METHODS FOR DERIVING AUTOLOGOUS AND HYPOIMMUNOGENIC HAIR FOLLICLE CONTAINING SHEETS IN VITRO

Abstract

The present disclosure relates to a bioengineering process to derive hair follicles in vitro from the in vitro disposition and differentiation of autologous pluripotent stem cells and dermal papilla stem cells. The present disclosure also relates to the in vitro bioengineering of hypoimmunogenic hair follicles from allogenic pluripotent stem cells and dermal papilla stem cells. The present disclosure also relates to bioengineering of autologous and allogenic hypoimmunogenic hair follicles and hair follicle containing sheets with asymmetric disposition of hair shafts. The present disclosure also relates to a bioengineering process to derive hair follicle containing sheets in vitro from a biodegradable supportive grid and said in vitro derived hair follicles. The present disclosure also relates to the controlled asymmetry of the hair shaft on said hair follicle containing sheets. The present disclosure also relates to the field of cosmetic materials and method for reconstructing hair follicle containing materials in vitro.

Claims

1. A bioengineered hair follicle comprising: one or more bioengineered dermal papillae stem cell-induced and pluripotent stem cell-derived hair follicles, and containing hair bulge, hair shaft, and protruding hair filament, also a cell-composed cortex containing stem cell-derived synthetic interfollicular epidermis, and intertwining extracellular matrix.

2. The dermal papillae stem cell of claim 1, wherein the cell is preferably a dermal papillae stem cell, or a dermal papillae stem cell line, and said cell is preferably genetically modified for unlimited expansion, or immortalized, or engineered to over-express extracellular matrix proteins or attachment proteins, or genetically engendered to eliminate immunogenicity and to avoid allogenic transplant rejection, or combinations of the above.

3. The extracellular matrix of claim 1, wherein the matrix is preferably composed of combinations of laminin, fibronectin, collagen, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, elastin, integrin, cadherin, selectin, connexins, claudins, occludins, and or chemically modified extracellular matrix proteins.

4. The bioengineered hair follicle and synthetic interfollicular epidermis of claim 1, (a) wherein the bioengineered hair follicle and synthetic interfollicular epidermis are derived from stem cells; wherein a stem cell may preferably be embryonic stem cells or induced pluripotent stem cell, with induced pluripotent stem cell being preferred; or (b) wherein the bioengineered hair follicle and synthetic interfollicular epidermis cell is preferably a genetically modified cell, wherein the modification results in its unlimited expansion, or immortalization, or constitutive expression, or inducible expression, or permanently encoding expression, or excisable encoding expression of telomerase, Ras, Abl, Akap13, Araf, Tim, Atf, Axl, Bcl, Braf, Brca, Brip, Cbl, Csflr, Dapk, Dek, Dusp, Egf, Egfr, Erbb, Erg, Ets, Ewsr, Fes, Fgf, Fgfr, Flcn, Fos, Frap, Fus, Hras, Gli, Gpc, Neu, Hgf, Irf, Junb, Kit, Kras, Lck, Lco, Mapk, Mcf, Mdm2, Met, Mlh, Mmd, Mos, Mras, Msh, Myb, Myc, Lmyc, Nmyc, Ele1, Nf1, Trk, Can, Ovc, Tp53, Palb2, Pax3, Pdgfb, Pim, Pml, Pms, Wip, Pten, Pvt, Raf, Craf, Rb, Rras, Mcf, Smad, Smurf, Src, Stat, Tdgf, Tgfbr, Erba, Tgf, Tif, Tnc, Trk, Tusc, Usp, Wnt, Wt, Vhl, and with excisability of coding sequence preferred, or genetically engendered to eliminate immunogenicity and to avoid allogenic transplant rejection, or combinations of the above.

5. (canceled)

6. The bioengineered hair follicle of claim 1, wherein (a) the bioengineered hair follicle is derived from dermal papillae stem cells; wherein a dermal papillae stem cell may preferably be Sox2-positive, Sox9-positive, Nestin-positive, P63-positive, CD133-positive, AP-positive, alpha-SMA-positive or combinations of the above; or (b) during manufacturing are preferably integrated and inserted into a biodegradable grid sheet that forces and facilitates the asymmetric positioning of protruding hair filaments to only one side of the biodegradable grid sheet.

