Accelerated development of new hair follicles in pluripotent stem cell-derived skin organoids

Abstract

Disclosed are methods of accelerating development of new hair follicles in pluripotent stem cell-derived skin organoids. In particular, disclosed are methods of accelerating development of new hair follicles in skin organoids by adding one or more growth factors, such as FGF10, FGF20, BIO, ruxolitinib, tofacitinib, LDN193189, vitamin E, GDNF, retinoic acid, or IGF2. The growth factors are added to a culture of skin organoid at critical windows of time of the skin organoid development, such as after (or upon) the formation of epidermis and dermis in the skin organoid.

Description

ACCELERATED DEVELOPMENT OF NEW HAIR FOLLICLES IN PLURIPOTENTSTEM CELL-DERIVED SKIN ORGANOIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore Provisional Application No. 1020240201 1 Y, filed 9 July 2024, the contents of it being hereby incorporated by reference in its entirety for all purposes.TECHNICAL FIELD

[0002] The present disclosure generally relates to methods of accelerating hair follicle development in stem cell-derived skin organoids.BACKGROUND

[0003] Severe bums, such as those involving large areas of the skin due to accidents, often result in the loss of not only the epidermis and dermis of the skin but also the underlying hair follicles, leading to long-term functional and aesthetic challenges. Skin grafts that are currently used to treat bum victims or patients with skin loss due to genetic diseases do not have hair follicles and other appendages, compromising quality of life after recovery. Current in vitro laboratory skin models also lack hair follicles and fail to replicate the cellular complexity of physiological skin.

[0004] Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have recently been shown to be able to differentiate and self-organize into skin organoids that are replete of skin appendages, including hair follicles. Such skin organoids have immense medical, research and industrial potential. For example, they can be used not just for grafting onto patients, but also as laboratory skin models to facilitate biological, drug and compound discovery efforts. However, the time required for maturation of skin organoids is considerably long, currently at about four months. If this timeframe can be reduced, workflows and processes in clinics and laboratories can become more efficient.

[0005] There is therefore a need to develop a method that can accelerate hair follicle development in skin organoids, to produce functional skin organoids with hair follicles within a shortened timeframe, for use in medical applications such as bum treatments.SUMMARY

[0006] In one aspect, the present disclosure refers to a method of inducing and / or stimulating the formation of a plurality of hair placodes on a skin organoid, comprising (a) adding one or more growth factors to a culture of skin organoid, after (or upon) the formation of epidermis and dermis in the skin organoid.

[0007] Tn one aspect, the present disclosure refers to a method of accelerating the formation of a plurality of hair placodes on a skin organoid, comprising (a) adding one or more growth factors to a culture of skin organoid, after (or upon) the formation of epidermis and dermis in the skin organoid.

[0008] In one aspect, the present disclosure refers to a method of producing a skin organoid with a plurality of hair placodes, comprising (a) adding one or more growth factors to a culture of skin organoid, after (or upon) the formation of epidermis and dermis in the skin organoid.

[0009] Tn one aspect, the present disclosure refers to a skin organoid produced by the method disclosed herein.

[0010] In one aspect, the present disclosure refers to a method of treating skin and / or hair loss in a subject, comprising grafting or implanting the skin organoid disclosed herein onto the subject.

[0011] In one aspect, the present disclosure refers to a method of screening a compound for treating skin-related and / or hair-related disease, comprising:(i) contacting or treating a skin organoid disclosed herein with a test compound;(ii) measuring a biological response of the skin organoid to the test compound, wherein the biological response is indicative of the therapeutic potential of the test compound in treating the skin-related and / or hair-related disease; and(iii) comparing the measured biological response to a control response to determine the efficacy of the test compound in treating the skin-related and / or hair-related disease.

[0012] In one aspect, the present disclosure refers to a method of screening a gene for skin and / or hair follicle development activity, comprising:(i) introducing a test gene into, and / or deleting, and / or base-editing a test gene in a skin organoid disclosed herein;(ii) culturing the skin organoid under conditions suitable for skin and / or hair follicle development;(iii) measuring the expression of marker(s) indicative of skin or hair follicle development; and(iv) comparing the expression level(s) of the marker(s) to a control to determine the activity of the test gene.

[0013] In one aspect, the present disclosure refers to a method of screening a compound for cosmetic application, comprising:(i) contacting a skin organoid disclosed herein with a test compound;(ii) measuring a cosmetic response of the skin organoid to the test compound; and(iii) comparing the measured cosmetic response to a control response to determine the efficacy of the test compound for cosmetic application.BRIEF DESCRIPTION OF DRAWINGS

[0014] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0015] Fig. 1 (comprised of Figs. 1A and IB) shows the effects of FGF treatment on skin organoids compared to untreated control skin organoids. Fig. 1A shows bright-field images of skin organoids that are either untreated (left) or treated with FGF 10 (right). Asterisks (*) denote hair placodes. Fig. IB is a graph showing the number of organoids with hair placodes scored at different days (D) post-differentiation of iPSCs. Organoids are non-treated (NO) or treated with FGF10, FGF20, or LDN.

[0016] Fig. 2 shows immunofluorescence images of cryo-sectioned organoids harvested on day 55. DAPI, KRT14, and SOX2 mark nucleus, basal keratinocyte, and dermal papilla respectively. Scale bar: 100 pm.

[0017] Fig. 3 shows immunofluorescence images of cryo-sectioned organoids harvested on day 100 The rows labelled (-) represent organoids without any additional treatment, whereas the rows labelled (+LDN) represent organoids treated with small molecule inhibitor of bone morphogenetic protein (BMP) type 1 receptors, LDN-193189, between day 21 and day 35. The specific antibodies and their target cell components are indicated at the top of the images. Scale bar: 100 pm.

[0018] Fig. 4 (comprised of Figs. 4A and 4B) shows a Uniform Manifold Approximation and Projection (UMAP) visualization of cell proportions from skin organoids harvested on all time points including D27, D48, and D63 (Fig. 4A) and cell distribution from individual samples of untreated, FGF10- and LDN-treated skin organoids harvested on D48 and / or D63 (Fig. 4B). Solid line circles indicate hair-related fibroblasts, whereas dotted line circles indicate hair- related keratinocytes.

[0019] Fig. 5 shows representative images for the definition of maturation index. The maturation index was defined by the most mature hair follicles in the organoid as hair placode (1), hair germ (2), hair peg (3), hair bulb (4), and hair shaft (5). Hair placodes (1) are shown in Fig. 1 and are characterized by their rounded structure. Hair germs (2) and pegs (3) are elongated structures visible from the side with differences in their length. Hair bulb (4) has a broader and rounder base, while hair shaft (5) displays as a visible fibrous structure within a hair bulb. Representative structural features are indicated with arrow heads. Scale bar: 500 gm.DEFINITION OF TERMS]0020] The following words and terms used herein shall have the meaning indicated:

[0021] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a primer” includes a plurality of primers, including mixtures and combinations thereof.

[0022] As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means + / - 5% of the stated value, or + / - 4% of the stated value, or + / - 3% of the stated value, or + / - 2% of the stated value, or + / - 1% of the stated value, or + / - 0.5% of the stated value.

[0023] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0024] The disclosure illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure.

[0025] The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subj ect matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0026] Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.DETAILED DESCRIPTION

[0027] The present disclosure provides methods of accelerating hair follicle development in stem cell-derived skin organoids. The present disclosure provides application of hair-inducing growth factors onto skin organoids derived from stem cells, such as human ESCs or human iPSCs, so as to reduce the time required for the skin organoids to differentiate and mature into skin, including development of hair follicles.

[0028] The term “organoid” or “organoids” refers to self-organized 3D tissues in vitro that resemble and / or possess the key functional, structural and / or biological complexity of an organ in vivo. As used herein, the term “skin organoid” refers to a skin-like cellular or tissue structure that may be derived from cells, such as but are not limited to, pluripotent stem cells, mesenchymal stem cells, adipose-derived stem cells, and cells isolated from skin tissues. Skinorganoids may be spheroidal, elongated, tubular or planar in shape, floated in liquid media, partially or fully embedded in natural- or synthetic-hydrogel, or exposed to liquid on one side with the other side facing to the air. Skin organoids may comprise epidermis and dermis (or epidermal and dermal layers), and may be capable of developing a plurality of skin appendages, including but not limited to, hair follicles (including hair placodes, hair germs, hair pegs, hair bulbs and hair shafts), sebaceous glands, and sweat gland.

