Assemblies of differentiated cells and related methods

Abstract

This disclosure relates to cell culture applications, and more specifically to cell culture applications involving assemblies of differentiated cells, such as the formation / derivation and dissociation thereof. Assemblies of differentiated cells may produce follicular units or structures resembling follicular units, which include one or more hair follicles. Depending on the nature of the cells from which the follicular units or structures resembling follicular units are derived, it may be possible to generate scalp-like follicles or non-scalp-like follicles. Expression of POU5F1 and / or SOX2 among cells from which the follicular units or structures resembling follicular units are derived correlates with the derivation of scalp-like follicles or non-scalp-like follicles.

Description

ASSEMBLIES OF DIFFERENTIATED CELLS AND RELATED METHODSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 609,134, filed December 12, 2023, the entire contents of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to cell culture applications, and more specifically to cell culture applications involving assemblies of differentiated cells, and still more specifically to the formation, dissociation, and culture thereof.BACKGROUND

[0003] Organism and tissue-level development occurs in the context of direct cell-cell contact and in the context of direct or indirect signaling between cells. Three-dimensional in vitro models, such as embryoid bodies and more recently organoids, have been employed to study mechanisms of normal and disease development. However, such three-dimensional in vitro models are not yet fully understood, and are not yet capable of reproducibly deriving all target tissue types. Regardless, three-dimensional in vitro models hold incredible medical promise, including for understanding mechanisms of development / disease, testing the effects of drugs and other treatment modalities, and regenerative medicine applications.

[0004] Follicular units, or multicellular bodies comprising one or more hair follicles, are an exemplary three-dimensional assembly of cells that hold great medical and cosmetic promise. In the context of researching and treating disorders or diseases relating to hair growth, an in vitro derivable source of hair follicles represents an important tool and a potentially limitless source of material to treat individuals suffering from (androgenic) alopecia, for example.

[0005] In the context of researching and treating disorders or diseases relating to skin (including hair follicles), an in vitro-derived assembly of differentiated cells representative of the complexity of skin (including cell types having keratinocyte, dermal fibroblast, and other potentials) may be an important tool and a potentially limitless source of material to treat burn victims, for example.

[0006] However, at present it is not known how to generate cell assemblies comprising hair follicles and / or representative cell types of the skin in vitro that are suited for scalp versus nonscalp applications. In the absence of efficient and effective approaches to derive hair follicles, and / or representative cell types of the skin, current treatment relies on sourcing either autologous or allogeneic material for direct transplantation / grafting, which can be costly andburdensome. Thus, there is a need for in vitro approaches and tools for generating cell assemblies comprising hair follicle(s), particularly those that are suited for applications involving the scalp or otherwise. In addition or in the alternative, there is also a need for in vitro approaches and tools for generating cell assemblies comprising cell types representative of skin.

[0007] Such in vitro approaches could be particularly advantageous if beginning from an unlimited or expandable cell source, such as from stem or progenitor cells, including but not limited to pluripotent stem cells (PSCs). Accordingly, robust and efficient methods / tools for deriving stem cell-derived assemblies of differentiated cells, particularly those comprising one or more hair follicles and / or those that produce cells having the potential to give rise to representative cell types of the skin, hold promise to enable the modeling and treatment of skin diseases and hair disorders, whether inherited or spontaneous, and provide hope for patient-specific cell therapy and regenerative medicine in the clinic.SUMMARY

[0008] In one aspect of this disclosure are provided assemblies of differentiated cells, which may be in-vitro derived. Assemblies of differentiated cells may be comprised in a skin organoid, preferably a hair-bearing skin organoid, and / or may be or comprise a follicular unit. Assemblies of differentiated cells may comprise one or more hair follicle, preferably more than one hair follicle.

[0009] Assemblies of differentiated cells of this disclosure may comprise a plurality of cell types among the assembly of differentiated cells, such as those cell types representative of the skin (and associated hair follicles / follicular units).

[0010] One or more cells of an assembly of differentiated cells may ectopically express POU5F 1 and / or SOX2, or a functional isoform or variant thereof. One or more cells ectopically expressing POU5F1 and / or SOX2, or a functional isoform or variant thereof, may be transgenic. If transgenic, such one or more cells may comprise at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof. In one embodiment, at least one exogenous copy of POU5F1 and / or SOX2, or the functional isoform or variant thereof, is encoded in a genomic landing pad.

[0011] In another aspect of this disclosure is provided a dissociated population of cells of an assembly of differentiated cells. Assemblies of differentiated cells may be comprised in a skin organoid, preferably a hair-bearing skin organoid, and / or may be or comprise a follicular unit. Assemblies of differentiated cells may comprise one or more hair follicle, preferably more than one hair follicle.

[0012] In one embodiment, one or more hair follicles is extracted from the assembly prior to dissociation.

[0013] A dissociated population of cells may comprise a plurality of cell types, wherein a first cell type has mesenchymal potential and a second cell type has keratinocyte potential.

[0014] One or more cells of an assembly of differentiated cells may ectopically express POU5F 1 and / or SOX2, or a functional isoform or variant thereof. One or more cells ectopically expressing POU5F1 and / or SOX2, or a functional isoform or variant thereof, may be transgenic. If transgenic, such one or more cells may comprise at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof. In one embodiment, at least one exogenous copy of POU5F1 and / or SOX2, or the functional isoform or variant thereof, is encoded in a genomic landing pad.

[0015] In another aspect of this disclosure are provided methods of deriving / forming an assembly of differentiated cells, which may be in-vitro methods. Assemblies of differentiated cells may be comprised in a skin organoid, preferably a hair-bearing skin organoid, and / or may be or comprise a follicular unit. Assemblies of differentiated cells may comprise one or more hair follicle, preferably more than one hair follicle.

[0016] Methods of deriving / forming an assembly of differentiated cells may comprise providing one or more stem or progenitor cells in a differentiating culture environment, and exposing the one or more stem or progenitor cells to the culture environment for a sufficient period of time to derive / form the assembly of differentiated cells as comprising a plurality of cell types.

[0017] One or more cells of an assembly of differentiated cells may ectopically express POU5F 1 and / or SOX2, or a functional isoform or variant thereof. One or more cells ectopically expressing POU5F1 and / or SOX2, or a functional isoform or variant thereof, may be transgenic. If transgenic, such one or more cells may comprise at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof. In one embodiment, at least one exogenous copy of POU5F1 and / or SOX2, or the functional isoform or variant thereof, is encoded in a genomic landing pad.

[0018] Methods may further comprise extracting a follicular unit or a structure resembling a follicular unit (or the hair follicles thereof) from the assembly, and optionally exposing / culturing the extracted follicular unit or the structure resembling a follicular unit (or the hair follicles thereof) in a culture environment, such as the differentiating culture environment.

[0019] A culture environment of any aspect of this disclosure may be serum-free and feeder cell-free.

[0020] In another aspect of this disclosure are provided methods of generating a population of differentiated cell types having one or both of mesenchymal (e.g. (dermal) fibroblast) and keratinocyte potential. Methods of this aspect may be in vitro methods.

[0021] Methods of generating a population of differentiated cell types may comprise providing a starting population of stem or progenitor cells, deriving / differentiating an assembly of differentiated cells from the starting population of stem or progenitor cells in a differentiating culture environment, and dissociating the assembly of differentiated cells to yield at least a first cell type having mesenchymal potential and a second cell type having keratinocyte potential, thereby generating a population of differentiated cell types.

[0022] Methods of this or any aspect may further comprise culturing at least a portion of a population of differentiated cell types in conditions supportive of mesenchymal cells (e.g. (dermal) fibroblasts) or keratinocytes.

[0023] Assemblies of differentiated cells may be comprised in a skin organoid, preferably a hair-bearing skin organoid, and / or may be or comprise a follicular unit. Assemblies of differentiated cells may comprise one or more hair follicle, preferably more than one hair follicle.

[0024] One or more cells of i) the assembly, ii) the population of differentiated cell types, or iii) both i) and ii) ectopically express POU5F1 and / or SOX2, or a functional isoform or variant thereof. One or more cells ectopically expressing POU5F1 and / or SOX2, or a functional isoform or variant thereof, may be transgenic. If transgenic, such one or more cells may comprise at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof. In one embodiment, at least one exogenous copy of POU5F1 and / or SOX2, or the functional isoform or variant thereof, is encoded in a genomic landing pad.

[0025] In any aspect, stem or progenitor cells from which an assembly of differentiated cells is derived are stem cells, preferably pluripotent stem cells.

[0026] In any aspect, an assembly of differentiated cells may be initiated by aggregating a starting or input population of stem or progenitor cells before or concurrent with exposure to a differentiating culture environment.

[0027] In one embodiment, this disclosure relates to the expansion of or the expansion conditions for keratinocytes rather than the expansion (conditions) for off-target or contaminating cell-types present in an assembly of differentiated cells. In one embodiment, this disclosure relates to the preferential or selective expansion of (epidermal) keratinocytes or (epidermal) keratinocyte-like cells from cells having (epidermal) keratinocyte potential, incomparison to other cell types of the skin, such as mesenchymal cells or mesenchymal like cells (e.g. fibroblasts).

[0028] In one embodiment, this disclosure relates to the expansion of or the expansion conditions for (dermal) fibroblasts rather than the expansion (conditions) for off-target or contaminating cell-types present in an assembly of differentiated cells. In one embodiment, this disclosure relates to the preferential or selective expansion of (dermal) fibroblasts or (dermal) fibroblast-like cells from cells having (dermal) fibroblast potential (e.g. mesenchymal cells), in comparison to other cell types of the skin, such as keratinocytes.

[0029] In any aspect, (generating) a population of differentiated cell types having at least (dermal) mesenchymal and keratinocyte potential, following dissociation of an assembly of differentiated cells, one or more cells of i) the assembly, ii) the population of differentiated cell types, or iii) both i) and ii) ectopically express POU5F1 and / or SOX2, or a functional variant thereof.

[0030] In another aspect are provided genomic landing pads comprising an exogenous polynucleotide. In one embodiment, a genomic landing pad is located on chr2q37.3. In one embodiment, a genomic landing pad is located on chr14q24.3. In one embodiment, a genomic landing pad is located on chr16q23.2.