7. The bioengineered hair follicle-producing dermal papillae stem cell of claim 1 is preferably a genetically modified cell, wherein the modification results in its unlimited expansion, or immortalization, or constitutive or inducible, or permanently encoding or excisable encoding expression of telomerase, Ras, Abl, Akap13, Araf, Tim, Atf, Axl, Bcl, Braf, Brea, Brip, Cbl, Csflr, Dapk, Dek, Dusp, Egf, Egfr, Erbb, Erg, Ets, Ewsr, Fes, Fgf, Fgfr, Flcn, Fos, Frap, Fus, Hras, Gli, Gpc, Neu, Hgf, Irf, Junb, Kit, Kras, Lck, Lco, Mapk, Mcf, Mdm2, Met, Mlh, Mmd, Mos, Mras, Msh, Myb, Myc, Lmyc, Nmyc, Ele1, Nf1, Trk, Can, Ovc, Tp53, Palb2, Pax3, Pdgfb, Pim, Pml, Pms, Wip, Pten, Pvt, Raf, Craf, Rb, Rras, Mcf, Smad, Smurf, Src, Stat, Tdgf, Tgfbr, Erba, Tgf, Tif, Tnc, Trk, Tusc, Usp, Wnt, Wt, Vhl, or combinations of the above, and with excisability of coding sequence preferred.

8. The synthetic interfollicular epidermis of claim 1, wherein the synthetic interfollicular epidermis is composed of one or more cell types; wherein a synthetic interfollicular epidermis composed cellular cortex may preferably be composed of keratinocytes, basal cells, spinous cells, granular cells, cornified cells, or combinations of the above.

9. The dermal papillae stem cells and stem cells of claim 1 and during manufacturing are preferably homogeneously distributed within the cellular aggregate; wherein said cells are encapsulated or intertwined in extracellular matrix, wherein the extracellular matrix is preferably composed of combinations of laminin, fibronectin, collagen, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, elastin, integrin, cadherin, selectin, connexins, claudins, occludins, and or chemically modified extracellular matrix proteins.

10. (canceled)

11. The biodegradable grid sheet of claim 6 composed of (a) biodegradable substrates blend, wherein said substrates blend components is selected from polyglycolic acid, polyacetic acid, epsilon-caprolactones, polydioxanones, lactides, hydrogels, or combinations of the above, or (b) one or more cavities that support and contain the bioengineered hair follicle comprising: one or more bioengineered dermal papillae stem cell-induced and pluripotent stem cell-derived hair follicles, and containing hair bulge, hair shaft, and protruding hair filament.

12. (canceled)

13. The cellular aggregates of claim 9, generated in a media blend, wherein said media blend components is selected from Calcium chloride, Ferric nitrate, Magnesium sulfate, Potassium chloride, Sodium bicarbonate, Sodium chloride, Sodium phosphate monobasic, L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, Choline chloride, Folic Acid, myo-Inositol, Niacinamide, D-Pantothenic Acid, Pyridoxal, Pyridoxine, Riboflavin, Thiamine, D-Glucose, Pyruvic Acid, L-Glutamine, L-proline, L-hydroxyproline, reduced glutathione, ascorbic acid, Iron saturated Transferrin, Insulin, albumin, L-alanine, L-asparagine, L-aspartate, L-glutamate, beta-mercaptoethanol, L-alanyl-glutamine, Y-27632, Rho-associated coiled-coil containing protein kinase inhibitor, or combinations of the above and/or during manufacturing, is generated in liquid by the combination of dermal papillae stem cells, stem cells and extracellular matrix.

14. The bioengineered hair follicle of claim 1 is preformed by and through a culture period within seven days and ninety days.

15. The bioengineered hair follicles containing sheet of claim 10 and during manufacturing, is generated by culturing the contained bioengineered hair follicles within a period of seven days and ninety days.