[0029] In one aspect, the present disclosure refers to a method of inducing and / or stimulating the formation of a plurality of hair placodes on a skin organoid, comprising (a) adding one or more growth factors to a culture of skin organoid, after (or upon) the formation of epidermis and dermis in the skin organoid. In one example, the phrase “inducing and / or stimulating the formation of a plurality of hair placodes” refers to initiating or enhancing the growth / development of a plurality of hair placodes (which are capable of developing into hair germs, hair pegs, hair bulbs, and / or hair shafts) of a hair follicle in the skin organoid.

[0030] In another aspect, the present disclosure refers to a method of accelerating the formation of a plurality of hair placodes on a skin organoid, comprising (a) adding one or more growth factors to a culture of skin organoid, after (or upon) the formation of epidermis and dermis in the skin organoid. As used herein, the term “accelerate”, or grammatical variants thereof, refers to an increase in the rate at which a given outcome is achieved or a process is completed in a subset of a population, relative or compared to the rate observed in a control population. The term “increase” refers to the relative alteration of a chosen trait or characteristic in a subset of a population, on a positive scale, in comparison to the same trait or characteristic as present in the whole population. In one example, acceleration indicates that the process progresses more rapidly, suggesting enhanced efficiency or faster progression toward the given outcome. In one example, enhanced efficiency or faster progression may entail reduced time taken to achieve the given outcome. The term “reduce”, or grammatical variants thereof, when used in relation to the time required to achieve a given outcome, refers to a shortening of the time required for achieving the given outcome in a subset of a population, relative or compared to the time taken for the same outcome in a control population. In one example, a reduction in time indicates a decrease in the duration needed to achieve the given outcome, suggesting a more efficient, or an accelerated or expedited process. Thus, in one example, addition of one or more growth factors to a culture of skin organoid, after (or upon) the formation of epidermis and dermis in the skin organoid, in the disclosed method enables formation of hair placodes on the skinorganoid at an increased rate and requiring reduced time, relative or compared to the rate and timing observed in a control population.

[0031] In yet another aspect, the present disclosure refers to a method of producing a skin organoid with a plurality of hair placodes, comprising (a) adding one or more growth factors to a culture of skin organoid, after (or upon) the formation of epidermis and dermis in the skin organoid.

[0032] In one example of the above methods, the skin organoid is contacted or treated with one or more growth factors after (or upon) the formation of the epidermis and dermis in the skin organoid. The layers may be detected and distinguishable for example by expression of specific cell markers. Conventional histological and immunohistochemical techniques may be used to confirm the presence of these layers In one example, the skin organoid does not comprise (or does not have) or is devoid of hair placodes, hair germs, hair pegs, hair bulbs, and / or hair shafts of a hair follicle prior to being contacted or treated with one or more growth factors.]0033] ‘ ‘Hair placode” or “hair placodes” refers to precursors to hair follicles, typically characterized by the localized thickening of the epithelium that mark the early stage of hair follicle morphogenesis. The structural change (or thickening) may be detectable under bright field microscopy on the surface of the skin bilayer. In one example, the shape of the hair placode(s) may be circular or elliptic with a diameter of about 20-200 um, and may protrude from the epidermal layer toward the dermal layer to form a dome shape Exemplary images of hair placodes are shown in Fig. 1 as disclosed herein. In one example, the hair placodes are a mid-way to the development of hair germs and / or hair pegs. In one example, the hair placodes, hair germs and / or hair pegs comprise dense cell populations of SOX2-positive dermal papillae cells on the bottom of the dome-shape and invaginating KRT5-positive epidermal cells inside the dome-shape In one example, the hair germs and / or hair pegs comprise dense cell populations of SOX2-positive dermal papillae cells on the bottom of the dome-shape. In one example, the hair placodes comprise invaginating KRT5-positive epidermal cells inside the dome-shape.

[0034] “Hair germ” or “hair germs” refers to structures that form following hair placode formation in the early developmental stage of hair follicles.

[0035] “Hair peg” or “hair pegs” refers to structures composed of epithelial cells in close contact with mesenchymal dermal papilla cells which develop at the intermediate stage in hairfollicle development, and are indicative of progressive follicular morphogenesis. Hair peg formation typically follows hair germ formation in development of hair follicles.

[0036] “Hair bulb” or “hair bulbs” refers to broadened and rounded base structures at the lower ends of hair follicles The hair bulb houses the dermal papilla, and is responsible for the production and growth of the hair shaft. Hair bulb formation typically follows hair peg formation in development of hair follicles.

[0037] ‘ ‘Hair shaft” or “hair shafts” refers to filamentous keratinized structures that emerge from the surface of the skin and are produced by cells within the hair follicle. Hair shaft formation typically follows hair bulb formation in development of hair follicles.

[0038] Therefore, the development of hair follicles begins with the formation of hair placodes, which then develop into hair germs, hair pegs, hair bulbs, hair shafts, which eventually mature into hair follicles. These various stages may sometimes overlap (i.e. not strictly separated) in the development process. “Hair follicle” or “hair follicles” are complex structures composed of multiple cell types and structures, including but not limited to, the outer root sheath, inner root sheath, dermal papilla, and matrix cells. In one example, the hair follicle may have an extended structure having multiple layers or components such as: a dermal sheath that is alphaSMA-positive dermal sheath, an outer root sheath that is K5-positive, a companion layer that is K75-positive, an inner root sheath that is K71-positive, a cortex / cuticle that is AE13- positive, a matrix that is Ki67-positive, and a dermal papilla that is SOX2-positive. In one example, a hair follicle comprises at least the extended structure and a K75-positive hairspecific keratinocyte layer.

[0039] In one example, the skin organoid of the method disclosed herein comprises less than 1% dermal papilla cells prior to step (a) i.e., prior to being contacted or treated with one or more growth factors. In one example, the skin organoid does not comprise (or does not have) or is devoid of dermal papilla cells prior to step (a) i.e., prior to being contacted or treated with one or more growth factors. In one example, the skin organoid may comprise dermal papilla cells after the skin organoid is contacted or treated with one or more growth factors. In one example, the hair placodes, hair germs and / or hair pegs of the skin organoid may comprise dermal papilla cells after the skin organoid is treated with one or more growth factors.

[0040] In one example, the skin organoid of the method disclosed herein is capable of forming a plurality of hair shafts. In another example, the skin organoid is capable of forming a plurality of hair shafts following long-term culture. In one example, the skin organoid is capable offorming a plurality of hair shafts following long-term culture of the skin organoid after (or upon) the formation of a plurality of hair placodes on the skin organoid In one example, the skin organoid is capable of forming a plurality of hair shafts following a long-term culture of about 50 days or longer after (or upon) the formation of a plurality of hair placodes on the skin organoid.

[0041] In one example, the skin organoid comprises two or more cell types (or at least two cell types). In another example, the skin organoid comprises more than two cell types. In one example, the skin organoid of the method disclosed herein comprises three or more cell types. In one example, the skin organoid of the method disclosed herein comprises four cell types. In one example, the cell types include, but are not limited to, melanocytes, sensory neurons, Schwann cells, Merkel cells, and any combinations thereof.

[0042] In one example of the disclosed methods, the skin organoid is prepared by step (b) culturing pluripotent stem cells in the presence of one or more recombinant proteins prior to step (a) to obtain the skin organoid, wherein the pluripotent stem cells differentiate to form the epidermis and dermis of the skin organoid. In one example, the one or more recombinant proteins include, but are not limited to, Y27632, BMP4, bFGF, and SB-431542.