[0031] An exogenous polynucleotide may comprise one or at least one transcriptional unit. In one embodiment, a transcriptional unit comprises an open reading frame encoding a peptide, polypeptide, or a protein. In the same or different embodiment, a transcriptional unit comprises an open reading frame encoding a transcription regulator. A transcription regulator may be a transcription factor, transcription co-factor, a transactivator, or the like.

[0032] A genomic landing pad may transcriptionally silent or inactive in a stem or progenitor cell, and / or transcriptionally active or open in a cell differentiated from the stem or progenitor cell. A stem or progenitor cell may be an undifferentiated stem or progenitor cell, preferably a pluripotent stem cell. A cell differentiated from a stem or progenitor cell is not particularly limited provided that a landing pad of this disclosure is in a transcriptionally active or open state. For example, a cell differentiated from a stem or progenitor cell may be ectodermal, endodermal, or mesodermal. Or, a cell differentiated from a stem or progenitor cell may be dermal, mesenchymal, epithelial, or a keratinocyte

[0033] In a specific embodiment, an exogenous polynucleotide encodes POU5F1. In a specific embodiment, an exogenous polynucleotide encodes SOX2.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0035] Figure 1 shows representative photographs of developing assemblies of differentiated cells comprising one or more hair follicles (e.g. follicular units) over the course of in vitro culture. Panel A) shows an intact day 70-120 hair-bearing skin organoid derived from a PSC line comprising at least one exogenous copy of POU5F1 (PSCP0UF51), as derived according to methods published by Lee et al. (Nature, 2020). Scale bar is 200 pm. Panel B) shows a time course following culture of a representative assembly of differentiated cells comprising one or more hair follicles (e.g. follicular unit) dissected from a skin organoid derived using a PSC line comprising only endogenous copies of POU5F1 (PSCCTRL). Panel C) shows a time course following culture of a representative assembly of differentiated cells comprising one or more hair follicles (e.g. follicular unit) dissected from a PSCP0UF51-derived organoid. Scale bar is 200 pm.

[0036] Figure 2 shows the characterization of POU5F1 and SOX2 integration sites among PSCP0U5F1and expression of POU5F1 and SOX2 among various cell types. Screen grabs from the UCSC Genome Browser indicate the integration sites of POU5F1 (A) and (B) and SOX2 (C) in PSCP0UF51. Plots quantifying gene expression of pluripotency and control markers among dissociated assemblies of differentiated cells (e.g. dissociated hair-bearing skin organoids) derived from PSCP0UF51or PSCCTRL(D). Bar graph quantifying RNAseq expression of endogenous (E) and exogenous (F) copies of POU5F1 among undifferentiated PSCP0UF51or dissociated assemblies of differentiated cells (e.g. dissociated hair-bearing skin organoids) derived from pscP0UF51, on the basis of detection of distinguishing SNPs. Summary of RNAseq reads mapped across the endogenous SOX2 locus of undifferentiated PSCP0UF51(spanning the UTRs and coding region) or across the exogenous SOX2 integration (lacking read alignment to UTRs) (G).

[0037] Figure 3 shows representative images of the multi-lineage potential of dissociated (hair-bearing) skin organoids. A PSC-derived skin organoid (comprising at least one assembly of differentiated cells comprising one or more hair follicles) was dissociated and seeded as a monolayer into either dermal fibroblast supportive conditions or keratinocyte supportive conditions. Scale bar is 200 pm.DETAILED DESCRIPTION

[0038] This disclosure relates to cell culture applications, and more specifically to cell culture applications involving assemblies of differentiated cells, and still more specifically to the formation, dissociation, and culture thereof.

[0039] In one aspect of this disclosure are provided methods of forming an assembly of differentiated cells comprising a plurality of cell types and / or one or more hair follicle.

[0040] In one aspect of this disclosure are provided methods of forming an assembly of differentiated cells comprising a plurality of cell types and one or more hair follicle.

[0041] In one aspect of this disclosure are provided methods of forming an assembly of differentiated cells comprising a plurality of cell types and / or one or more hair follicle, wherein one or more cells of the assembly ectopically express POU5F1 and / or SOX2.

[0042] In any aspect, an assembly of differentiated cells formed in accordance with the disclosed methods may be a follicular unit or a structure resembling a follicular unit.

[0043] In one aspect of this disclosure are provided are provided assemblies of differentiated cells comprising a plurality of cell types and one or more hair follicle.

[0044] In one aspect of this disclosure are provided assemblies of differentiated cells comprising a plurality of cell types and / or one or more hair follicle, wherein one or more cells of the assembly ectopically express POU5F1 and / or SOX2.

[0045] In any aspect, an assembly of differentiated cells may be a follicular unit or a structure resembling a follicular unit.

[0046] In one aspect of this disclosure are provided dissociated assemblies of differentiated cells, that is assemblies of differentiated cells having been dissociated, comprising a plurality of cell types within an assembly of differentiated cells.

[0047] In one aspect of this disclosure are provided dissociated assemblies of differentiated cells, that is assemblies of differentiated cells having been dissociated, comprising a plurality of cell types within an assembly of differentiated cells, wherein one or more cells thereof ectopically express POU5F1 and / or SOX2 or a functional variant thereof.

[0048] In one aspect of this disclosure are provided dissociated assemblies of differentiated cells, that is assemblies of differentiated cells having been dissociated, comprising a plurality of cell types within an assembly of differentiated cells, wherein a first cell type thereof has dermal fibroblast potential and a second cell type thereof has keratinocyte potential.

[0049] In one aspect of this disclosure are provided methods of generating a population of differentiated cell types.

[0050] In one aspect of this disclosure are provided methods of generating a population of differentiated cell types having at least dermal fibroblast and keratinocyte potential. Such population may comprise at least a first cell type having dermal fibroblast potential and / or at least a second cell type having keratinocyte potential.

[0051] In one aspect of this disclosure are provided methods of generating a population of differentiated cell types having at least dermal fibroblast and keratinocyte potential following dissociation of an assembly of differentiated cells.

[0052] In one aspect of this disclosure are provided methods of generating a population of differentiated cell types having at least dermal fibroblast and keratinocyte potential following dissociation of an assembly of differentiated cells, wherein one or more cells of i) the assembly, ii) the population of differentiated cell types, or iii) both i) and ii) ectopically express POU5F1 and / or SOX2 or a functional variant thereof.

[0053] In any aspect, the assembly, subjected to dissociation, may correspond to a skin organoid or a follicular unit.

[0054] In one aspect of this disclosure are provided genomic landing pads comprising an exogenous polynucleotide.

[0055] In one aspect of this disclosure are provided genomic landing pads comprising an exogenous polynucleotide located on one or more of chromosome 2q37.3, chromosome 14q24.3, or chromosome 16q23.2.

[0056] In one aspect of this disclosure are provided genomic landing pads comprising an exogenous polynucleotide located on one or more of chromosome 2q37.3, chromosome 14q24.3, or chromosome 16q23.2, encoding a transcription regulator, such as POU5F1 and / or SOX2.

[0057] Where used in this disclosure, the term “stem cell” or pluralized variations thereof, refers to a cell capable of both self-renewal (in response to certain cues) and also capable of differentiating to a more specialized cell type (in response to certain other cues). A stem cell may be tissue- or lineage- restricted. Or, a stem cell may be pluripotent, in which case it may be referred to as a “pluripotent stem cell” or “PSC”, and is capable of self-renewal and giving rise to all three germ layers and cell types respectively downstream thereof. Types of PSC includes embryonic stem cells and induced pluripotent stem cells, and they may be obtained, derived or induced from any source species, but in this disclosure PSCs are preferably mammalian, and still more preferably human. PSC are an important model to study differentiation mechanisms, to model diseases, and offer significant medical opportunities.

[0058] Where used in this disclosure, the term “assembly of differentiated cells” refers to a three-dimensional structure of a plurality of differentiated cells, and thus may likewise be considered an aggregate, a multi-cellular structure, a construct, or the like. An assembly of differentiated cells may comprise more than one type of cell, and in such cases the different types of cells may self-organize. An assembly of differentiated cells may correspond to an organoid, or may be comprised in an organoid. If an organoid, methods of generating them have been previously described. In a specific embodiment related to this disclosure, an organoid may correspond to a skin organoid, or a hair-bearing skin organoid. If comprised in an organoid, such an assembly of differentiated cells may correspond to a distinct or discrete structure, which may or may not be dissectible therefrom. In a specific embodiment related to this disclosure, an assembly of differentiated cells may correspond to follicular unit, or a structure resembling a follicular unit.

[0059] Where used in this disclosure, the term “follicular unit” refers to a structure ordinarily found within the skin of animals, such as mammals. A follicular unit typically comprises - dermal fat, sebaceous gland, and at least one hair follicle. Follicular units of the scalp may comprise more than one hair follicle, or hair shaft. Non-scalp follicular units, such as of the arms or legs, may comprise a single hair follicle, or hair shaft. Thus, follicular units of the scalp and non-scalp follicular units may be distinguished by the number of hair follicles, or hair shafts. If generated in vitro, a follicular unit of this disclosure may or may not comprise all of the constituent cell types of a classical follicular unit; however, such in vitro derived assembly / structure may nevertheless closely resemble a classical follicular unit, and may be referenced as such in this disclosure.

[0060] Where used in this disclosure, the term “ectopically expressing” refers to a cell or tissue that expresses a gene or protein out of context. The gene or protein may be expressed at a level or quantity not ordinarily observed in a given cell type or tissue type. For example, in the ordinary course, a cell or tissue at one stage of development may express (or may not express) a given gene or protein, but a cell or tissue at a different stage of development may no longer express (or may begin expressing) the gene or protein. Thus, when such cell or tissue begin or cease expressing a gene or protein at out-of-context levels, such gene or protein may be considered ectopically expressed. In the context of this disclosure, PSC ordinarily express POUF51 and as they differentiate the expression of POU5F1 may be downregulated or silenced; however, PSC-derived cells (e.g. an assembly of differentiated cells, and / or a population of differentiated cell types) ectopically expressing POU5F1 will express this gene / protein even after differentiation. A gene or protein may be induced or become ectopically expressed in any way known in the art. For example, said gene or protein may be expressed from an expression vector, such as a plasmid, a non-integrating virus or sequence,or an integrating virus or sequence. In one embodiment, ectopic (e.g. out of context) expression of an integrated virus or sequence may depend on the state of surrounding chromatin, such as chromatin that is ordinarily in a condensed (or closed) or in an open configuration. Alternatively, the expression of an endogenous and / or transgenic copy of said gene may be induced or silenced / downregulated by the presence of a second factor, such as a transcription regulator (e.g an activator / repressor). In one embodiment, an activator / repressor is a protein or peptide. In one embodiment, an activator / repressor is a small molecule. In one embodiment, an activator / repressor is a hormone or a hormone analog. In one embodiment, an activator / repressor is a vitamin or vitamin analog.