16. (canceled)

17. The cellular aggregates of claim 9 are matured in a media blend to facilitate the patterning and paracrine signalling between dermal papillae stem cells and stem cells, wherein said media blend components is selected from Calcium chloride, Cupric sulfate, Ferrous sulfate, Magnesium chloride, Potassium chloride, Sodium bicarbonate, Sodium chloride, Sodium phosphate dibasic, Zinc Sulfate, L-alanine, L-asparagine, L-arginine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, D-biotin, Choline chloride, Folic acid, myo-Inositol, Niacinamide, D-Pantothenic Acid, Pyridoxine, Riboflavin, Thiamine, Vitamin B12, D-Glucose, Hypoxanthine, Linoleic acid, Putrescine, Pyruvic acid, Thioctic acid, Thymidine, Sodium bicarbonate, Magnesium sulfate, Potassium nitrate, Sodium phosphate monobasic, Sodium selenite, HEPES, Arachidonic acid, Cholesterol, DL-alpha-tocopherol acetate, Ethyl alcohol, Linoleic acid, Linolenic acid, Myristic acid, Oleic acid, Palmitic acid, Palmitoleic acid, Polyoxyethylene-polyoxypropylene copolymer, Stearic acid, Tween, albumin, insulin, transferrin, 1-thioglycerol, FGF2, BMP4, 4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide, 4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1.Math.5-a]pyrimidin-3-yl]-quinoline, dorsomorphin, L-alanyl-glutamine, extracellular matrix, or combinations of the above.

18. The bioengineered hair follicles of claim 1 are derived in a media blend to facilitate the maturation of cellular aggregates, patterning and paracrine signalling between dermal papillae stem cells and stem cells and maturation of hair follicles, wherein said media blend components is selected from Ammonium molybdate, Ammonium metavanadate, Cupric sulfate, Ferrous sulfate, Manganese sulfate, Magnesium sulfate, Nickel chloride, Sodium metasilicate, Sodium selenite, Sodium phosphate dibasic, Stannous chloride, L-aspartic acid, L-glutamic acid, L-glutamine, Calcium chloride, Ferric nitrate, Magnesium chloride, Potassium chloride, Sodium bicarbonate, Sodium chloride, Sodium phosphate monobasic, Zinc sulfate, L-alanine, L-asparagine, L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, Choline chloride, Folic Acid, Niacinamide, D-Pantothenic Acid, Pyridoxal, Riboflavin, Thiamine, Vitamin B12, inositol, D-Glucose, HEPES, Pyruvic acid, D-biotin, myo-Inositol, Hypoxanthine, Linoleic acid, Putrescine, DL-Thioctic Acid, Thymidine, Transferrin, Insulin, Progesterone, Catalase, reduced glutathione, Superoxide dismutase, T3, L-carnitine, Ethanolamine, D+-galactose, Corticosterone, Linolenic acid, DL alpha tocopherol, DL alpha tocopherol acetate, Oleic acid, Pipecolic acid, albumin, L-alanyl-glutamine, beta-mercaptoethanol, FGF2, BMP4, 4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide, 4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline, dorsomorphin, L-alanyl-glutamine, or combinations of the above.

19. A bioengineered and biodegradable hair follicles containing sheet comprising: bioengineered hair follicles of claim 1 and/or a biodegradable grid comprising the bioengineered hair follicles of claim 1, wherein during manufacturing are preferably integrated and inserted into a biodegradable grid sheet that forces and facilitates the asymmetric positioning of protruding hair filaments to only one side of the biodegradable grid sheet.