[0043] In one example, the pluripotent stem cells used in the methods disclosed herein are ESCs or iPSCs. In one example, the pluripotent stem cells used in the methods of the present disclosure are mammalian pluripotent stem cells. In one example, the mammalian pluripotent stem cells include, but are not limited to, human pluripotent stem cells, rabbit pluripotent stem cells, non-human primate pluripotent stem cells, canine pluripotent stem cells, porcine pluripotent stem cells, bovine pluripotent stem cells, murine pluripotent stem cells (such as mouse or rat pluripotent stem cells), feline pluripotent stem cells, and ovine pluripotent stem cells. In one example, the mammalian pluripotent stem cells are human pluripotent stem cells. In one example, the human pluripotent stem cells are human ESCs or human iPSCs. In one example, the human pluripotent stem cells are human iPSCs. In one example, the human ESCs or human iPSCs can differentiate into various skin cell lineages, including, but not limited to, fibroblasts, keratinocytes, and melanocytes. In one specific example, the human ESCs or human iPSCs can differentiate into fibroblasts.

[0044] The pluripotent stem cells may be differentiated into the skin organoids using conventional cell culture methods and media. This includes any suitable cell culture protocol (including, but not limited to, cell culture conditions such as pH, temperature, CO2concentration, humidity, stirring speed (if applicable), aeration (if applicable) etc.) as well as parameters such as, but not limited to, the types of cell plate coating, culture plate or well size, and whether the cells are suspended or attached to the plate surface. It also includes any range of suitable cell culture media, substrate and / or matrices to support the growth of the cells.

[0045] Once the pluripotent cells have differentiated into skin organoid, the methods disclosed herein comprise adding one or more growth factors into the culture of the skin organoid as follows:• On Day 21 (and Day 28), removing all the medium from the cells, and adding 500 pL of fresh medium containing the appropriate concentrations of the growth factors.• On Day 24 and Day 26, removing half of the medium (=250 pL), and adding an equal volume of the medium containing the same concentrations of growth factors to supply nutrients for the cells without changing the growth factor concentrations.• On Day 31 and Day 33, removing half of the medium, and adding an equal volume of the medium without any growth factors.• On Day 35, replacing all the medium with fresh medium without any growth factors.

[0046] In one example, once the pluripotent cells have differentiated into skin organoid, and after (or upon) the development (or formation) of epidermis and dermis in the skin organoid, the methods disclosed herein comprise adding one or more growth factors into the culture of the skin organoid as follows:• On Day 21 (and Day 28), removing all the medium from the cells, and adding 500 pL of fresh medium containing the appropriate concentrations of the growth factors.• On Day 24 and Day 26, removing half of the medium (=250 pL), and adding an equal volume of the medium containing the same concentrations of growth factors to supply nutrients for the cells without changing the growth factor concentrations.• On Day 31 and Day 33, removing half of the medium, and adding an equal volume of the medium without any growth factors.

[0047] Tn one example, the one or more growth factors used in the methods of the present disclosure may include, but are not limited to, small molecules, peptides, proteins, nucleosides, bifunctional molecules, multifunctional molecules, biologies, antibodies and hormones. Combinations of any two or more of these growth factors may be used. In one example, these growth factors may be added to the culture media singly or in combinations. In one example,contact or treatment of skin organoids with the one or more growth factors accelerates the development of hair follicles on the skin organoids.

[0048] In one example, the one or more growth factors used in step (a) of the method disclosed herein are selected from the group consisting of: (i) a fibroblast growth factor (FGF), wherein optionally the FGF is selected from the group consisting of a FGF7 subfamily member and a FGF9 subfamily member, wherein optionally the FGF7 subfamily member is selected from the group consisting of FGF3, FGF7, FGF10 and FGF22, wherein optionally the FGF9 subfamily member is selected from the group consisting of FGF9, FGF16 and FGF20, wherein optionally the FGF7 subfamily member is FGF10 and the FGF9 subfamily member is FGF20; (ii) an inhibitor of bone morphogenetic protein (BMP) receptor, wherein optionally the inhibitor of BMP receptor is LDN-193189; (iii) a bifunctional or multifunctional compound which inhibits glycogen synthase kinase-3 (GSK3) and activates Wnt signaling pathway, wherein optionally the bifunctional or multifunctional compound is BIO; (iv) a glial cell line-derived neurotrophic factor (GDNF); (v) retinoic acid; (vi) vitamin E, wherein optionally the vitamin E is a- tocopherol, y-tocopherol, P-tocopherol, 8-tocopherol, a-tocotrienol, y-tocotrienol, p- tocotrienol, and 8-tocotrienol, wherein optionally the vitamin E is a-tocopherol; (vii) an inhibitor of Janus kinase (JAK), wherein optionally the inhibitor of JAK is selected from the group consisting of ruxolitinib and tofacitinib; (viii) an insulin-like growth factor (IGF), wherein optionally the IGF ligand is IGF2; and (ix) any combination of (i) to (viii). A person skilled in the art may appreciate that certain growth factors may potentially be substituted with similar growth factors from the same subfamily, or with other growth factors known to exhibit comparable functional and / or physiological properties for use in the methods of the present disclosure. Examples of comparable functional and / or physiological properties may include activation of similar signalling pathway, binding to shared receptors, or promoting similar cellular outcomes such as proliferation, differentiation, or morphogenesis depending on the specific context and application of the method.

[0049] In one example, the one or more growth factors used in step (a) include, but are not limited to, recombinant proteins referred to as fibroblast growth factor 10 (FGF 10) and fibroblast growth factor 20 (FGF20); a small molecule inhibitor of bone morphogenetic protein (BMP) type I receptors referred to as LDN193189; an inhibitor of class I and II mammalian histone deacetylase (HDAC) referred to as trichostatin A; a bifunctional molecule referred to as BIO, which is an inhibitor of GSK3a / (3 and activator of WNT signalling pathway; and insulin-like growth factor 2 (IGF2).

[0050] In one example, the (i) the concentration of the FGF added in step (a) is about 0.1-200 ng / ml, wherein optionally the concentration of the FGF is about 10-100 ng / ml; (ii) the concentration of the inhibitor of BMP receptor added in step (a) is about 2-550 ng / ml, wherein optionally the concentration of the inhibitor of BMP receptor is about 20-200 ng / ml; (iii) the concentration of the bifunctional or multifunctional compound added in step (a) is about 1- 2000 nM, wherein optionally the concentration of the bifunctional or multifunctional compound is about 10-100 nM; (iv) the concentration of the GDNF added in step (a) is about 1-5000 ng / ml, wherein optionally the concentration of the GDNF is about 50-200 ng / ml; (v) the concentration of the retinoic acid added in step (a) is about 10-5000 nM, wherein optionally the concentration of the retinoic acid is about 200-2000 nM; (vi) the concentration of the vitamin E added in step (a) is about 0 1 -200 uM, wherein optionally the concentration of the vitamin E is about 5-20 uM, (vii) the concentration of the inhibitor of JAK added in step (a) is about 1-5000 nM, wherein optionally the concentration of the inhibitor of JAK is about 100- 1000 nM; and (viii) the concentration of the IGF added in step (a) is about 1-5000 ng / ml, wherein optionally the concentration of the IGF is about 100-1000 ng / ml.

[0051] In one example, the growth factor(s) added in step (a) of the methods disclosed herein is selected from the group consisting of or consisting essentially of: FGF10, FGF20, BIO, ruxolitinib, tofacitinib, LDN193189, vitamin E, GDNF, retinoic acid; and IGF2. In one example, the one or more growth factors used in the methods disclosed herein is selected from the group consisting of FGFlO, FGF20, BIO, LDN193189, vitamin E, ruxolitinib, tofacitinib, IGF2 and any combination thereof.