[0061] Where used in this disclosure, the term “population of differentiated cell types” or “differentiated population of PSC” refers to a population of cells that emerged from one or more stem or progenitor cell or undifferentiated PSC, and as a result of being exposed to various internal or external cues have become more specialized than the parental stem cell or undifferentiated PSC. Unless terminally differentiated, an arising population of differentiated cell types may still comprise cells that retain some capacity for self-renewal, albeit with a reduced capacity to generate the breadth of cells as the parental stem cell or undifferentiated PSC. In the context of this disclosure, a population of differentiated cell types may comprise two or more different cell types that are more specialized compared to the parental stem / progenitor cell or undifferentiated PSC from which they were derived. An exemplary differentiated cell type of the population may be a keratinocyte, a keratinocyte-like cell, or a cell having keratinocyte potential (e.g. a progenitor of keratinocytes). While keratinocytes may be ectodermal, endodermal, or mesodermal in origin, in the context of skin and hair follicles such keratinocyte, keratinocyte-like cell, or a cell having keratinocyte potential is likely ectodermal in origin, or more specifically epidermal. An exemplary differentiated cell type of the population may be a mesenchymal lineage cell, such as a fibroblast / fibrocyte, a fibroblast / fibrocyte-lite cells, or a cell having fibroblast / fibrocyte potential (e.g. a progenitor). Despite an ability to undergo mesenchymal to epithelial transition (or vice versa), in the context of skin and hair follicles a fibroblast / fibrocyte, fibroblast / fibrocyte-lite cells, or cell having fibroblast / fibrocyte potential (e.g. a progenitor of fibroblast) is likely mesodermal or neural crest in origin, or more specifically mesenchymal.

[0062] A population of differentiated cell types may be obtained from an assembly of differentiated cells, such as a follicular unit or an assembly resembling a follicular unit, and / or from a stem cell (e.g. PSC)-derived skin organoid, preferably a hair-bearing skin organoid. PSC-derived skin organoids may be generated in any way known in the art, including as published by Lee et al. (Nature, 2020). In one embodiment, a population of differentiated cell types corresponds to those cell types as obtained by dissociating an assembly of differentiatedcells, such as a follicular unit or an assembly resembling a follicular unit, and / or from a stem cell (e.g. PSC)-derived skin organoid, preferably a hair-bearing skin organoid. While preferably mammalian, and more preferably human, a population of differentiated cell types may correspond to any source species.

[0063] Where used in this disclosure, the term “keratinocyte potential” or “epidermal keratinocyte potential” refers to the characteristic of a cell to differentiate to the keratinocyte lineage. In the context of skin, keratinocytes differentiate from an underlying basal layer and migrate upward toward the surface 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 the expression of TP63, keratin 5 (“K5”), and keratin 14 (“K14”). Keratinocytes are ordinarily present in various animals, and particularly within the skin thereof, but also in the esophagus and elsewhere. In humans and other animals, keratinocytes make up the majority of the outer layer of the skin (i.e. the epidermis). As such, the terms “keratinocytes” and “epidermal keratinocytes” may be used interchangeably herein. In humans, keratinocytes may make up about 90% or more of epidermal skin cells. In vivo, keratinocytes have at least some ability to proliferate before they differentiate and / or senesce and / or become shed.

[0064] Where used in this disclosure, the term “mesenchymal potential” refers to the characteristic of a cell to differentiate to the mesenchymal lineage. In the context of skin, cells having mesenchymal potential (e.g. mesenchymal stem cells) may give rise to dermal mesenchymal cells, such as dermal fibroblasts, dermal papilla cells, and dermal sheath cells. Cells having mesenchymal potential and / or (dermal) fibroblasts are characterized by the expression of one or more stage-specific markers. Examples of markers include CD90, PDGFR, FGFR, CD29, CD164, COL1A1 and ELN. Cells having mesenchymal potential, such as those that may give rise to (dermal) fibroblasts, are ordinarily present in various animals, and particularly within the dermis thereof. In humans and other animals, dermal fibroblasts make up the majority of the skin connective tissue and interact with epidermal cells during hair development and in interfollicular skin. Thus, cells having mesenchymal potential and / or (dermal) fibroblasts may serve an important role in support of follicular units and hair follicle development, such as by generating extracellular matrix.

[0065] Where used in this disclosure, the term “landing pad” or “genomic landing pad” refers to a region of a chromosome that has accepted or may accept an integration of an exogenous nucleotide sequence without resulting in insertional mutagenesis and / or materially disrupting or interfering with the expression of (nearby) endogenous genes. A landing pad of this disclosure may undergo changes to its chromatin conformation (e.g. euchromatin to heterochromatin, or vice versa) depending on the stage of development or responsiveness toan internal or external signal. By way of example, a landing pad may be transcriptionally inactive or silent (and possibly in a transcriptionally “closed” chromatin context) in a stem cell, such as a PSC, and may be transcriptionally active (and possibly in an “open” chromatin context).Methods

[0066] In one aspect of this disclosure are provided methods of forming / deriving an assembly of differentiated cells comprising a plurality of cell types and / or one or more hair follicle. An assembly of differentiated cells may form / derive when providing one or more stem or progenitor cells in a differentiating culture environment, and exposing the one or more stem or progenitor cells to the culture environment for a sufficient period of time to form the assembly of differentiated cells comprising a plurality of cell types.

[0067] Stem or progenitor cells are not particularly limited provided that they are capable of giving rise / differentiating to a plurality of cell types. A stem or progenitor cell of this disclosure may correspond to an undifferentiated PSC, preferably a human PSC. Or, a stem or progenitor cell of this disclosure may correspond to a cell type downstream of a PSC, but that may nevertheless give rise / differentiate into a plurality of cell types. In this context, a non- PSC stem or progenitor cell may be differentiated from a PSC or may be obtained / isolated from a subject.

[0068] Where methods of this disclosure form / derive an assembly of differentiated cells comprising a plurality of cell types and one or more hair follicle, a stem or progenitor cell that is not a PSC (but may be derived from a PSC) may correspond to an epidermal stem cell or a hair follicle stem cell.

[0069] Providing an appropriate starting cell type or types may not be sufficient to form / derive an assembly of differentiated cells comprising a plurality of cell types and / or one or more hair follicle, and the methods of this disclosure may further rely on an appropriate differentiating culture environment. In a specific example, a differentiating culture environment will bias the one or more stem or progenitor cells toward an ectodermal and / or epidermal and / or mesenchymal fate in order to yield an assembly of differentiated cells comprising one or more hair follicle.

[0070] Exposure of one or more stem or progenitor cells to a differentiating culture environment for a sufficient period of time to form / derive the assembly may elapse between 1 and 26 weeks. In some embodiments, the one or more stem or progenitor cells are exposed to the differentiating culture environment for 1 or more weeks, 2 or more weeks, 3 or more weeks, 4 or more weeks, 5 or more weeks, 6 or more weeks, 7 or more weeks, 8 or more weeks, 9 or more weeks, 10 or more weeks, 11 or more weeks, 12 or more weeks, 13 or moreweeks, 14 or more weeks, 15 or more weeks, 16 or more weeks, 17 or more weeks, 18 or more weeks, 19 or more weeks, 20 or more weeks, 21 or more weeks, 22 or more weeks, 23 or more weeks, 24 or more weeks, or 25 or more weeks.

[0071] Forming / deriving the assembly may involve a plurality of different culture environments, such as an initial induction environment followed by a maturation environment. If relying on an initial induction environment and a maturation environment each stage may be about the same duration or different durations. In one embodiment, a maturation stage (in a maturation environment) is longer than an induction stage (in an induction environment). For example, an induction stage may elapse 1-4 weeks, and a maturation stage may elapse 2-15 weeks, or longer.

[0072] Methods of forming / deriving an assembly of differentiated cells may comprise preaggregating one or more stem or progenitor cells either before exposure to a differentiating culture environment or while exposed to a differentiating culture environment. Preaggregating cells typically can be performed in between 1-5 days, and aggregation may be facilitated by the selection of cultureware, such as in a microwell device (e.g. an AggreWell™ device) or in a u-bottom microplate.

[0073] Thus, following methods of this to disclosure to form / derive an assembly of differentiated cells comprising a plurality of cell types and one or more hair follicle, such hair follicle may be comprised in an aggregate of cells. While an aggregate of cells (e.g. an assembly of differentiated cells) may take any form it may be, or may be comprised in, a skin organoid, preferably a hair-bearing skin organoid. A skin organoid (e.g. a hair-bearing skin organoid) may be derived according to published methods of Lee, as referenced herein. A skin organoid (e.g. a hair-bearing skin organoid) may be derived using a commercially available kit or reagents, as commercialized by STEMCELL Technologies. In addition or in the alternative, while an aggregate of cells (e.g. an assembly of differentiated cells) may take any form it may be, or may comprise, a follicular unit or a structure resembling a follicular unit.

[0074] Based on the foregoing, the skilled person will know the factors to include in such a differentiating culture environment. Briefly, stem or progenitor cell (e.g. PSC) aggregates may be exposed for approximately 1 week in a first indication condition (e.g. FGF2, TGFbeta inhibition and BMP) and a second induction condition (e.g. FGF2 and BMP inhibition). Following, self-assembly may progress in a maturation condition in the absence of any of the foregoing factors.

[0075] Methods of this disclosure may further comprise extracting a hair follicle, as may be comprised in a follicular unit or a structure resembling a follicular unit, or from an assembly of differentiated cells (e.g. a skin organoid). Extraction may be by any conventional means inthe art, such as by dissection using a scalpel or fine-tipped forceps. After extracting a hair follicle, as may be comprised in a follicular unit or a structure resembling a follicular unit, it may be incubated or cultured in a culture environment (e.g. a differentiation culture environment) as previously described for between about 1 to 14 days, or more.