20. The bioengineered hair follicles of claim 1, and biodegradable hair follicles containing grid sheet wherein during manufacturing are preferably integrated and inserted into a biodegradable grid sheet that forces and facilitates the asymmetric positioning of protruding hair filaments to only one side of the biodegradable grid sheet and are manufactured and produced by successively culturing them on media blends wherein (a) said media blend components is selected from Calcium chloride, Ferric nitrate, Magnesium sulfate, Potassium chloride, Sodium bicarbonate, Sodium chloride, Sodium phosphate monobasic, L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, Choline chloride, Folic Acid, myo-Inositol, Niacinamide, D-Pantothenic Acid, Pyridoxal, Pyridoxine, Riboflavin, Thiamine, D-Glucose, Pyruvic Acid, L-Glutamine, L-proline, L-hydroxyproline, reduced glutathione, ascorbic acid, Iron saturated Transferrin, Insulin, albumin, L-alanine, L-asparagine, L-aspartate, L-glutamate, beta-mercaptoethanol, L-alanyl-glutamine, Y-27632, Rho-associated coiled-coil containing protein kinase inhibitor, or combinations of the above; (b) matured in a media blend to facilitate the patterning and paracrine signalling between dermal papillae stem cells and stem cells, wherein said media blend components is selected from Calcium chloride, Cupric sulfate, Ferrous sulfate, Magnesium chloride, Potassium chloride, Sodium bicarbonate, Sodium chloride, Sodium phosphate dibasic, Zinc Sulfate, L-alanine, L-asparagine, L-arginine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, D-biotin, Choline chloride, Folic acid, myo-Inositol, Niacinamide, D-Pantothenic Acid, Pyridoxine, Riboflavin, Thiamine, Vitamin B12, D-Glucose, Hypoxanthine, Linoleic acid, Putrescine, Pyruvic acid, Thioctic acid, Thymidine, Sodium bicarbonate, Magnesium sulfate, Potassium nitrate, Sodium phosphate monobasic, Sodium selenite, HEPES, Arachidonic acid, Cholesterol, DL-alpha-tocopherol acetate, Ethyl alcohol, Linoleic acid, Linolenic acid, Myristic acid, Oleic acid, Palmitic acid, Palmitoleic acid, Polyoxvethylene-polyoxypropylene copolymer, Stearic acid, Tween, albumin, insulin, transferrin, 1-thioglycerol, FGF2, BMP4, 4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide, 4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline, dorsomorphin, L-alanyl-glutamine, extracellular matrix, or combinations of the above; or (c) media blend to facilitate the maturation of cellular aggregates, patterning and paracrine signalling between dermal papillae stem cells and stem cells and maturation of hair follicles, wherein said media blend components is selected from Ammonium molybdate, Ammonium metavandate, Cupric sulfate, Ferrous sulfate, Manganese sulfate, Magnesium sulfate, Nickel chloride, Sodium metasilicate, Sodium selenite, Sodium phosphate dibasic, Stannous chloride, L-aspartic acid, L-glutamic acid, L-glutamine, Calcium chloride, Ferric nitrate, Magnesium chloride, Potassium chloride, Sodium bicarbonate, Sodium chloride, Sodium phosphate monobasic, Zinc sulfate, L-alanine, L-asparagine, L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, Choline chloride, Folic Acid, Niacinamide, D-Pantothenic Acid, Pyridoxal, Riboflavin, Thiamine, Vitamin B12, inositol, D-Glucose, HEPES, Pyruvic acid, D-biotin, myo-Inositol, Hypoxanthine, Linoleic acid, Putrescine, DL-Thioctic Acid, Thymidine, Transferrin, Insulin, Progesterone, Catalase, reduced glutathione, Superoxide dismutase, T3, L-camitine, Ethanolamine, D+-galactose, Corticosterone, Linolenic acid, DL alpha tocopherol, DL alpha tocopherol acetate, Oleic acid, Pipecolic acid, albumin, L-alanyl-glutamine, beta-mercaptoethanol, FGF2, BMP4, 4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide, 4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline, dorsomorphin, L-alanyl-glutamine, or combinations of the above.

21. The hypoimmunogenic dermal papilla stem cells of claim 2 comprising reduced expression and/or deletion one or more genes of Major Histocompatibility Antigen Class I (HLA-I) including HLA-A, HLA-B, HLA-C; reduced expression and/or deletion one or more genes of Major Histocompatibility Antigen Class II (HLA-II) including HLA-DP, HLA-DR, HLA-DQ; reduced expression and/or deletion of B2M, CIITA, NLRC5 and combinations thereof; increased expression or activity of CD47, PD-L1, CTLA4-lg, IL-35, HLA-E, HLA-G, C1-inhibitor and combinations thereof.

22. The hypoimmunogenic bioengineered hair follicles of claim 1, or hypoimmunogenic biodegradable hair follicles containing grid sheet wherein during manufacturing are preferably integrated and inserted into a biodegradable grid sheet that forces and facilitates the asymmetric positioning of protruding hair filaments to only one side of the biodegradable grid sheet composed of the hypoimmunogenic dermal papilla stem cells comprising reduced expression and/or deletion one or more genes of Major Histocompatibility Antigen Class I (HLA-I) including HLA-A, HLA-B, HLA-C: reduced expression and/or deletion one or more genes of Major Histocompatibility Antigen Class II (HLA-II) including HLA-DP, HLA-DR, HLA-DQ; reduced expression and/or deletion of B2M, CIITA, NLRC5 and combinations thereof; increased expression or activity of CD47, PD-L1, CTLA4-lg, IL-35, HLA-E, HLA-G, C1-inhibitor and combinations thereof and/or hypoimmunogencic pluripotent stem cells selected from the group consisting of embryonic stem cells or induced pluripotent stem cells.