[0052] In one example, the methods of the present disclosure comprise (c) reducing the concentration of the one or more growth factors. In one example, the methods of the present disclosure comprise (c) reducing the concentration of the one or more growth factors by about 0.5-fold after performing step (a). In one example, the concentration of the one or more growth factors is reduced by about 0.5 fold two days after performing step (a). In one specific example, the concentration of the one or more growth factors is reduced by about 0.5 fold every two days until Day 35 of culture. In one example, the step (c) of reducing the concentration of the one or more growth factors minimizes the risk of overstimulation and / or non-specific stimulation of the organoids due to prolonged exposure to growth factor treatment, while maintaining normal differentiation following growth factor treatment. As used throughout the present disclosure, the term “normal differentiation” refers to a biologically regulated process by whichundifferentiated cells progress into specialized cell types with expected cellular maturation and physiological cellular activity profiles.

[0053] In one example, the methods of the present disclosure comprise d) further reducing the concentration of the one or more growth factors after performing step (c). In one example, the methods of the present disclosure comprise d) further reducing the concentration of the one or more growth factors by about 0.5-fold after performing step (c). In one example, the concentration of the one or more growth factors is reduced by about 0.5 fold two days after performing step (c). In one specific example, the concentration of the one or more growth factors is reduced by about 0.5 fold every two days until Day 35 of culturing. In one example, the step (d) of further reducing the concentration of the one or more growth factors after performing step (c) further minimizes the risk of overstimulation and / or non-specific stimulation of the organoids due to prolonged exposure to growth factor treatment, while maintaining normal differentiation following growth factor treatment and step (c).

[0054] In one example, the one or more growth factors used in the methods of the present disclosure reduce the time taken for the formation of the plurality of hair placodes on the skin organoid by at least about 2 days compared to a control skin organoid, wherein optionally the one or more growth factors reduce the time taken for the formation of the plurality of hair placodes on the skin organoid by about 2-19 days compared to the control skin organoid, wherein the control skin organoid is not treated with the one or more growth factors after (or upon) the formation of epidermis and dermis in the control skin organoid In one example, the one or more growth factors used in the methods of the present disclosure reduce the time taken for the formation of the plurality of hair placodes on the skin organoid by at least 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, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, or about 19 days. In some examples, the one or more growth factors used in the methods of the present disclosure reduce the time taken for the formation of the plurality of hair placodes on the skin organoid by more than 19 days. Reduction in time taken for the formation of the plurality of hair placodes on the skin organoid may differ depending on the cell types and culture conditions used. In one example, a control skin organoid is a skin organoid that is not treated with one or more growth factors after (or upon) the formation of epidermis and dermis in the control skin organoid. In one example, the plurality of hair placodes were formed in the control skin organoid between Day 42 and Day 65 of differentiation. In one example, the plurality of hair placodes were formed in the controlskin organoid on Day 47. In one example, in order to compare the reduction in time / day for the formation of hair placodes on a skin organoid treated with growth factor(s) compared to a control skin organoid, a robust criterion is used to determine the time point or day of hair placodes formation in each control / treatment group. In one example, the total number of skin organoids in each control / treatment group is 12 (n=12). In one example, the time point or day where at least 25% of the total number of skin organoids in each control / treatment group that had formed hair placodes was taken to be the time point or day of hair placodes formation for that particular control / treatment group.

[0055] In one example, the methods of the present disclosure comprise (e) further adding one or more growth factors to the culture after (or upon) the formation of the plurality of hair placodes on the skin organoid, to accelerate the maturation of the plurality of hair placodes into a plurality of hair germs, hair pegs, hair bulbs, and / or hair shafts.

[0056] In one example, the one or more growth factors used in the methods of the present disclosure to be further added in step (e) are selected from the group consisting of: (i) a fibroblast growth factor (FGF), wherein optionally the FGF is selected from consisting of FGF3, FGF7, FGF9, FGF10, FGF16, FGF20 and FGF22; (ii) an inhibitor of bone morphogenetic protein (BMP) receptor, wherein optionally the inhibitor of BMP receptor is LDN-193189; (iii) a Wnt protein, wherein optionally the Wnt protein is Wnt3A; (iv) a glial cell line derived neurotrophic factor (GDNF); (v) retinoic acid; (vi) vitamin E, wherein optionally the vitamin E is a-tocopherol; (vii) a nucleoside, wherein optionally the nucleoside is adenosine, and (viii) any combination of (i) to (vii). In one example, the one or more growth factors used in the methods of the present disclosure to be further added in step (e) are selected from the group consisting of: (i) FGF10; (ii) vitamin E, wherein optionally the vitamin E is a- tocopherol; and (iii) any combination of (i) to (ii).

[0057] In one example of the methods of the present disclosure, (i) the concentration of the FGF added in step (e) is about 0. 1-200 ng / ml, wherein optionally the concentration of the FGF is about 10-100 ng / ml; (ii) the concentration of the inhibitor of BMP receptor added in step (e) is about 2-550 ng / ml, wherein optionally the concentration of the inhibitor of BMP receptor is about 20-200 ng / ml; (iii) the concentration of the Wnt protein added in step (c) is about 10- 10000 ng / ml, wherein optionally the concentration of the Wnt protein is about 100-1000 ng / ml; (iv) the concentration of the GDNF added in step (e) is about 1-5000 ng / ml, wherein optionally the concentration of the GDNF is about 50-200 ng / ml; (v) the concentration of the retinoic acidadded in step (e) is about 10-5000 nM, wherein optionally the concentration of the retinoic acid is about 200-2000 nM; (vi) the concentration of the vitamin E added in step (e) is about 0.1- 200 uM, wherein optionally the concentration of the vitamin E is about 5-20 uM; and (vii) the concentration of the nucleoside added in step (c) is about 0 01-100 mM, wherein optionally the concentration of the nucleoside is about 1-10 mM. In one example of the methods of the present disclosure, (i) the concentration of the FGF added in step (e) is about 0.1-200 ng / ml, wherein optionally the concentration of the FGF is about 10-100 ng / ml; and (ii) the concentration of the vitamin E added in step (e) is about 0.1-200 uM, wherein optionally the concentration of the vitamin E is about 5-20 uM.

[0058] In one example, the methods of the present disclosure comprise (f) reducing the concentration of the one or more growth factors after performing step (e). In one example, the methods of the present disclosure comprise reducing the concentration of the one or more growth factors by about 0.5 fold after performing step (e). In one example, the concentration of the one or more growth factors is reduced by about 0.5 fold two days after performing step (e). In one example, the one or more growth factors is reduced by about 0.5 fold two days after performing step (e), and is thereafter removed from culture two days later. In one example, the step (f) of reducing the concentration of the one or more growth factors after performing step(e) minimizes the risk of overstimulation and / or non-specific stimulation of the organoids due to prolonged exposure to growth factor treatment, while maintaining normal differentiation following growth factor treatment.

[0059] In one example, the methods of the present disclosure comprise further reducing the concentration of the one or more growth factors after performing step (f). In one example, the methods of the present disclosure comprise (g) further reducing the concentration of the one or more growth factors by about 0.5 fold after performing step (f). In one example, the concentration of the one or more growth factors is reduced by about 0.5 fold two days after performing step (f). In one example, the one or more growth factors is reduced by about 0.5 fold two days after performing step (f), and is thereafter removed from culture two days later. Fold reduction and time period for reduction / removal of the growth factors may differ depending on the cell type and culture conditions used for culturing. In one example, step (g) of further reducing the concentration of the one or more growth factors after performing step(f) further minimizes the risk of overstimulation and / or non-specific stimulation of the organoids due to prolonged exposure to growth factor treatment, while maintaining normal differentiation following growth factor treatment and step (f).

[0060] In one example, addition of the one or more growth factors used in the methods of the present disclosure reduce the time taken for the maturation of the plurality of hair placodes on the skin organoid into a plurality of hair germs, hair pegs, hair bulbs, and / or hair shafts by at least about 2 days compared to a control skin organoid, wherein optionally addition of the one or more growth factors reduces the time taken for the maturation of the plurality of hair placodes on the skin organoid into a plurality of hair germs, hair pegs, hair bulbs, and / or hair shafts by about 2-28 days compared to the control skin organoid, wherein the control skin organoid is not further treated with the one or more growth factors after (or upon) the formation of the plurality of hair placodes on the control skin organoid. In one example, addition of the one or more growth factors used in the methods of the present disclosure reduce the time taken for the maturation of the plurality of hair placodes on the skin organoid into a plurality of hair germs, hair pegs, hair bulbs, and / or hair shafts by at least 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, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, or about 28 days. In one example, addition of the one or more growth factors used in the methods of the present disclosure reduce the time taken for the maturation of the plurality of hair placodes on the skin organoid into a plurality of hair germs, hair pegs, hair bulbs, and / or hair shafts by more than about 28 days. Reduction in time taken for the maturation of the plurality of hair placodes on the skin organoid into a plurality of hair germs, hair pegs, hair bulbs, and / or hair shafts may differ depending on the cell types and culture conditions used.