[0076] In another aspect of this disclosure are provided methods of generating a population of differentiated cell types comprising at least two different differentiated cell types. A population of (at least two) differentiated cell types may arise when providing a starting population of stem or progenitor cells, and deriving an assembly of cells from the starting population in a differentiating culture environment.

[0077] As described hereinabove, stem or progenitor cells are not particularly limited. A stem or progenitor cell of this disclosure may correspond to an undifferentiated PSC, preferably a human PSC. Or, a stem or progenitor cell of this disclosure may correspond to a cell type downstream of a PSC, but that may nevertheless give rise / differentiate into a plurality of cell types. For example, a stem or progenitor cell that is not a PSC (but may be derived from a PSC) may correspond to an epidermal stem cell or a hair follicle stem cell, or a cell type that can give rise to both an epidermal stem cell or a hair follicle stem cell. Or, a stem or progenitor cell that is not a PSC (but may be derived from a PSC) may correspond to a cell type that gives rise to (dermal) fibroblasts, such as a mesenchyme progenitor or stem cell. Or, a stem or progenitor cell that is not a PSC (but may be derived from a PSC) may correspond to a cell type that gives rise to both (i) an epidermal stem cell and / or a hair follicle stem cell, and (ii) (dermal) fibroblasts, such as a mesenchyme progenitor or stem cell.

[0078] As described hereinabove, the differentiating culture environment to form / derive an assembly of differentiated cells is not particularly limited, and the description above may apply to methods of generating a population of differentiated cell types comprising at least two different differentiated cell types. As above, an assembly of differentiated cells may correspond to a follicular unit or a structure resembling a follicular unit. In addition or in the alternative, as above, an assembly of differentiated cells may correspond to a skin organoid, or a hair-bearing skin organoid, as described in Lee et al. (Nature, 2020).

[0079] Methods of this disclosure may further comprise dissociating an assembly of differentiated cells (e.g. follicular unit, structure resembling a follicular unit, skin organoid). Following, at least two differentiated cell types may be decoupled from the context of the assembly, and thus more readily addressable in downstream assays / steps. Numerous dissociation reagents are commercially available, such as Dispase™, Accutase™, a trypsinbased solution, or any other suitable enzyme or cocktail of enzymes. An assembly of differentiated cells (e.g. follicular unit, structure resembling a follicular unit, skin organoid) maybe dissociated at various time points during its (in vitro) development, such as following induction but before maturation, or during or after maturation (e.g. in the range of day 28 - day 150, such as on day 28, 35, 42, 49, 56, 63, 70, 77, 84, 91 , 98, 105, 112, 119, 126, 133, 140, or 147). Without being bound by theory, cell types having appropriate potential may be present within an assembly of differentiated cell prior to becoming matured, and such cells may persist through to maturity.

[0080] Following dissociation of an assembly of differentiated cells, methods of this disclosure may further comprise culturing at least a portion of a population of differentiated cell types in conditions supportive of a target cell type.

[0081] Differentiated cell types within an assembly of differentiated cells (or within a population following dissociation) are not particularly limited. In the context of a dissociated follicular unit (or a structure resembling a follicular unit) and / or a skin organoid (or a hairbearing skin organoid), a first cell type may correspond to a keratinocyte (e.g. epidermal keratinocyte) or a cell having keratinocyte potential (e.g. progenitor of keratinocytes). A second cell type among the population of cells of a dissociated assembly may correspond to mesenchymal cells (e.g. (dermal) fibroblasts), or a cell having mesenchymal potential (e.g. a progenitor of (dermal) fibroblasts such as a mesenchymal stem or progenitor cells). Thus, one, some, or all of the differentiated cells types within an assembly of differentiated cells (or within a population following dissociation) may be terminally differentiated, have some degree of differentiation capacity (whether multi-potent or not), or any combination thereof.

[0082] In one embodiment, a population of differentiated cell type(s) comprises a cell type that is not terminally differentiated and retains some degree of multi-potency, such as comprising (dermal) fibroblast potential and / or keratinocyte potential. In one embodiment, a population of differentiated cell types comprises different populations of differentiated cells, whether terminally differentiated (e.g. keratinocytes or keratinocyte-like cells, or fibroblasts or fibroblast-like cells) and / or having some differentiation capacity, such as a first cell type having (dermal) fibroblast potential (e.g. dermal mesenchymal cells) and / or a second cell type having keratinocyte potential. In one embodiment, a population of differentiated cells (e.g. a differentiated population of PSC) may comprise both multi-potent cells and cells capable of only giving rise to (dermal) fibroblasts (e.g. (dermal) mesenchymal cells) and / or keratinocytes (e.g progenitor of keratinocytes).

[0083] Differentiated cell types within an assembly of differentiated cells (or within a population following dissociation) may comprise i) one or more epidermal stem cells, ii) (dermal) mesenchymal stem or progenitor cells, iii) keratinocytes and / or one or more progenitors of keratinocytes or keratinocyte-like cells, iv) (dermal) fibroblasts and / or one ormore progenitors of (dermal) fibroblasts or (dermal) fibroblast-like cells, or v) any combination of the foregoing.

[0084] Differentiated cell types within an assembly of differentiated cells (or within a population following dissociation) may comprise i) one or more holoclones, ii) one or more meroclones, iii) one or more paraclones, or iv) any combination of the foregoing. In one embodiment, differentiated cell types within an assembly of differentiated cells (or within a population following dissociation) may comprise more than one holoclones and more than one meroclones. In one embodiment, differentiated cell types within an assembly of differentiated cells (or within a population following dissociation) may comprise more holoclones and / or meroclones than paraclones.

[0085] As indicated above, cells of a dissociated assembly of differentiated cells may be cultured in conditions supportive of a cell type of interest. If a target cell type (e.g. a first cell type of interest) is fibroblasts, such as dermal fibroblasts, then at least a portion of a dissociated assembly of differentiated cells will be cultured in conditions supportive of (dermal) fibroblasts and / or cells having (dermal) fibroblast potential (e.g. dermal mesenchymal cells). An exemplary culture condition that supports maintenance / proliferation of such cells may involve culturing in MesenCultTM-branded media, or any other culture medium that supports mesenchymal cells. In addition or in the alternative, a (dermal) fibroblast medium may expand cells having (dermal) fibroblast potential and / or (dermal) fibroblasts (e.g. dermal fibroblasts and / or epidermal fibroblast-like cells).

[0086] A medium that supports the culture of (dermal) mesenchymal cells (e.g. (dermal) fibroblasts or fibroblast-like cells) may comprise a basal medium supplemented with one or more growth factors, such as an FGF (~1-50 ng / mL) and / or a PDGF (~1-50 ng / mL). Basal media are well known in the art, and may include RPMI, DMEM, F-12, MCDB153, DMEM / F- 12, Adv DMEM, Adv DMEM / F-12. Basal medium typically include carbohydrates, amino acids, trace elements, lipids, buffers, salts, and the like.

[0087] Culturing in conditions supportive of (dermal) mesenchymal cells (e.g. (dermal) fibroblasts) may confer a selective advantage to such cells or cells having such potential, at the expense of other cells among a dissociated assembly of differentiated cells (e.g. keratinocytes, keratinocyte-like cells, cells having keratinocyte potential). In addition or in the alternative, culturing in conditions supportive of (dermal) mesenchymal cells (e.g. (dermal) fibroblasts) may both stimulate differentiation / expansion of (dermal) fibroblast cells or cells having (dermal) fibroblast potential, while also differentiating keratinocytes, keratinocyte-like cells, or cells having keratinocyte potential to or toward (dermal) fibroblasts.

[0088] Fibroblasts, such as dermal fibroblasts, should exhibit elongated or spindled cell morphology, and when culturing at least a portion of cells of a dissociated assembly of differentiated cells under conditions supportive of (dermal) fibroblasts few or no cobblestone and polygonal epithelial cells (e.g. keratinocytes, keratinocyte-like cells, or other off-target cell types) should be observed. In one embodiment, media of this disclosure will support 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 mesenchymal cell (e.g (dermal) fibroblast) population doublings.

[0089] If a target cell type (e.g. a second cell type of interest) is keratinocytes, then at least a portion of a dissociated assembly of differentiated cells will be cultured in conditions supportive of keratinocytes and / or cells having keratinocyte potential. An exemplary culture condition that supports keratinocyte maintenance / proliferation may involve culturing in DermaCult™- branded media, or any other culture medium that supports epidermal cells. In addition or in the alternative, a keratinocyte medium may expand cells having keratinocyte potential, and / or keratinocytes or keratinocyte-like cells (e.g. epidermal keratinocytes).

[0090] A medium that may support the maintenance / expansion of (epidermal) keratinocytes or keratinocyte-like cells) may comprise a basal medium supplemented with one or more of an inhibitor of transformation growth factor (TGF) signaling, a gamma secretase inhibitor, and an agent that disrupts cytoskeletal structure. Basal media are well known in the art, and may include RPMI, DMEM, F-12, MCDB153, DMEM / F-12, Adv DMEM, Adv DMEM / F-12. Basal medium typically include carbohydrates, amino acids, trace elements, lipids, buffers, salts, and the like.

[0091] If present, an inhibitor of transformation growth factor (TGF) signaling may be any factor that inhibits signaling through a TGF. In one embodiment, an inhibitor of transformation growth factor (TGF) signaling is a small molecule, such as a small molecule inhibitor. In one embodiment, an inhibitor of transformation growth factor (TGF) signaling is a protein. In one embodiment, an inhibitor of TGF signaling is an inhibitor of TGF-beta 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. An effective concentration may range between about 0.1 pM to 50 pM, or between about 0.25 pM to 25 pM, or between about 0.5 pM to 10 pM.

[0092] A gamma secretase inhibitor may be any factor that inhibits signaling through a gamma secretase. In one embodiment, a gamma secretase inhibitor is a small molecule, such as a small molecule inhibitor. In one embodiment, a gamma secretase inhibitor is a protein. In one embodiment, a gamma secretase inhibitor is one or more of DAPT, RO4929097,Semagacestat (LY450139), Avagacestat (BMS-708163), Dibenzazepine (YO-01027), LY411575, L-685,458, Crenigacestat (LY3039478). An effective concentration may range between about 0.5 pM to 25 pM, or between about 1 pM to 10 pM.