23. The manufacturing process of claim 20 for the generation of bioengineered hair follicles and biodegradable hair follicles containing sheet, wherein the process is implemented with; (a) autologous embryonic stem cells or induced pluripotent stem cells, dermal papillae stem cell, or a dermal papillae stem cell line, and said cell is preferably genetically modified for unlimited expansion, or immortalized, or engineered to over-express extracellular matrix proteins or attachment proteins, or genetically engendered to eliminate immunogenicity and to avoid allogenic transplant rejection, or combinations thereof or to derive autologous bioengineered hair follicles and autologous bioengineered hair follicles containing sheets yielding non-immunogenic hair follicles supply source for cosmetic industry; or (b) allogenic hypoimmunogenic cells comprising reduced expression and/or deletion one or more genes of Major Histocompatibility Antigen Class I (HLA-I) including HLA-A, HLA-B, HLA-C reduced expression and/or deletion one or more genes of Major Histocompatibility Antigen Class II (HLA-I I) including HLA-DP, HLA-DR, HLA-DO; reduced expression and/or deletion of B2M, CIITA, NLRC5 and combinations thereof; increased expression or activity of CD47, PD-L1, CTLA4-lg, IL-35, HLA-E, HLA-G, C1-inhibitor and combinations thereof; and/or embryonic stem cells or induced pluripotent stem cells to derive allogenic hypoimmunogenic bioengineered hair follicles and allogenic hypoimmunogenic bioengineered hair follicles containing sheets integrated during manufacture with bioengineered hair follicle comprising: one or more bioengineered dermal papillae stem cell-induced and pluripotent stem cell-derived hair follicles, and containing hair bulge, hair shaft, and protruding hair filament, also a cell-composed cortex containing stem cell-derived synthetic interfollicular epidermis, and intertwining extracellular matrix and inserted into a biodegradable grid sheet that forces and facilitates the asymmetric positioning of protruding hair filaments to only one side of the biodegradable grid sheet yielding hypoimmunogenic hair follicles supply source for cosmetic industry.

24. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0073] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0074] In the figures, similar components and/or features may have the same reference label. Further, various components of the same type may be distinguished by following the reference label with a second label that distinguishes among the similar components. If only the first reference label is used in the specification, the description is applicable to any one of the similar components having the same first reference label irrespective of the second reference label.

[0075] FIG. 1 illustrates a proposed biodegradable grid scaffold item 15 and biodegradable hair follicles containing sheet item 24 in accordance with an exemplary embodiment of the present disclosure.

a—item 15 grid of biodegradable substrates blend
b—item 27 cavities included in item 15
c—item 22 bioengineered hair follicle
d—item 24 bioengineered hair follicles containing sheet
e—mature cell aggregate item 17

[0076] FIG. 2 illustrates a proposed three-dimensional cell aggregate item 17 and bioengineered hair follicle item 22 in accordance with an exemplary embodiment of the present disclosure.

a—item 17 three dimensional cell aggregate
b—item 22 bioengineered hair follicle

[0077] FIG. 3 illustrates a proposed culture condition timeline with media blend sequences of item 18, item 19 and item 20. Also shows the manufacturing steps of loaded biodegradable grid item 23, and bioengineered hair follicles containing sheet item 24 in accordance with an exemplary embodiment of the present disclosure.

a—item 18 culture media blend
b—item 19 culture media blend
c—item 20 culture media blend
d—item 23 loaded biodegradable grid item 15 containing cell aggregate item 17
e—item 24 bioengineered hair follicles containing sheet, containing bioengineered hair follicles item 22

[0078] FIG. 4 illustrates immunological responses that activate and inhibit allogenic transplant rejection

[0079] FIG. 5 illustrates a proposed hypoimmunogenic hair follicle-producing three-dimensional cell aggregate item 17 in accordance with an exemplary embodiment of the present disclosure.

i—dermal papilla stem cell item 25
ii—pluripotent stem cell item 27
iii—three dimensional cell aggregate item 17 composed of cellular mixtures of item 25 and item 27, and extracellular matrix item 16

[0080] FIG. 6 Experimental timeline of de novo hair derivation. Staring with from iPSC and DPSC cultures EB-assembly was performed in U-Bottom ULA plates. After formation the EBs were subjected to CDM for a two-step patterning process.