[0061] In one aspect, the present disclosure refers to a skin organoid produced by the methods disclosed herein. In one example, the skin organoid produced by the methods disclosed herein is capable of forming a plurality of hair shafts, which can develop into hair follicles unlike skin organoids produced by conventional methods which lack hair follicles and other appendages. In one example, the skin organoid produced by the methods disclosed herein is capable of forming - in an accelerated manner - a plurality of hair shafts, which can develop into hair follicles unlike skin organoids produced by conventional methods which lack hair follicles and other appendages. In one example, the skin organoid produced by the methods disclosed herein contain skin-associated cell types including, but not limited to, melanocytes, sensory neurons, Schwann cells, and Merkel cells, and possess hair follicles with multiple layers, including inner root sheath, companion layer, outer root sheath, and dermal sheath, unlike skin organoidsproduced by conventional methods which are replete of such cell types and lack hair follicles and other appendages. The skin organoid produced by the methods disclosed herein may therefore be useful in various applications, such as medical, cosmetic, research and industrial applications.

[0062] In one aspect, the present disclosure refers to a method of treating skin and / or hair loss in a subject, comprising grafting or implanting the skin organoid disclosed herein onto the subject. In one example, the subject to be treated may include individuals with burn injuries, chronic wounds, hair loss conditions (such as those due to disease like alopecia, hormonal changes, nutritional deficiencies etc.), trauma, genetic skin diseases, adverse skin drug diseases, blistering diseases (EB), or other skin-related disorders (such as skin infections, skin cancers, skin ulcers or bedsores, etc ). In one example, the subject to be treated may include individuals who have undergone cosmetic skin / hair restoration. In this context, the term “treatment” or “treating” refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. “Grafting” or “implanting” refers to any and all procedures by which the skin organoid can be transferred, attached, inserted, or otherwise placed onto or into a biological subject for therapeutic or medical purpose, such as for treating subjects suffering from any of the above conditions.

[0063] In one aspect, the present disclosure refers to a method of screening a compound for treating skin-related and / or hair-related disease, comprising: (i) contacting or treating a skin organoid disclosed herein with a test compound, (ii) measuring a biological response of the skin organoid to the test compound, wherein the biological response is indicative of the therapeutic potential of the test compound in treating the skin-related and / or hair-related disease, and (iii) comparing the measured biological response to a control response to determine the efficacy of the test compound in treating the skin-related and / or hair-related disease. “Contacting” or “treating” in this context refers to the application of a test compound to the skin organoid by any means, such as topical application, injection (e g. intradermal, subcutaneous, etc.), immersion, etc. In one example, the method of screening a compound may refer to a process used to evaluate a substance (such as a compound, drug, or molecule) for its potential biological activity or therapeutic effect e.g., by identifying compounds that may have beneficial effects on specific targets, pathways, or disease processes. Exemplary biological responses of the skin organoid to the test compound that may be measured include changes incell viability, morphology, gene expression, and functional properties, wherein a change may be indicative of an effect of the test compound on the skin organoid

[0064] In one aspect, the present disclosure refers to a method of screening a gene for skin and / or hair follicle development activity, comprising: (i) introducing a test gene into, and / or deleting, and / or base-editing a test gene in a skin organoid disclosed herein; (ii) culturing the skin organoid under conditions suitable for skin and / or hair follicle development; (iii) measuring the expression of marker(s) indicative of skin or hair follicle development; and (iv) comparing the expression level(s) of the marker(s) to a control to determine the activity of the test gene. In one example, the method of screening a gene may refer to a process of testing / identifying a specific gene to determine its effect on a biological process, such as skin and hair follicle development e.g., whether the gene play a role in regulating or influencing the formation, growth, or regeneration of skin and hair structures. Exemplary screening methods that may be applied to the skin organoids of the present disclosure include, but are not limited to, gene knockdown / knockout assays, gene overexpression assays, microarray or RNA-seq high-throughput screening, and gene editing / activation screens (e.g., CRISPR).

[0065] In one aspect, the present disclosure refers to a method of screening a compound for cosmetic application, comprising: (i) contacting a skin organoid disclosed herein with a test compound; (ii) measuring a cosmetic response of the skin organoid to the test compound; and (iii) comparing the measured cosmetic response to a control response to determine the efficacy of the test compound for cosmetic application “Cosmetic application” includes any and all non-therapeutic or non-medical uses intended to enhance, improve, or restore overall aesthetic of a biological subject e.g., enhancing hair growth or density for aesthetic purposes without necessarily treating or preventing a disease. The methods may be useful, for example, in devising personalized cosmetic strategies for consumer care. Measuring the cosmetic response of the skin organoid to the test compound may include measuring changes in skin hydration, elasticity, or pigmentation. In one example, the method of screening a compound for cosmetic application may refer to a process of evaluating a compound to determine its potential efficacy and safety for use in cosmetic products e.g., assess the compound’s effects on skin health, appearance, and other cosmetic properties. Exemplary screening methods include, but are not limited to, HTS for skin benefits, anti-aging screening, pigmentation assays, toxicity and irritation testing.

[0066] The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the present disclosure. This includes the generic description of the present disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0067] Other embodiments are within the following claims and non-limiting examples.EXAMPLES

[0068] Methods

[0069] Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

[0070] The protocol was previously described in “Lee J, et al. Generation and characterization of hair -bearing skin organoids from human pluripotent stem cells. Nat Protoc. 2022 May;17(5):1266-1305. doi: 10.1038 / s41596-022-0068l-y” . Certain modifications to the protocol e g., changing the media used from Day 21 to Day 28 were made in the methods of the present disclosure.

[0071] Table 1

[0072] Differentiation day (-2)1. Media preparation for one 96-well plate a. 45 mL E8 + 20Y was prepared using: . E8 (+N) 45 mL. Y-27632 (10 mM) 90 pL b. 17 mL E8 + 10 Y was prepared using:. E8 17 mL. Y 17 uL 2. Accutase was pre-warmed to RT.3. Cell dissociation: Spent media was removed from the 6-well plate and the well was washed twice with 2.5 mL dPBS. 500 pL accutase was added to each well and incubated for 3 - 4 mins at 37°C. Once the cells were detached, a P1000 tip was used to briefly and gentlypipette the cell suspension to break the cell clumps into single cells. The cell suspension was transferred to a 15 mL conical tube and 2 mL / well of E8 + 10Y media was added to the cell suspension. Brief and gentle pipetting was performed on the cell suspension to break the cells into single cells, and the conical tube was centrifuged at 230 g for 5 min and 30 s at RT. The supernatant was removed and 1 mL of E8 + 10Y media was added to resuspend the pellet.4. Cell aggregation: The 35 pm mesh cell strainer was equilibrated using 1 mL E8 + 10Y. The cell suspension was transferred to the strainer in a dropwise manner. The tube that formerly contained the cell suspension was rinsed with 1 mL E8 + 10Y and the suspension was transferred to the strainer in a dropwise manner. The snap cap of the cell strainer was removed. The number of live cells was determined. 22 mL of the 5.0 x 10A4 cells / mL cell suspension was prepared based on the cell count using E8 + 20Y medium and 100 pL cell suspension was seeded into each well (5000 cells / well). The plates were centrifuged at 110 g for 6 mins at RT.5. The plates were incubated in a 37°C incubator for 24 h.