[0093] An agent that disrupts cytoskeletal structure may be any factor that disrupts the cytoskeleton. In one embodiment, an agent that disrupts cytoskeletal structure is a small molecule, such as a small molecule inhibitor. In one embodiment, an agent that disrupts cytoskeletal structure is a protein. In one embodiment, an agent that disrupts cytoskeletal structure is a Rho / Rock kinase inhibitor. In one embodiment, an agent that disrupts cytoskeletal structure is one or more of Y-27632, Thiazovivin, Fasudil (HA-1077), GSK429286A, RKI-1447, Y-27632, H-1152 dihydrochloride, Azaindole 1 (TC-S 7001). An effective concentration may range between about 0.5 pM to 25 pM, or between about 1 pM to 10 pM.

[0094] Culturing in conditions supportive of (epidermal) keratinocytes may confer a selective advantage to such cells or cells having such potential, at the expense of other cells among a dissociated assembly of differentiated cells (e.g. fibroblasts, fibroblast-like cells, cells having fibroblast potential). In addition or in the alternative, culturing in conditions supportive of (epidermal) keratinocytes may both stimulate differentiation / expansion of (epidermal) keratinocytes or cells having (epidermal) keratinocyte potential, while also differentiating mesenchymal cells (e.g. fibroblasts or fibroblast-like cells, or cells having fibroblast potential) to or toward (dermal) keratinocytes.

[0095] Keratinocytes, such as epidermal keratinocytes, should exhibit cobblestone and polygonal epithelial cell morphology, and when culturing at least a portion of cells of a dissociated assembly of differentiated cells under conditions supportive of (epidermal) keratinocytes few or no elongated or spindled cells (e.g. fibroblasts, fibroblast-like cells, or other off-target cell types) should be observed. In one embodiment, media of this disclosure will support 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 (epidermal) keratinocyte population doublings.

[0096] As described above, a starting population of stem or progenitor cells may be preaggregated (as described above) before deriving an assembly of differentiated cells (e.g. a follicular unit or a structure resembling a follicular unit, or a skin organoid, such as a hairbearing skin organoid). Pre-aggregation of a starting cell population may be done as described above, or essentially as described above.

[0097] In any method of this disclosure, assemblies of differentiated cells (e.g. follicular units, structures resembling follicular units, skin organoids), and more particularly thedifferentiation / derivation culture environment, may be a serum-free environment and / or a feeder cell-free environment. In place of feeder-cells, methods (e.g. the differentiation culture environment) may comprise an appropriate substrate, such as an extracellular matrix (e.g. Matrigel™) or one or more extracellular matrix proteins, such as a laminin, fibronectin, vitronectin, collagen, etc. In one embodiment, methods (e.g. the differentiation culture environment) may be performed in the absence of Matrigel™, such as in a low attachment, an ultra-low attachment, or a non-tissue culture treated plate of any size (e.g. 96-well, 24-well, or 6 well, etc). Depending on the nature of such plates, it may be desirable to coat with a surfactant, such as Anti-adherence Rinse Solution (STEMCELL Technologies), prior to seeding one or more stem or progenitor cells, or an aggregate or assembly of differentiated cells.

[0098] In any method of this disclosure, assemblies of differentiated cells (e.g. follicular units, structures resembling follicular units, skin organoids) may comprise a single hair shaft / follicle, or may comprise a plurality of hair shafts / follicles. Unexpectedly it was discovered that expression levels of POU5F1 and / or SOX2, or a functional isoform or variant thereof, may influence whether assemblies comprise one hair shaft / follicle or a plurality of hair shafts / follicles. Stated another way, ectopic expression of POU5F1 and / or SOX2, or a functional isoform or variant thereof, may influence whether or not an assembly of differentiated cells (e.g. follicular units, structures resembling follicular units, skin organoids) comprise one hair shaft / follicle or a plurality of hair shafts / follicles.

[0099] If forming or deriving an assembly (e.g. aggregate) of differentiated cells, one or more cells of such assembly may not ectopically express POU5F1 and / or SOX2, or a functional isoform or variant thereof, and should typically be expected to comprise one hair shaft / follicle. Thus, an expression level of POU5F1 and / or SOX2, or a functional isoform or variant thereof, among cells of such assemblies may be comparable to background expression levels of POU5F1 SOX2 among skin cells, such as ectodermal lineage cells, mesenchymal cells, epidermal stem cells, keratinocytes, or the like, or to PSC having been differentiated to such lineages.

[0100] If forming or deriving an assembly (e.g. aggregate) of differentiated cells, one or more cells of such assembly may ectopically express POU5F 1 and / or SOX2, or a functional isoform or variant thereof, and should typically be expected to comprise more than one hair shaft / follicle. Thus, an expression level of POU5F1 and / or SOX2, or a functional isoform or variant thereof, among cells of such assemblies may be higher than background expression levels of POU5F1 and / or SOX2 among skin cells, such as ectodermal lineage cells, mesenchymal cells, epidermal stem cells, keratinocytes, or the like, or to PSC having been differentiated to such lineages.

[0101] One or more cells of an assembly ectopically expressing POU5F1 and / or SOX2, or a functional isoform or variant thereof, may be transgenic and / or comprise at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof. If transgenic for and / or comprising an exogenous copy of POU5F 1 and / or SOX2, or a functional isoform or variant thereof, its genomic context within one or more cells of an assembly of differentiated cells may be at least partly responsible for ectopic expression. Without being bound by theory, during the process of reprogramming an induced pluripotent stem cell using, for example, retroviral vectors carrying the Yamanaka factors, a POU5F1 and / or SOX2 gene may have integrated into a region of chromatin that becomes accessible (e.g. open, or transcriptionally active) among differentiated cells of an assembly (e.g. follicular unit, skin organoid). Conversely, among the stem or progenitor cells from which an assembly of differentiated cells is formed or derived, the same region of chromatin may be inaccessible (e.g closed, or transcriptionally silent). Thus, such a chromosomal locus may be considered a “genomic landing pad”, characterized by open (e.g. transcriptionally active or accessible) chromatin among specific lineages of cells, which in a specific context may be one or more of ectodermal, mesenchymal, epidermal, keratinocytes, or the like, and closed (e.g. transcriptionally inactive, silenced, or inaccessible) chromatin among different lineages of cells, which in a specific context may be upstream stem or progenitor cells of the foregoing lineages (such as PSC). Accordingly, at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof, may be encoded in a genomic landing pad.

[0102] Assemblies of differentiated cells (e.g. follicular units, structures resembling follicular units, skin organoids) or dissociated cells of such assemblies (e.g. keratinocytes, keratinocyte- like cells, cells having keratinocyte-potential, and / or fibroblasts, fibroblast-like cells, cells having fibroblast potential) formed / derived / generated / expanded via methods and / or media of this disclosure may be used for therapeutic or cosmetic purposes.

[0103] In applications relating to skin grafting, such assemblies and / or populations of dissociated cells may be combined with an appropriate scaffold to prepare an in vitro-derived skin graft. Thus, in certain aspects, this disclosure may relate to methods of treating burn victims or other patients in need of treatment, or to methods of preparing skin grafts, or to uses of assemblies and / or populations of dissociated cells to treat burn victims or other patients in need of therapeutic or cosmetic intervention.

[0104] In related applications, such assemblies and / or populations of dissociated cells may be deployed or used to study the development of skin, and or the biology of various skin- related diseases. In particular, such assemblies and / or populations of dissociated cells as comprised in an in vitro-derived skin graft may be used in methods to test toxicity and / orefficacy and / or safety of compounds or other substances intended for use on the skin of subjects.

[0105] Assemblies of differentiated cells (e.g. follicular units, structures resembling follicular units, skin organoids), or hair follicles as comprised therein and extracted / isolated therefrom, of this disclosure may be used in hair transplantation methods, such as to treat conditions in patients in need thereof (e.g. androgenic alopecia), or for cosmetic hair transplantation. In particular, if transplantation in the scalp of an individual is intended, the unexpected finding of multi-shaft hair follicles may be especially advantageous due to the fact follicular units of the scalp tend to have multiple hair follicles in contrast to follicular units of other parts of the body. Thus, this disclosure contemplates methods of restoring hair, by forming / deriving and extracting follicular units in accordance with the foregoing, and transplanting a plurality of follicular units into affected areas of an individual. Injection devices for transplanting follicular units are known in the art.

[0106] In any aspect, methods of this disclosure are further advantageous because they may rely on a limitless source of cells (e.g. stem or progenitor cells, such as PSC) and are in vitro methods.Cell compositions or features

[0107] In another aspect of this disclosure is provided an assembly of differentiated cells, which may be formed / derived and / or extracted as described above. An assembly of differentiated cells may comprise a plurality of cell types, all of which or one or more of which may be in a differentiated state in comparison to the cell(s) from the assembly was derived.

[0108] As described above, an assembly of differentiated cells may derive from one or more stem or progenitor cells. Preferably, an assembly of differentiated cells is in vitro derived from one or more stem or progenitor cells, such as a PSC.

[0109] Also as described above, an assembly of differentiated cells may also derive from one or more pre-aggregated stem or progenitor cells. Preferably, an assembly of differentiated cells is pre-aggregated in vitro from one or more stem or progenitor cells, such as a PSC.

[0110] A plurality of cell types (of an assembly of differentiated cells) may correspond to more than one terminally differentiated cell types, or a mixture of terminally differentiated cell types and upstream progenitors of the terminally differentiated cell types.

[0111] In the context of an assembly of differentiated cell types that is or is comprised in a skin organoid, such as a hair-bearing skin organoid, a plurality of cell types comprised therein include one or more of, but are not limited to, epidermal stem or progenitor cells, hair follicle stem cells, mesenchymal progenitors or stem / stromal cells, cells having (epidermal)keratinocyte potential, (epidermal) keratinocytes or keratinocyte-like cells, or cells having (dermal) fibroblast potential, (dermal) fibroblasts or fibroblast-like cells.