[0081] FIG. 7 Day 5 (30% iPSC)—DPSC go to the center, like previously, indicated by the GFP negative area of the EB (arrow)

[0082] FIG. 8 Day 35 (20180502): Cond. 1, 90% iPSC, 6

[0083] FIG. 9 Day 74 (20180610), organoid No. 16.

Bottom: GFP-positive (no direct involvement of DPSC)

[0084] FIG. 10 Day 78 (20180614), 90%, Cond. 1, Organoid No. 13

[0085] FIG. 11. Day 82, Cond. 1, 50%, No. 16

[0086] FIG. 12 Day 82, 90%, Cond. 1, No. 13

[0087] FIG. 13 Day 82, 90%, Cond. 2, No. 13

[0088] FIG. 14 Day 92, 90%, Cond. 1, No. 13

[0089] FIG. 15 Day 92, 95%, Cond. 3, No. 32

[0090] Middle: GFP; Bottom: merge (Fixation o/n 20180629 in 4% PFA, 1×PBS)

EXPERIMENTAL SECTION

[0091] Based on the available literature Lee et al 2018 (doi: 10.1016/j.celrep.2017.12.007) the inventor developed a method to also derive human de novo hair follicle containing organoids. Lee et al 2018 was based on Koehler et al 2013 (doi: 10.1038/nature12298). Consequently, the inventor assumed that an approach based on a similar publication from Koehler 2017 (doi: 10.1038/nbt.3840) could lead to a similar success when using mature human induced pluripotent stem cells (iPSC) as a starting population. Since the inventor also had patient derived dermal papilla stem cells (DPSC) extracted from FUE-hair follicles available, the inventors assumed these cells could have a positive effect on potential de novo hair follicle assembly. In the following months, the inventor tested a variety of different combinations based on Koehler 2017 and our own protocol, to screen for potential successful combinations. Taking in to account the mouse versus human developmental timeframe, the inventor expected the de novo HF assembly to be respectively slower. Here the inventor outlines one first successful combination (FIG. 6) resulting in the first, yet characterized, organoids containing HF within a time-frame of 70-80 days of maturation. Interestingly, this approach however, was particularly successful in combinations containing DPSC. iPSC-only conditions as shown in all available papers made it barely beyond the EB-state. To be able to discriminate between the combination of iPSC and DPSC, the inventor used GFP-tagged iPSC (A13337, Gibco). During the first steps of embryoid body (EB) formation the cells were visually clearly separable, with the DPSC centered inside, which is maybe not surprising due to their mesodermal origin, and the iPSC engulfing the DPSC. Quickly the GFP+iPSC based cells overgrew and throughout the whole maturation of the organoid clearly GFP-negative cells were not observable anymore.

Organoid Preparation and Cultivation Protocol:

[0092] (i) Have (GFP-/for DPSC mix approach) wild-type iPSC in expansion culture [0093] (ii) When the cells are 80% confluent, aspirate the E8 medium (and wash the cells three times with PBS at RT). [0094] (iii) Cells were dissociated with StemPro Accutase (Invitrogen, cat. no. A1110501) for ˜5 min at 37 C [0095] (iv) Collect the dissociated cells into 1 ml of E8-Ri (ectodermal differentiation)—or desired EB forming media medium and transfer them into a 2-ml microcentrifuge tube. [0096] (v) Break the cell clumps into single cells by pipetting with a P1000 tip. Pellet the cells by centrifugation at 200 rcf for 5 min at RT. [0097] (vi) Completely remove the supernatant and resuspend the cell pellet in 1 ml of EB-Ri Y27632 (5 uM) medium. [0098] (vii) Forcefully pipette 1 ml of EB-Ri medium through a cell-strainer-top test tube to prime the strainer. [0099] (viii) Pipette the 1 ml of ES cell suspension dropwise onto the cell strainer. Next, pipette 1 ml of fresh EB-Ri medium dropwise onto the cell strainer. There should be 3 ml in the test tube. [0100] (i) Mix the cell suspension by pipetting with a P1000 tip, and then determine the concentration of cells with a haemocytometer. [0101] (j) Dilute the appropriate volume of cell suspension in xy ml of fresh EB-Ri medium (5 uM Ri) to acquire a final concentration of 50,000 cells per ml—to achieve 5,000 (used 10 k) cells per organoid (at least 10 ml for a complete 96er plate). Invert the tube several times to mix. [0102] (k) Pour the cell suspension into a reservoir and aliquot 100 μl of cell suspension into each well of two 96-well plates with a multichannel pipette. [0103] (l) Centrifuge the U-bottom plate at 200 rcf for 3 min. [0104] Use 1300 ul total volume [0105] NOTE: Here some of the extracted patient DPSC can be mixed in to mimic and induce HF growth [0106] Which will be a mix of GFP-iPSC (A13337, p14) and unlabelled primary DPSC (004A, p6).