[0073] Differentiation day (-1)1. Media preparation. a. 33 mL E8 without Y solution was prepared.. E8 (+N) 44 mL2. 100 pL E8 media wo Y was added to each well, to bring the total volume to 200 pL / well.3. 1000 pL of Matrigel was thawed on ice overnight at 4°C to be ready for the next day.

[0074] Differentiation day 01. Media preparation. a E6 (+N) 56 mL was prepared. b. 20 mL 2.x E6SFB (5.0 ng / mL final) was prepared using:. E6 (+N) 20 mL• Matrigel 2% 800 pL. SB (10 mM) 40 pL• bFGF (200 pg / mL) 0.8 pL (4 ng / mL). BMP4 (100 pg / mL) 2 pL2. All the aggregates were collected into a petri dish using a multichannel pipette and all the aggregates were collected into a 2 mL round bottom tube. The E8 media were removed and the aggregates were washed 3 times using 1 mL E6 media. 1 mL of E6 was added to the tube containing the aggregates and all the aggregates were transferredto a petri dish containing 10 mL E6SFB.3. Individual cell aggregates in 100 pL E6SFB were transferred into each well in a new 96-well plate.4. The plate was then incubated in a 37°C incubator with 5% CO2.

[0075] Differentiation day 31 . Media preparation a. 9 mL E6LF was prepared using:. E6 (+N) 9 mL. LDN (10 mM) 0.9 pL, 1 pM (200 nM)• bFGF (200 pg / mL) 11.25 pL, 250 ng / mL (50 ng / mL)2. 25 uL of E6LF was added into each well to bring the total volume / well to 125 pL3. The plates were gently tapped to mix the media.4. The plates were then incubated in a 37°C incubator with 5% CO2.

[0076] Differentiation day 61. 24 mL E6 (+N) media was prepared and prewarmed.2. 75 pL was added into each well to bring the total volume to 200 pL / well.3. The plates were gently tapped to mix the media.4. The plates were then incubated in a 37°C incubator with 5% CO2.

[0077] Differentiation day 91 . 32 mL E6 (+N) at RT was prepared and prewarmed.2. 100 pL of spent media was removed and 100 pL fresh media was added into each well.3. The plates were gently tapped to mix the media.4. The plates were incubated in a 37°C incubator with 5% CO2.

[0078] Differentiation day 111. 1000 pL of Matrigel was thawed on ice overnight at 4°C.

[0079] Differentiation day 121. Media preparation a. OMM (200 mL) was prepared using:. Advanced DMEM / F 12 100 mL• Neurobasal media 100 mL• GlutaMAX supplement 2 mL• B27 wo Vit A 2 mL. N2 1 mL• 2-Mercaptoethanol 364 pL• Normocin (100 pg / mL) 400 pL b. 70 mL OMM1.25%M was prepared as follows:• 875 pL of OMM was removed and 875 pL Matrigel was added into the OMM medium. c. Advanced DMEM / F12 media (4 mL)2. A petri dish and 24-well low-attachment plate were prechilled on ice.3. All the aggregates were collected into a petri dish using a multichannel pipette and all the aggregates were collected into 6 cm dish and then collected into a 2 mL round bottom tube. The media was removed and the aggregates were washed 3 times using 1 mL Advanced DMEM / F12 media. 1 mL OMM was added to the tube containing the aggregates and all the aggregates were transferred to a petri dish containing 8(+8) mL OMM. Individual cell aggregates were transferred in 100 pL OMM into each well in a 24-well plate supplemented with 400 pL OMM1.25%M.4. Each well was gently swirled to make sure that each well is completely covered by medium, and aggregates were not floating on the surface of the medium.5. The plate was placed on an orbital shaker inside the incubator and subjected to constant agitation at a speed of 65 rpm.6. The plate was then incubated in a 37°C incubator with 5% CO2.

[0080] Differentiation day 141. 500 pL of Matrigel was thawed on ice overnight at 4°C.

[0081] Differentiation day 151. 45 mL OMM1%M was prepared and prewarmed on ice.2. 450 pL spent media was removed and 450 pL fresh media was added to each well.3. The plate was gently swirled to evenly mix the medium and to ensure that the aggregates were not floating on the surface of the medium.4. The plates were then incubated in a 37°C incubator with 5% CO2.

[0082] Differentiation day 181. 45 mL OMM was prepared.2. 450 pL spent media was removed and 450 pL fresh media was added to each well.3. The plate was gently swirled to evenly mix the medium and to ensure that the aggregates were not floating on the surface of the medium.4. The plate was then incubated in a 37°C incubator with 5% CO2.5. Media change schedule for the first time window screening up to day 47 were as follows:• Day 21: 500 pL OMM + Growth factors• Day 24, Day 26: 250 pL OMM + Growth factors• Day 28: 500 uL OMM + Growth factors• Day 31, Day 33: 250 pL OMM• Day 35: 500 pL OMM. Day 38, Day 40: 250 pL OMM• Day 42: 500 pL OMM• Day 45, Day 47: 250 pL OMMThe culture media was replaced according to the schedule defined at point (5) above for the first time window screening. On Day 21, all the medium was removed, and 500 pL of fresh OMM containing the appropriate concentrations of growth factors was added. On Day 24 and Day 26, half of the medium (250 pL) was replaced with an equal volume of fresh medium containing the same concentrations of growth factors, ensuring nutrient supply without altering growth factor concentrations. On Day 28, all the medium was replaced again with 500 pL of fresh OMM containing the same concentrations of growth factors to maintain the initial levels. From Day 31, the media was gradually diluted with non-growth factor-containing medium; half of the medium (250 pL) was replaced on Day 31 and Day 33, reducing the growth factor concentration by half. On Day 35, all the medium was replaced with fresh OMM without any growth factors. Subsequent media changes continued on Days 38, 40, 42, 45, and 47, where either half (250 pL) or the full volume (500 pL) of OMM was replaced, maintaining cell conditions for the duration of the experiment. This protocol was designed as part of the first time window screening to regulate growth factor exposure over time during the first time window screening.

[0083] OMM + Growth factors

[0084] All the media used on Day 21 to Day 28 were prepared on the Day 21 for each batch of experiment and stored at 4°C until use. The growth factors are listed below.

[0085] Table 26. Media change schedule for the second time window screening up to day 77 were as follows: . Day 21 : 500 pL OMM + FGFIO• Day 24, Day 26: 250 pL OMM + FGF10. Day 28: 500 uL OMM + FGF10• Day 31, Day 33: 250 pL OMM• Day 35: 500 pL OMM . Day 38, Day 40: 250 pL OMM• Day 42: 500 pL OMM• Day 45, Day 47: 250 pL OMM• Day 49 top up 1 mL OMM + Growth factors• Day 52, Day 54: 500 pL OMM + Growth factors • Day 56: 1 mL OMM + Growth factors• Day 59, Day 61 : 500 pL OMM• Day 63 : 1 mL OMM• Day 66, Day 68: 500 pL OMM• Day 70: 1 mL OMM. Day 73, Day 75: 500 pL OMMThe culture media was replaced according to the schedule defined at point (6) above for the second time window screening. On Day 49, the volume was topped up with 1 mL of OMM containing the appropriate concentration of growth factors. On Day 52 and Day 54, 500 pL of medium was replaced with an equal volume of fresh medium containing the same concentrations of growth factors, ensuring nutrient supply without altering growth factor concentrations. On Day 56, all the medium was replaced with 1 mL of fresh OMM containing the same concentrations of growth factors to maintain the initial levels. On Days 59 and 61, the media was diluted with non -growth factorcontaining medium; half of the medium (500 pL) was replaced on each of these days, reducing the growth factor concentration by half. Subsequent media changes continued on Days 63, 66, 68, 70, 73, and 75, where either half (500 pL) or the full volume (1 mL) of OMM was replaced, maintaining cell conditions for the duration of the experiment. This protocol ensured controlled growth factor exposure and gradual depletion over time during the second time window screening

[0086] OMM + Growth factors

[0087] All the media used on Day 49 to Day 56 were prepared on Day 49 for each batch of experiment and stored at 4°C until use. The growth factors were listed below.