[0112] In the context of an assembly of differentiated cell types that is or comprises a follicular unit or a structure resembling a follicular unit, a plurality of cell types comprised therein include one or more of, but are not limited to, epidermal stem or progenitor cells, hair follicle stem cells, mesenchymal progenitors or stem / stromal cells, cells having (epidermal) keratinocyte potential, (epidermal) keratinocytes or keratinocyte-like cells, or cells having (dermal) fibroblast potential, (dermal) fibroblasts or fibroblast-like cells.

[0113] If an assembly of differentiated cells is either a) a skin organoid or comprised in a skin organoid, or b) a follicular unit a structure resembling a follicular unit, or comprises a follicular unit or a structure resembling a follicular unit, it may comprise one or more hair follicle. In one embodiment, an assembly of differentiated cells comprises one hair follicle. In one embodiment, an assembly of differentiated cells comprises more than one hair follicle.

[0114] In another aspect of this disclosure are provided dissociated assemblies of differentiated cells, which may be formed / derived and dissociated as described above. Optionally, a follicular unit or one or more hair follicles thereof may be extracted prior to dissociation.

[0115] A dissociated assembly of differentiated cells may comprise a plurality of cell types, all of which or one or more of which may be in a differentiated state in comparison to the cell(s) from which the assembly was derived. A plurality of cell types (of an assembly of differentiated cells) may correspond to more than one terminally differentiated cell type, or a mixture of terminally differentiated cell types and upstream progenitors of the terminally differentiated cell types.

[0116] In the context of an assembly of differentiated cell types that is or is comprised in a skin organoid, such as a hair-bearing skin organoid, a plurality of cell types comprised therein include one or more of, but are not limited to, epidermal stem or progenitor cells, hair follicle stem cells, mesenchymal progenitors or stem / stromal cells, cells having (epidermal) keratinocyte potential, (epidermal) keratinocytes or keratinocyte-like cells, or cells having (dermal) fibroblast potential, (dermal) fibroblasts or fibroblast-like cells. In one embodiment, a first cell type of a plurality of cell types (as had been comprised in an assembly of differentiated cells prior to dissociation) is a (dermal) fibroblast or possesses a (dermal) fibroblast potential (e.g. (dermal) mesenchymal cell or mesenchymal-like cell). In the same or different embodiment, a second cell type of a plurality of cell types (as had been comprised in an assembly of differentiated cells prior to dissociation) is a (epidermal) keratinocyte or possesses a (epidermal) keratinocyte potential. In a specific embodiment, a first cell type ofa plurality of cell types (as had been comprised in an assembly of differentiated cells prior to dissociation) is a (dermal) fibroblast or possesses a (dermal) fibroblast potential (e.g. a (dermal) mesenchymal cell or mesenchymal-like cell), and a second cell type thereof is a (epidermal) keratinocyte or possesses a (epidermal) keratinocyte potential.

[0117] In the context of an assembly of differentiated cell types that is or comprises a follicular unit or a structure resembling a follicular unit, a plurality of cell types comprised therein include one or more of, but are not limited to, epidermal stem or progenitor cells, hair follicle stem cells, mesenchymal progenitors or stem / stromal cells, cells having (epidermal) keratinocyte potential, (epidermal) keratinocytes or keratinocyte-like cells, or cells having (dermal) fibroblast potential, (dermal) fibroblasts or fibroblast-like cells. In one embodiment, a first cell type of a plurality of cell types (as had been comprised in an assembly of differentiated cells prior to dissociation) is a (dermal) fibroblast or possesses a (dermal) fibroblast potential (e.g. (dermal) mesenchymal cell or mesenchymal-like cell). In the same or different embodiment, a second cell type of a plurality of cell types (as had been comprised in an assembly of differentiated cells prior to dissociation) is a (epidermal) keratinocyte or possesses a (epidermal) keratinocyte potential. In a specific embodiment, a first cell type of a plurality of cell types (as had been comprised in an assembly of differentiated cells prior to dissociation) is a (dermal) fibroblast or possesses a (dermal) fibroblast potential (e.g. (dermal) mesenchymal cell or mesenchymal-like cell), and a second cell type thereof is a (epidermal) keratinocyte or possesses a (epidermal) keratinocyte potential.

[0118] As described above, an assembly of differentiated cells may derive from one or more stem or progenitor cells. Preferably, an assembly of differentiated cells is in vitro derived from one or more stem cells, such as a PSC.

[0119] Also as described above, an assembly of differentiated cells may also derive from one or more pre-aggregated stem or progenitor cells. Preferably, an assembly of differentiated cells is pre-aggregated in vitro from one or more stem cells, such as a PSC.

[0120] If an assembly of differentiated cells is either a) a skin organoid or comprised in a skin organoid, or b) a follicular unit a structure resembling a follicular unit, or comprises a follicular unit or a structure resembling a follicular unit, it may comprise one or more hair follicle. In one embodiment, an assembly of differentiated cells comprises one hair follicle. In one embodiment, an assembly of differentiated cells comprises more than one hair follicle.

[0121] In any aspect, one or more cells of an assembly of differentiated cells or a dissociated assembly of differentiated cells may ectopically express POU5F1 and / or SOX2, or a functional isoform or variant thereof. Such one or more cells may be transgenic, such as to encode a direct or indirect transcription regulator of POU5F1 and / or SOX2, or to comprise at least oneexogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof. If an assembly of differentiated cells arises out of one or more cell ectopically expressing POU5F1 and / or SOX2, or a functional isoform or variant thereof, then such assembly may be stem cell (e.g. PSC)-derived.

[0122] Unexpectedly it was discovered that expression levels of POU5F1 and / or SOX2, or a functional isoform or variant thereof, may influence whether assemblies comprise one hair shaft / follicle or a plurality of hair shafts / follicles. Stated another way, ectopic expression of POU5F 1 and / or SOX2, or a functional isoform or variant thereof, may influence whether or not an assembly of differentiated cells (e.g. follicular units, structures resembling follicular units, skin organoids) comprises one hair shaft / follicle or a plurality of hair shafts / follicles.

[0123] If an assembly (e.g. aggregate) of differentiated cells comprises or is derived from one or more cells not ectopically expressing POU5F1 and / or SOX2, or a functional isoform or variant thereof, it should typically be expected to comprise one hair shaft / follicle. Thus, an expression level of POU5F1 and / or SOX2, or a functional isoform or variant thereof, among cells of such assemblies may be comparable to background expression levels of POU5F1 and / or SOX2 among cells of the skin, such as ectodermal lineage cells, (dermal) mesenchymal cells, epidermal stem cells, keratinocytes, dermal fibroblasts, or the like, or to PSC-derived analogs / variants thereof.

[0124] If an assembly (e.g. aggregate) of differentiated cells comprises or is derived from one or more cells ectopically expressing POU5F1 and / or SOX2, or a functional isoform or variant thereof, it should typically be expected to comprise more than one hair shaft / follicle. Thus, an expression level of POU5F1 and / or SOX2, or a functional isoform or variant thereof, among cells of such assemblies may be higher than background expression levels of POU5F1 and / or SOX2 among cells of the skin, such as ectodermal lineage cells, (dermal) mesenchymal cells, epidermal stem cells, keratinocytes, dermal fibroblasts, or the like, or to PSC-derived analogs / variants thereof.

[0125] One or more cells of an assembly or a dissociated assembly ectopically expressing POU5F1 and / or SOX2, or a functional isoform or variant thereof, may be transgenic and / or comprise at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof. If transgenic for and / or comprising an exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof, its genomic context within one or more cells of an assembly of differentiated cells may be at least partly responsible for ectopic expression. Without being bound by theory, during the process of reprogramming an induced pluripotent stem cell using, for example, retroviral vectors carrying the Yamanaka factors, a POU5F1 and / or SOX2 gene may have integrated into a region of chromatin that is accessible (e.g.open, or transcriptionally active) among differentiated cells of an assembly (e.g. follicular unit, skin organoid). Conversely, among the stem or progenitor cells from which an assembly of differentiated cells is formed or derived, the same region of chromatin may be inaccessible (e.g closed, or transcriptionally silent). Thus, such a chromosomal locus may be considered a “genomic landing pad”, characterized by open (e.g. transcriptionally active or accessible) chromatin among specific lineages of cells, which in a specific context may be one or more of ectodermal, (dermal) mesenchymal, epidermal, keratinocytes, or the like, and closed (e.g. transcriptionally inactive, silenced, or inaccessible) chromatin among different lineages of cells, which in a specific context may be upstream stem or progenitor cells of the foregoing lineages (such as PSC). Accordingly, at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof, may be encoded in a genomic landing pad.

[0126] Thus, in another aspect of this disclosure is provided one or more genomic landing pad, preferably in the human genome (and any syntenic region of a non-human genome). References to the human genome herein regard assembly hg38 (UCSC Genome Browser).

[0127] A genomic landing pad may comprise an exogenous polynucleotide, which polynucleotide may be coding or non-coding, and further comprises at minimum the necessary regulatory sequences to transcribe an associated coding or non-coding transcript. Thus, an exogenous polynucleotide may comprise at least one transcriptional unit.

[0128] An at least one transcriptional unit of an exogenous polynucleotide in a genomic landing pad may encode a peptide, polypeptide, or a protein. Such at least one transcriptional unit may encode a transcriptional regulator, such as a transcriptional activator or repressor. In one embodiment, a transcriptional regulator may be a transcription factor or a transcription co-factor. In one embodiment, an at least one transcriptional unit encodes a post- transcriptional regulator, such as a complementary RNA (e.g. siRNA, miRNA, piRNA, or the like).

[0129] In the context of this disclosure, a genomic landing pad may comprise an exogenous polynucleotide that encodes POU5F1 and / or SOX2, or a functional isoform or variant thereof, or encodes a factor that activates endogenous POU5F1 and / or SOX2 expression. However, it is stressed that such exogenous polynucleotide is not limited to encoding POU5F1 and / or SOX2 or a factor that activates endogenous POU5F1 and / or SOX2 expression. Rather, a genomic landing pad of this disclosure could encode for any transcriptional unit that is desirably expressed among cell types in which the landing pad is in a transcriptionally accessible state (e.g. open or active chromatin) rather than in a transcriptionally inaccessible sate (e.g. closed or silenced chromatin).

