was clearly more K14 expression in DermaCult™ compared to the commercial media tested. + In particular, K14 cells were generated using DermaCult™. + The percentage of cells was consistent across keratinocytes generated from each of the PSC lines tested.

[0107] Figure 10A shows representative images of TP63 expression among keratinocytes differentiated from two ES cell lines (H1 and H9) and two iPS cell lines (WLS-1C and STiPS F022) in either EpiLife HKGS, EpiLife EDGS, CnT-57, and DermaCult™. The images suggest that the DermaCult™ formulation is superior to the commercial formulation, and to confirm these observations, the resulting PSC-derived keratinocytes were quantified (Figure 10B). Cells were quantified as described above with respect to Figure 9, and there was clearly more TP63 expression in DermaCult™ compared to the commercial media tested. + In particular, TP63 cells were generated using DermaCult™. + The percentage of cells was consistent across keratinocytes generated from each of the PSC lines tested.

[0108] The ability of the commercial medium to proliferate PSC-derived keratinocytes could be somewhat restored by supplementing with one or more small molecule inhibitors, but not to the level of DermaCult™ (Figure 11). Specifically, HKGS medium was supplemented with A83-01, DAPT, Y-27632, or all three of A83-01, DAPT, and Y-27632. Figure 10 shows the appearance of colonies when ES cell lines (H9) and iPS cell lines (1C) were cultured in HKGS supplemented with Y-27632 or all three of the aforementioned factors. These findings were confirmed in a separate experiment in which 1C iPS cells were cultured in the aforementioned media conditions and analyzed for p63 expression to distinguish keratinocytes from other cell types, such as fibroblasts, contained in PSC-derived skin organoids (Figure 12). Exposure of cells from dissociated (hair-containing) skin organoids to Y-27632 or all three of the aforementioned factors resulted in the appearance of a comparable number of p63+ cells compared to the absence of p63+ cells appearing after culture in the presence of HKGS alone.

Claims

1. 1. A method for growing epidermal keratinocytes, comprising: contacting a differentiated population of pluripotent stem cells (PSCs) with a growth medium comprising a basal medium and one or more of an inhibitor of transforming growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure; and culturing the differentiated population of PSCs in the growth medium to generate expanded epidermal keratinocytes.

2. The method of claim 1 , wherein the growth medium comprises two or more of the inhibitor of TGF signaling, the gamma secretase inhibitor, and the agent that disrupts the cytoskeletal structure.

3. The method of claim 1 , wherein the growth medium is serum- and / or bovine pituitary extract-free.

4. The method of claim 1 , wherein the contacting step and the culturing step are carried out under feeder cell-free conditions.

5. 10. The method of claim 1, wherein the contacting and culturing steps are carried out on or within a support or coating comprising one or more extracellular matrix proteins.

6. 2. The method of claim 1, further comprising dissociating the PSC-derived skin organoids to obtain a differentiated population of the PSCs.

7. The method of claim 6, wherein the PSC-derived skin organoids are formed under serum- and / or feeder cell-free conditions.

8. 7. The method of claim 6, wherein the PSC-derived skin organoid is a PSC-derived hair-containing skin organoid.

9. 7. The method of any one of claims 1 to 6, further comprising producing more epidermal keratinocytes than contaminating cell types when the contacting and culturing steps are carried out in the growth medium compared to a basal medium that is not supplemented with one or more of the inhibitor of TGFB signaling, the gamma secretase inhibitor, and the agent that disrupts cytoskeletal structure.

10. The method according to any one of claims 1 to 6, wherein the inhibitor of TGF signaling is an inhibitor of TGFβ signaling.

11. The method of any one of claims 1 to 6, wherein the inhibitor of TGFβ signaling is one or more of A83-01, A77-01, and SB431542.

12. The method according to any one of claims 1 to 6, wherein the gamma secretase inhibitor is DAPT.

13. The method according to any one of claims 1 to 6, wherein the agent that disrupts the cytoskeletal structure is a Rho / Rock kinase inhibitor.

14. The method of claim 13, wherein the Rho / Rock kinase inhibitor is Y-27632.

15. An epidermal keratinocyte growth medium, comprising a basal medium supplemented with one or more of an inhibitor of transforming growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure.

16. The medium of claim 15 , wherein the inhibitor of TGF signaling is an inhibitor of TGFβ signaling.

17. The medium according to claim 15, wherein the inhibitor of TGFβ signaling is one or more of A83-01, A77-01, and SB431542.

18. The medium of claim 15, wherein the gamma secretase inhibitor is DAPT.

19. The medium according to claim 15 , wherein the agent that disrupts the cytoskeletal structure is a Rho / Rock kinase inhibitor.

20. The medium according to claim 19, wherein the Rho / Rock kinase inhibitor is Y-27632.

21. The medium according to any one of claims 15 to 20, wherein the medium allows epidermal keratinocytes to grow without contact with feeder cells.

22. The medium according to any one of claims 15 to 20, wherein the epidermal keratinocytes are derived from a differentiated population of PSCs.

23. The medium according to any one of claims 15 to 20, wherein the medium is free of serum and / or BPE.

24. The medium according to any one of claims 15 to 20, wherein the medium does not contain an exogenously added inhibitor of TGF signaling.

25. The medium according to any one of claims 15 to 20, wherein the medium does not contain an exogenously added gamma secretase inhibitor.

26. The medium of claim 15 , wherein the medium supports the growth of the epithelial cells.

Images