[0088] Table 3

[0089] Results

[0090] FGF treatment accelerates hair placode formation in skin organoids

[0091] Skin organoids at 21 days of age were treated with FGF 10 or LDN193189 for a duration of 15 days, first at 1-100 ng / ml and 20-200 ng / ml respectively for 10 days, then with concentrations reduced by half every 2 days until Day 35. First, it was found that the untreated control had varied maturation speed within the same batch of 12 organoids as demonstrated in Fig. 1A and Fig. IB Here, it is defined that the untreated control condition formed hair placodes on day 47 as a representative time point, and defined that the probability of hair placode-forming organoid (25% in Fig. IB) was the threshold to say that the organoids in one condition formed hair placodes on a certain day as a batch. With FGF10 treatment, appearance of hair placodes could be observed as early as Day 28, which was an improvement of 19 days over untreated control organoids (Fig. IB). With LDN193189 treatment, appearance of hair placodes was observed on Day 40, which was earlier by 7 days than that without any additives. Similarly, treatment with 11 other compounds (in addition to FGF10 and LDN193189), which are known to affect hair induction or growth in mouse studies, was tested and summarized in Table 4. The dose-responses of FGF 10, LDN193189, and BIO were investigated (Table 5).

[0092] Table 4 - Effects of various growth factors on acceleration of the appearance of hair placodes in skin organoids.|0093| The effect of the acceleration was quantified as the reduced time frame required to form the hair placodes compared to the untreated control. The results are further qualitatively categorized into very effective (©, more than or equal to 5 days improvement), effective (o, less than 5 days improvement), and no effect (X) Wnt or W: Wnt3A, BIO or B: BIO, LDN or L: LDN193189, GDNF: glial cell line derived neurotrophic factor, RA orR: Retinoic acid, Rx: Ruxolitinib, Tf: Tofacitinib.

[0094] Table 5 - Dose-responses of typical growth factors on acceleration of the appearance of hair placodes in skin organoids.The occurrence of hair placode formation by day 48

[0095] The effect of the acceleration was indirectly quantified by counting the numbers of organoids which possessed at least one hair placode on them on day 48 after differentiation The left columns indicate the concentrations of the growth factors, while the right columns indicate the occurrence of hair placodes formation

[0096] FGF10 and BIO promote hair placode formation, validated by SOX2-positive dermal papillae cells in the organoids

[0097] Robust responses were detected for FGF10 and BIO in a wide range of concentrations, although 600 ng / ml or higher concentration of LDN193189 drastically reduced the numbers of organoids with hair placode formation on Day 48 The formation of functional hair placodes was validated by immunofluorescence staining for SOX2, which is a specific marker protein for dermal papillae (Fig. 2).

[0098] Optimal LDN concentrations support full maturation comparable to untreated control organoids and contain all major skin cell types

[0099] When the organoids were cultured with appropriate concentrations of growth factors, such as 200 ng / ml LDN193189, they grew into fully matured organoids comparable to the control skin organoids that were not treated with the growth factors (Fig. 3). Specifically, the organoids contained all the minor cell types, such as melanocytes, sensory neurons, Schwann cells, and Merkel cells, and possessed hair follicles with multiple layers, including inner root sheath, companion layer, outer root sheath, and dermal sheath (Fig. 4A). Single cell analysis also revealed that hair-related keratinocytes and fibroblasts were expanded after FGF10 or LDN 193189 treatment (Fig 4B). These results indicate that the growth factor treatment accelerated the maturation process of skin organoids without any defects in long-term culturingand effectively supports organoid maturation into skin organoids physiologically similar to untreated control skin organoids.

[0100] Targeted treatment with six candidate compounds at a second window of time further accelerates hair follicle maturation in skin organoids

[0101] To further reduce the time taken for skin organoids to develop hair follicles, the inventors targeted a second window of time to treat skin organoids with candidate compounds. This period was between Day 48 and Day 100, the time taken for hair placodes to develop and fully mature into hair follicles with hair shafts. Skin organoids were first treated with FGF10 for a duration of 15 days from Day 21 to Day 35 as described above. Upon the formation of hair placodes, the skin organoids were treated with 6 candidate compounds, namely FGF7, FGF10, Ruxolitinib, Trichostatin A, Vitamin E, and BIO. These were chosen based on their known abilities to modulate hair development, growth and regeneration. The concentrations of these compounds were then halved three days later, further halved two days thereafter, then removed two days later. The morphological changes were tracked and observed for signs of accelerated hair follicle maturation until Day 100. To do so, the inventors defined a maturation index score of 1 to 5 based on hair follicle developmental stages that enabled semiquantification and comparison of the speed of hair follicle development (Fig. 5). The result revealed that FGF10 and Vitamin E treatment increased the maturation index, suggesting an acceleration of skin organoid maturation (Table 6). Taken together, the methods of the present disclosure enable accelerated hair follicle development, growth and maturation using various combinations of additives in two defined (critical) time windows of skin and organoid development.

[0102] Table 6 - Effects of various growth factors on acceleration of the maturation of hair placodes in skin organoids.

[0103] The effect of the acceleration was quantified as the maturation index at day 77 compared to the untreated control. The results are further qualitatively categorized into effective (o) and non-effective (X).

[0104] Discussion

[0105] The present disclosure describes methods to accelerate hair follicle development in pluripotent stem cell-derived skin organoids (e.g., from iPSCs).

[0106] To differentiate iPSCs into skin organoids using conventional methods, iPSCs are initially treated with a series of recombinant proteins which direct their specialization into the two major skin compartments, namely epidermis and dermis, by 21 days. Thereafter, it typically takes at least 40 days for hair precursors, termed hair placodes, to develop, and at least 40 days more for hair placodes to fully mature into hair follicles.

[0107] To reduce the time taken for skin organoids to develop hair follicles, the inventors targeted a window of time between Day 21 and Day 60 for treatment with additives that are known to modulate hair follicle development, growth and regeneration. Manipulation of skin organoids during this window, after major skin compartments have formed but not hair placodes, ensures that skin development is not disrupted, while providing an opportunity for hair placodes to form earlier.|00108| The present disclosure describes for the first time a method of accelerating hair follicle development in pluripotent stem cell-derived skin organoids by adding growth factors at two specific windows of skin and organoid development.

[0109] The most important features of the present disclosure disclosed herein include:• The method disclosed herein allows accelerated process of hair follicle development from a skin organoid that contains all the minor cell types, such as melanocytes, sensory neurons, Schwann cells, and Merkel cells. The treated skin organoid possessed hair follicles with multiple layers, including inner root sheath, companion layer, outer root sheath, and dermal sheath. This is an indication that the resulting hair follicles are structurally complete, indicating their physiological relevance.• The method disclosed herein involves using specific growth factors, added at specific window s of time resulting in accelerated process of hair follicle development in treated skin organoids compared to control untreated skin organoids.

[0110] The methods of the present disclosure have the following advantages:• Compared to conventional skin grafts used for treating burn subj ects (which do not have hair follicles and other appendages), the skin grafts made from the methods of thepresent disclosure have developed hair follicles that can improve the quality of life of patients after treatment.• Compared to the time required for maturation of skin organoids using conventional methods (currently at about 4 months long), the identification of the two critical time windows of skin and organoid development for addition of specific growth factors allows accelerated process of hair follicle development from a skin organoid.

Claims

CLAIMSWhat is claimed is:

1. A method of inducing and / or stimulating the formation of a plurality of hair placodes on a skin organoid, comprising (a) adding one or more growth factors to a culture of skin organoid, after (or upon) the formation of epidermis and dermis in the skin organoid.

2. A method of accelerating the formation of a plurality of hair placodes on a skin organoid, comprising (a) adding one or more growth factors to a culture of skin organoid, after (or upon) the formation of epidermis and dermis in the skin organoid.

3. A method of producing a skin organoid with a plurality of hair placodes, comprising (a) adding one or more growth factors to a culture of skin organoid, after (or upon) the formation of epidermis and dermis in the skin organoid.