[0130] It has been unexpectedly discovered that one or more genomic landing pad located on chromosome 2q37.3, chromosome 14q24.3, or chromosome 16q23.2 of the human genome (hg38) are in a transcriptionally active state among cells of an assembly of differentiated cells (formed / derived as described herein) and / or of dissociated assemblies of differentiated cells (formed / derived and dissociated as described herein). Thus, exogenous polynucleotides introduced into one or more of the foregoing genomic landing pads are transcribed among cells of an assembly of differentiated cells (formed / derived as described herein) and / or of dissociated assemblies of differentiated cells (formed / derived and dissociated as described herein), while the same exogenous polynucleotides are not transcribed among cells from which the assemblies of differentiated cells are derived (e.g. stem cells, such as PSC).

[0131] Regarding the putative genomic landing pad on chromosome 2q37.3, it may more precisely be located to the 4thintron of the COPS8 gene. Still more precisely, such genomic landing pad may located to Chr2:237,092, 171 -237,092,774 ± 100 bp, or ± 200 bp, or ± 300 bp, or ± 400 bp, or ± 500 bp, or ± 600 bp, or ± 700 bp, or ± 800 bp, or ± 900 bp, or ± 1 kb, or ± 1.5 kb, or ± 2 kb.

[0132] Regarding the putative genomic landing pad on chromosome 14q24.3, it may more precisely be located to ~30 kb from the gene PGF toward the centromere, and ~15 kb from the gene EIF2B2 toward the telomere. Still more precisely, such genomic landing pad may located to Chr14:74988538-74989141 ± 100 bp, or ± 200 bp, or ± 300 bp, or ± 400 bp, or ± 500 bp, or ± 600 bp, or ± 700 bp, or ± 800 bp, or ± 900 bp, or ± 1 kb, or ± 1 .5 kb, or ± 2 kb, or ± 2.5 kb, or ± 3 kb, or ± 3 kb, or ± 3.5 kb, or ± 4 kb, or ± 4.5 kb, or ± 5 kb.

[0133] Regarding the putative genomic landing pad on chromosome 16q23.2, it may more precisely be located to ~80 kb from the gene ARLNC1 toward the centromere, and ~15 kb from the gene CMC2 toward the telomere. Still more precisely, such genomic landing pad may located to Chr16:80935502-80936106 ± 100 bp, or ± 200 bp, or ± 300 bp, or ± 400 bp, or ± 500 bp, or ± 600 bp, or ± 700 bp, or ± 800 bp, or ± 900 bp, or ± 1 kb, or ± 1 .5 kb, or ± 2 kb, or ± 2.5 kb, or ± 3 kb, or ± 3 kb, or ± 3.5 kb, or ± 4 kb, or ± 4.5 kb, or ± 5 kb.

[0134] A genomic landing pad of this disclosure may be transcriptionally silent in a stem or progenitor cell and / or transcriptionally active in a cell differentiated from such stem or progenitor cell. In one embodiment, a stem or progenitor cell is an undifferentiated stem cell, preferably a PSC. In one embodiment, a cell differentiated from a stem or progenitor cells is an ectodermal lineage cell, or a mesodermal lineage cell, or an endodermal lineage cell. Preferably, a cell differentiated from a stem or progenitor cells is an ectodermal lineage cell or a mesodermal lineage cell, in which case a cell differentiated from a stem or progenitor cellsis epidermal, mesenchymal, epithelial, a (epidermal) keratinocyte or progenitor thereof, or a (dermal) fibroblast or a progenitor thereof.

[0135] Interestingly, the putative genomic landings pads on chromosomes 14 and 16 are localized to repeat-rich regions of the genome, and the integration site of the exogenous polynucleotide be located within or within about 100 bp of a fossil LTR and / or LINE sequence. In contrast, the putative landing pad on chromosome 2 is within an intron of a widely- expressed gene, and possibly a constitutively expressed gene.

[0136] In a specific embodiment, an assembly of differentiated cells is or comprises a follicular unit (formed / derived and / or extracted as described above), and such assembly comprises one or more of: a plurality of cell types among the assembly of differentiated cells; one or more cells of the assembly ectopically expressing POU5F 1 and / or SOX2, or a functional isoform or variant thereof; and one or more hair follicle.

[0137] Thus, if such an assembly comprises one hair shaft / follicle, it likely does not comprise one or more cells ectopically expressing POU5F1 and / or SOX2. Accordingly, an expression level of POU5F1 and / or SOX2 among cells of such an assembly cells should be comparable to background levels of POU5F1 and / or SOX2 expressed by applicable cell types (e.g. cells of the skin and / or hair follicles, or stem cells differentiated to such fate(s)).

[0138] Or, if such as an assembly comprises a plurality of hair s haft / fol I icl es, it likely comprises one or more cells ectopically expressing POU5F1 and / or SOX2. Accordingly, an expression level of POU5F1 and / or SOX2, or a functional isoform or variant thereof, among cells of such an assembly cells should be higher than background levels of POU5F1 expressed by applicable cell types (e.g. cells of the skin and / or hair follicles, or stem cells differentiated to such fate(s)).

[0139] In one specific embodiment, a dissociated assembly of differentiated cells which comprised or comprises a follicular unit, the assembly comprising: a plurality of cell types including at least i) a first cell type that is a (dermal) fibroblast, or has (dermal) fibroblast potential (e.g. (dermal mesenchymal cell), and / or ii) a second type of cell that is a (epidermal) keratinocyte or has (epidermal) keratinocyte potential, wherein one or more cells of the dissociated assembly ectopically express POU5F1 and / or SOX2, or a functional isoform or variant thereof.

[0140] The following non-limiting examples are illustrative of the present disclosure.ExamplesExample 1: Maintaining pluripotent stem cells

[0141] Pluripotent stem cells (PSCs) were maintained in PSC maintenance media (mTeSRI™, TeSRTM-E8TM, mTeSR™ Plus, or another TeSRTM-branded medium) (STEMCELL Technologies) and passaged in accordance with the manufacturer’s recommended maintenance culture and passaging protocols. Cultures were dissociated using standard reagents such as ReLeSR™ (STEMCELL Technologies) and Accutase™ (STEMCELL Technologies). Following, between 1000-3500 PSCs were aggregated in each microwell of an AggreWell™ microwell device (STEMCELL Technologies), such as an AggreWell™ 800 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 mTeSRI™, TeSR™-E8™, or mTeSR™ Plus (STEMCELL Technologies) to form aggregates.Example 2: Forming skin organoids

[0142] The aggregates of Example 1 were cultured under non-adherent conditions in an AggreWell™ or a 96-well plate for about 12 days in a skin organoid induction medium, such as STEMdiff™ Skin Organoid Induction Medium (STEMCELL Technologies). Following, the developing skin organoids were transferred into STEMdiff™ Skin Organoid Maturation Medium (STEMCELL Technologies) for about 6 weeks or longer, adding or changing the medium every 3-4 days.

[0143] Figure 1A shows a representative image of a hair-bearing skin organoid formed as described in Examples 1 and 2. Skin organoids may be considered mature when hair follicles or clusters of hair follicles are observed on an outer surface thereof pointing toward the cell culture medium, as can be seen in Figure 1A.Example 3: Forming assemblies of differentiated cells comprising hair follicles

[0144] Assemblies of differentiated cells, comprising one or more hair follicles (e.g. follicular units) arise within the skin organoids, were formed as described in Example 2, and dissected / excised using fine-tipped forceps. The isolated assemblies were placed in an appropriate culture medium, such as STEMdiff™ Skin Organoid Maturation Medium, and cultured for at least 9 days. Hair follicles of such dissected assemblies were observed to lengthen when cultured in the appropriate culture medium for a period of more than one day (data not shown).

[0145] Figure 1 B shows the in vitro development of an assembly of differentiated cells derived from a control stem cell line comprising only endogenous copies of POU5F1 and SOX2 (PSCCTRL), revealing that such presumptive follicular units exhibit a characteristic dermal sheath, a single inverted cup, and a single hair shaft emerging therefrom (see white arrow heads). In contrast, an assembly of differentiated cells derived from an iPSC stem cell line additionally comprising at least one exogenous copy of POU5F1 and SOX2 (PSCP0UF51)exhibited presumptive follicular units having a characteristic dermal sheath, a single inverted cup and a single hair shaft emerging therefrom on day 1 in culture (see white arrow heads) (Figure 1C). However, as culture progressed, a bulge (while visible at day 1) developed into a structure resembling a second follicular unit fused with the initial structure. By day 5 it is apparent that the arising structure comprises a second inverted cup and a second hair shaft. This observation was only observed among assemblies of differentiated cells comprising hair follicles (e.g. follicular units) dissected from PSCP0UF51-derived skin organoids, and not among follicular units dissected from PSCCTRL-derived skin organoids.Example 4: Characterizing assemblies of differentiated cells comprising multiple hair follicles

[0146] As described in Example 3, assemblies of differentiated cells comprising one or more hair follicle could be formed and dissected, and the nature of the arising assemblies differed depending on the stem cell line from which they were derived. Thus, the genomes of the parental cell lines were assessed by sequencing and PCR, and it was confirmed that PSCCTRLdid not include an exogenous copy of POU5F1 and SOX2 (data not shown) while pscP0UF51included at least two copies of exogenous POU5F1 and one exogenous copy of SOX2. PCR using gDNA isolated from pscP0UF51confirmed the integration sites of POU5F1 and SOX2 expression constructs, used for the initial reprogramming to an iPSC: a first site located on chr2q37.3 (SOX2 integration), a second site located on chr14q24.3 (POU5F1 integration), and a third site located on chr16q23.2 (POU5F1 integration) of the hg38 reference genome assembly (USCS Genome Browser) (Figure 2A) and (Figure 2B).

[0147] Assessment of the first POU5F1 integration site (chr14q24.3) using the tools available on the UCSC Genome Browser revealed that it lies ~30 kb from the gene PGF toward the centromere and ~15 kb from the gene EIF2B2 toward the telomere. No RefSeq or predicted transcripts appeared to overlap the integration site. However, the integration site appeared to be within or nearby a fossil LTR and LINE sequence (Figure 2A). Further analysis revealed that the integration site is associated with a transcriptionally active chromatin signature in keratinocytes, NHEK (data not shown).