4. The method of any one of claims 1-3, wherein the skin organoid comprises less than 1% dermal papilla cells prior to step (a), wherein optionally the skin organoid does not comprise dermal papilla cells prior to step (a).

5. The method of any one of claims 1-4, wherein the skin organoid is capable of forming a plurality of hair shafts.

6. The method of any one of claims 1-5, wherein the skin organoid comprises three or more cell types.

7. The method of any one of claims 1-6, comprising (b) culturing pluripotent stem cells in the presence of one or more recombinant proteins prior to step (a) to obtain the skin organoid, wherein the pluripotent stem cells differentiate to form the epidermis and dermis.

8. The method of claim 7, wherein the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), wherein optionally the pluripotent stem cells are human ESCs or human iPSCs, wherein optionally the pluripotent stem cells are human iPSCs, and wherein optionally the human ESCs or human iPSCs differentiate into fibroblasts.

9. The method of any one of claims 1-8, wherein the one or more growth factors are selected from the group consisting of:(i) a fibroblast growth factor (FGF), wherein optionally the FGF is selected from the group consisting of a FGF7 subfamily member and a FGF9 subfamily member, wherein optionally the FGF7 subfamily member is selected from the group consisting of FGF3, FGF7, FGF10 and FGF22, wherein optionally the FGF9 subfamily member is selected from the group consisting of FGF9, FGF16 and FGF20, wherein optionally the FGF7 subfamily member is FGF10 and the FGF9 subfamily member is FGF20;(ii) an inhibitor of bone morphogenetic protein (BMP) receptor, wherein optionally the inhibitor of BMP receptor is LDN-193189;(iii) a bifunctional or multifunctional compound which inhibits glycogen synthase kinase-3 (GSK3) and activates Wnt signaling pathway, wherein optionally the bifunctional or multifunctional compound is BIO;(iv) a glial cell line-derived neurotrophic factor (GDNF);(v) retinoic acid;(vi) vitamin E, wherein optionally the vitamin E is a-tocopherol;(vii) an inhibitor of Janus kinase (JAK), wherein optionally the inhibitor of JAK is selected from the group consisting of ruxolitinib and tofacitinib;(viii) an insulin-like growth factor (IGF), wherein optionally the IGF ligand is IGF2, and(ix) any combination of (i) to (viii).

10. The method of claim 9, wherein:(i) the concentration of the FGF added in step (a) is about 0.1-200 ng / ml, wherein optionally the concentration of the FGF is about 10-100 ng / ml;(ii) the concentration of the inhibitor of BMP receptor added in step (a) is about 2-550 ng / ml, wherein optionally the concentration of the inhibitor of BMP receptor is about 20-200 ng / ml;(iii) the concentration of the bifunctional or multifunctional compound added in step (a) is about 1-2000 nM, wherein optionally the concentration of the bifunctional or multifunctional compound is about 10-100 nM;(iv) the concentration of the GDNF added in step (a) is about 1 -5000 ng / ml, wherein optionally the concentration of the GDNF is about 50-200 ng / ml;(v) the concentration of the retinoic acid added in step (a) is about 10-5000 nM, wherein optionally the concentration of the retinoic acid is about 200-2000 nM;(vi) the concentration of the vitamin E added in step (a) is about 0. 1-200 uM, wherein optionally the concentration of the vitamin E is about 5-20 uM;(vii) the concentration of the inhibitor of JAK added in step (a) is about 1-5000 nM, wherein optionally the concentration of the inhibitor of JAK is about 100-1000 nM; and(viii) the concentration of the IGF added in step (a) is about 1-5000 ng / ml, wherein optionally the concentration of the IGF is about 100-1000 ng / ml.

11. The method of claim 9 or claim 10, wherein the one or more growth factors are selected from the group consisting of FGF10, FGF20, BIO, LDN193189, vitamin E, ruxolitinib, tofacitinib, IGF2 and any combination thereof.

12. The method of claim 9 or claim 10, wherein the growth factor(s) added in step (a) is selected from the group consisting of or consisting essentially of:(i) FGF10;(ii) FGF20;(iii) BIO;(iv) ruxolitinib;(v) tofacitinib;(vi) LDN193189;(vii) vitamin E;(viii) GDNF;(ix) retinoic acid; and(x) IGF2.

13. The method of any one of claims 1-12, comprising (c) reducing the concentration of the one or more growth factors by about 0.5-fold after performing step (a).

14. The method of claim 13, comprising (d) further reducing the concentration of the one or more growth factors by about 0.5-fold after performing step (c).

15. The method of any one of claims 1-14, wherein the one or more growth factors reduce the time taken for the formation of the plurality of hair placodes on the skin organoid by at leastabout 2 days compared to a control skin organoid, wherein optionally the one or more growth factors reduce the time taken for the formation of the plurality of hair placodes on the skin organoid by about 2-19 days compared to the control skin organoid, wherein the control skin organoid is not treated with the one or more growth factors after (or upon) the formation of epidermis and dermis in the control skin organoid.

16. The method of any one of claims 1-15, comprising (e) further adding one or more growth factors to the culture after (or upon) the formation of the plurality of hair placodes on the skin organoid, to accelerate the maturation of the plurality of hair placodes into a plurality of hair germs, hair pegs, hair bulbs, and / or hair shafts.

17. The method of claim 16, wherein the one or more growth factors to be further added in step (e) are selected from the group consisting of:(i) FGF10;(ii) vitamin E, wherein optionally the vitamin E is a-tocopherol; and(iii) any combination of (i) to (ii).

18. The method of claim 17, wherein:(i) the concentration of the FGF10 added in step (e) is about 0.1-200 ng / ml, wherein optionally the concentration of the FGF10 is about 10-100 ng / ml; and(ii) the concentration of the vitamin E added in step (e) is about 0.1-200 uM, wherein optionally the concentration of the vitamin E is about 5-20 uM.

19. The method of any one of claims 16-18, comprising (f) reducing the concentration of the one or more growth factors by about 0.5 fold after performing step (e).

20. The method of claim 19, comprising (g) further reducing the concentration of the one or more growth factors by about 0.5 fold after performing step (f).

21. The method of any one of claims 16-20, wherein the one or more growth factors reduce the time taken for the maturation of the plurality of hair placodes on the skin organoid into a plurality of hair germs, hair pegs, hair bulbs, and / or hair shafts by at least about 2 days compared to a control skin organoid, wherein optionally the one or more growth factors reduce the time taken for the maturation of the plurality of hair placodes on the skin organoid into aplurality of hair germs, hair pegs, hair bulbs, and / or hair shafts by about 2-28 days compared to the control skin organoid, wherein the control skin organoid is not further treated with the one or more growth factors after (or upon) the formation of the plurality of hair placodes on the control skin organoid22. A skin organoid produced by the method of any one of claims 1-21.

23. A method of treating skin and / or hair loss in a subject, comprising grafting or implanting the skin organoid of claim 22 onto the subject.

24. A method of screening a compound for treating skin-related and / or hair-related disease, comprising:(i) contacting or treating the skin organoid of claim 22 with a test compound;(ii) measuring a biological response of the skin organoid to the test compound, wherein the biological response is indicative of the therapeutic potential of the test compound in treating the skin-related and / or hair-related disease; and(iii) comparing the measured biological response to a control response to determine the efficacy of the test compound in treating the skin-related and / or hair-related disease.

25. A method of screening a gene for skin and / or hair follicle development activity, comprising:(i) introducing a target gene into, and / or deleting, and / or base-editing a test gene in the skin organoid of claim 22;(ii) culturing the skin organoid under conditions suitable for skin and / or hair follicle develop ent;(iii) measuring the expression of marker(s) indicative of skin or hair follicle development; and(iv) comparing the expression level(s) of the marker(s) to a control to determine the activity of the test gene.

26. A method of screening a compound for cosmetic application, comprising:(i) contacting the skin organoid of claim 22 with a test compound;(ii) measuring a cosmetic response of the skin organoid to the test compound; and(iii) comparing the measured cosmetic response to a control response to determine the efficacy of the test compound for cosmetic application.

Images