[0148] Assessment of the second POU5F1 integration site (chr16q23.2) using the same tools available on the UCSC Genome Browser revealed that it lies ~80 kb from the gene ARLNC1 toward the centromere and ~15 kb from the gene CMC2 toward the telomere. No RefSeq or predicted transcripts appeared to overlap the integration site. However, the integration site appeared to be within or nearby fossil LTR and SINE sequence (Figure 2B). Further analysis did not identify that the integration site is associated with a transcriptionally active chromatin signature in any assessed cell type (data not shown).

[0149] Assessment of the SOX2 integration site (chr2q37.3) using the tools available on the UCSC Genome Browser revealed that it lies within the 4thintron of the COPS8 gene, which appeared to be widely transcribed across a number of diverse tissues and potentially constitutively expressed (Figure 2C). Further analysis revealed that the integration site is associated with a transcriptionally active chromatin signature in keratinocytes, NHEK (data not shown).

[0150] RNAseq was undertaken to investigate if POU5F1 and / or SOX2 is expressed among various PSC lines, including PSCCTRLand PSCP0U5F1, and various cell populations differentiated therefrom. Interestingly, it was shown that among four different PSC lines used to derive skin organoids, only the assemblies of differentiated cells comprising one or more hair follicles derived from PSCP0U5F1exhibited ectopic expression of POU5F1 and SOX2 (Figure 2D) and not in PSCCTRL. Ectopic expression of POU5F1 and SOX2 in only PSCP0U5F1was also confirmed against four control keratinocyte cell populations (neonatal keratinocytes: HEKn3 and HPEK, and adult keratinocytes: HEKa2 and HPEKa). Figure 2D also confirms that populations of differentiated cells from all 4 PSC lines, including pscP0U5F1, exhibited comparable expression levels of a different reprogramming factor, KLF4, and also a gene known to be expressed among cells of the skin, KRT14. Likewise, expression of these genes was comparable among the four control keratinocyte populations.

[0151] On the basis of two SNPs that distinguish the exogenous copies of POU5F1 from the endogenous copies of PSCP0U5F1, RNAseq further confirmed that among undifferentiated PSCP0U5F1only the endogenous copies of POU5F1 were expressed (Figure 2E), while among cells isolated from PSCP0U5F1-derived assemblies of differentiated cells comprising one or more hair follicles only one or both exogenous copies of POU5F1 were expressed (Figure 2F). While SOX2 did not encode any SNPs that could distinguish between exogenous and endogenous copies, analysis of RNA seq data confirmed that undifferentiated cells expressed SOX2 transcripts comprising both UTRs and the coding region, while among cells of the differentiated assemblies, sequence reads only aligned to the coding region of SOX2 and not its UTRs (Figure 2G) suggesting that only the exogenous copy of SOX2 was materially contributing to SOX2 expression.

[0152] Overall, ectopic expression of POU5F1 correlates with the phenomenon of formation of multi-hair follicular units, and such ectopic expression may be a consequence of the genomic locus into which this reprogramming factor has inserted.Example 5: Plating dissociated cells of in vitro-derived cell assemblies under adherent conditions

[0153] After the skin organoids of Example 2 have matured, preferably presenting as hairbearing skin organoids (e.g. hair placode present skin organoids), they may be dissociated overnight at 37 °C in Dispase™ (STEMCELL Technologies). Dissociated organoids are plated as a monolayer into a tissue culture treated culture plate or flask at an appropriate density, such as one to X dissociated organoid per well of a 6-well plate up to a T25 flask. The cell culture medium may be selected as appropriate for the downstream application.

[0154] If intending to culture (epidermal) keratinocytes from such a dissociated assembly of differentiated cells, DermaCult™ (STEMCELL Technologies), or another appropriate, medium may be used. After initially seeding the differentiated population of PSCs in DermaCult™, colonies of keratinocytes may emerge in as few as about 3 days (Figure 3). If intending to culture (dermal) fibroblasts from such a dissociated assembly of differentiated cells, a MesenCult™-branded (STEMCELL Technologies), or another appropriate, medium may be used. After initially seeding the differentiated population of PSCs in a MesenCult™-branded medium, (dermal) mesenchymal cells emerge as sparse cells before stretching out over the culture substrate (Figure 3).

[0155] Thus, the foregoing describes an improved way to generate PSC-derived (epidermal) keratinocytes, or keratinocyte-like cells, and / or (dermal) fibroblasts, or fibroblast-like cells, and highlights the unexpected discovery that a dissociated assembly of differentiated cells (e.g. of a dissociated skin organoid) provides a surprisingly effective starting point for expanding relatively pure cultures of PSC-derived keratinocytes and / or PSC-derived (dermal) fibroblasts.

Claims

CLAIMS1) An assembly of differentiated cells, comprising: a) a plurality of cell types among the assembly of differentiated cells; b) one or more cells of the assembly ectopically expressing POU5F1 and / or SOX2, or a functional isoform or variant thereof; and c) one or more hair follicle.2) The assembly of differentiated cells of claim 1 , wherein the assembly of differentiated cells: a) is comprised in a skin organoid, preferably a hair-bearing skin organoid; and / or b) is or comprises a follicular unit, preferably comprising more than one hair follicle.3) The assembly of differentiated cells of claim 1 or 2, wherein the one or more cells ectopically expressing POU5F1 and / or SOX2, or the functional isoform or variant thereof: a) are transgenic; and / or b) comprise at least one exogenous copy of POU5F1 and / or SOX2, or the functional isoform or variant thereof; and / or c) are derived from a stem or progenitor cell, preferably a pluripotent stem cell.4) The assembly of differentiated cells of claim 3, wherein the at least one exogenous copy of POU5F1 and / or SOX2, or the functional isoform or variant thereof is encoded in a genomic landing pad.5) A method of deriving an assembly of differentiated cells, comprising a) providing one or more stem or progenitor cells in a differentiating culture environment; and b) exposing the one or more stem or progenitor cells to the culture environment for a sufficient period of time to derive the assembly of differentiated cells comprising a plurality of cell types, wherein one or more cells of the assembly ectopically express POU5F1 and / or SOX2, or a functional isoform or variant thereof, and wherein the assembly comprises one or more hair follicle.6) The method of claim 5, wherein the assembly of differentiated cells: a) is comprised in a skin organoid, preferably a hair-bearing skin organoid; and / or b) is or comprises a follicular unit, preferably comprising more than one hair follicle; and / or c) is derived from a pluripotent stem cell.7) The method of claim 5 or 6, wherein the culture environment is serum-free.8) The method of any one of claims 5 to 7, wherein the one or more cells ectopically expressing POU5F1 or a functional isoform or variant thereof:a) are transgenic; and / or b) comprise at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof.9) The method of claim 8, wherein the at least one exogenous copy of POU5F 1 and / or SOX2, or a functional isoform or variant thereof is encoded in a genomic landing pad.10) A dissociated population of cells of an assembly of differentiated cells, the dissociated population of cells comprising a plurality of cell types, wherein a first cell type has mesenchymal potential and a second cell type has keratinocyte potential.11) The dissociated population of cells according to claim 10, wherein the assembly is a skin organoid, preferably a hair-bearing skin organoid.12) The dissociated population of cells according to claim 10 or 11 , wherein at least one follicular unit is comprised in the assembly of differentiated cells, and the at least one follicular unit comprises one or more hair follicle.13) The dissociated population of cells according to any one of claim 10 to 12, wherein one or more cells of the dissociated assembly ectopically express POU5F1 and / or SOX2, or a functional isoform or variant thereof.14) The dissociated population of cells according to claim 13, wherein the at least one exogenous copy of POU5F 1 and / or SOX2, or a functional isoform or variant thereof is encoded in a genomic landing pad.15) A method of generating a population of differentiated cell types having one or both mesenchymal and keratinocyte potential, the method comprising: a) providing a starting population of stem or progenitor cells, preferably pluripotent stem cells; b) deriving an assembly of differentiated cells from the starting population of stem or progenitor cells in a differentiating culture environment, the assembly of differentiated cells comprising a plurality of differentiated cell types; and c) dissociating the assembly of differentiated cells to yield at least a first cell type having mesenchymal potential and a second cell type having keratinocyte potential, thereby generating a population of differentiated cell types.16) The method of claim 15, further comprising culturing at least a portion of the population of differentiated cell types in conditions supportive of mesenchymal cells or keratinocytes.17) The method of claim 15 or 16, wherein the assembly of cells is a skin organoid, preferably a hair-bearing skin organoid.18) The method of any one of claims 15 to 17, wherein one or more cells of i) the assembly, ii) the population of differentiated cell types, or iii) both i) and ii) ectopically express POU5F1 and / or S0X2, or a functional isoform or variant thereof.19) The method of claim 18, wherein the starting population of stem or progenitor cells comprise at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof.20) The method of claim 19, wherein the at least one exogenous copy of POU5F1 and / or SOX2, or a functional isoform or variant thereof is encoded in a genomic landing pad.21) A genomic landing pad comprising an exogenous polynucleotide, wherein the genomic landing pad is located on chromosome 2q37.3, 14q24.3 or on chromosome 16q23.2.22) The genomic landing pad of claim 21 , wherein the exogenous polynucleotide comprises at least one transcriptional unit.23) The genomic landing pad of claim 21 or 22, wherein the at least one transcriptional unit comprises an open reading frame encoding: a) a peptide, polypeptide, or a protein; and / or b) a transcription regulator.24) The genomic landing pad of claim 23, wherein the transcription regulator is POU5F1 and / or SOX2, or a functional isoform or variant thereof.25) The genomic landing pad of any one of claims 21 to 24, wherein the genomic landing pad is: a) transcriptionally silent in a stem or progenitor cell; and / or b) transcriptionally active in a cell differentiated from the stem or progenitor cell.26) The genomic landing pad of claim 25, wherein the stem or progenitor cell is an undifferentiated stem or progenitor cell, preferably a pluripotent stem cell.27) The genomic landing pad of claim 25 or 26, wherein the cell differentiated from the stem or progenitor cell is: a) ectodermal, endodermal, or mesodermal; or b) dermal, mesenchymal, epithelial, or a keratinocyte.

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