TABLE-US-00001 100% iPSC 95% iPSC (1225 iPSC/65 ul DPSC) 90% (1170 iPSC/130 ul DPSC) 85% (1105 iPSC/195 ul DPSC) 80% (1040 iPSC/260 ul DPSC) 50% (750 iPSC/750 ul DPSC) 40% (720 iPSC/780 ul DPSC) 30% (390 iPSC/910 ul DPSC) [0107] (m) Place the plates in a 37° C. incubator with 5.0% CO.sub.2 for 24 h. [0108] (n) After 24h, very carefully replace medium by fresh EB-medium, without Rock-inhibitor [0109] (o) After another 24h, replace the medium by CDM containing the different combinations of patterning factors. [0110] (p) After 4 days add 25 ul of CDM containing factors as indicated. [0111] (q) After 4 days add 25 of fresh CDM w.o growth factors [0112] (r) At day 12 of pre-patterning the resulting patterned EBs were moved to 12 well ULA plates and incubated in 500 ul hOMM. Plates were incubated at dynamic conditions, at 50 rpm shaking. [0113] (s) 50% media change was performed every other day using fresh hOMM.

Visual Progression in FIG. 7.

[0114] Day 8: hOMM cultivation only, media change 50% every other day (500 ul OMM volume).

[0115] Day 12 20% DPSC (DPSC not visible anymore, organoids form similar structures, theoretically too early):

[0116] Similar to previous approaches

[0117] Day 35 (20180502):

[0118] Cond. 1, 90% iPSC, 6:

[0119] FIG. 8.

[0120] Day 74 (20180610), organoid No. 16:

[0121] FIG. 9

[0122] Day 78 (20180614), 90%, Cond. 1, Organoid No. 13,

[0123] FIG. 10

[0124] Day 82, Cond. 1, 50%, No. 16

[0125] FIG. 11.

[0126] Day 82, 90%, Cond. 1, No. 13

[0127] FIG. 12

[0128] Day 82, 90%, Cond. 2, No. 13

[0129] FIG. 13

[0130] Day 92, 90%, Cond. 1, No. 13

[0131] FIG. 14

[0132] Day 92, 95%, Cond. 3, No. 32

[0133] FIG. 15

SUMMARY

[0134] It takes around 80 Days to get the first Hair follicles. Survivability in the conditions with DPSC mixed in is good, allowing the development of Hair follicles de novo in the dish in some compositions.

[0135] The protocol is flexible allowing the de novo formation in different BMP4 conditions.

Material Overview:

EB-Formation Medium

EB Medium (50 mL):

[0136]

TABLE-US-00002 40 mL KnockOut ™ DMEM (TS: 10829018) 10 mL KnockOut ™ Serum Replacement (10828010) 500 μL Normocin (Ant-nr-1) 500 μL GlutaMAX ™ Supplement (35050061) 500 μL MEM Non-Essential Amino Acids Solution (11140050) 100 μL β-mercaptoethanol (100 uM from 50 mM) (31350010)

Chemically Defined Medium

[0137]

TABLE-US-00003 Supplementary Table 1 Chemically-Defined Differentiation Medium (CDM) Stock Final Volume Component Supplier Cat. No. Concentration Concentration used Ham's F12 Gibco 31765-035 — 49% (v/v) 100 ml GlutaMax IMDM GlutaMax Gibco 31980-030 — 49% (v/v) 100 ml Chemically- Gibco 11905-031 100x 1x 2 ml Defined Lipid BSA Sigma A1470 — 5 mg/ml 1 g Insulin Sigma I9278 10 mg/ml 7 μg/ml 140 μl Transferrin Sigma T8158 20 mg/ml 15 μg/ml 150 μl 1-thioglycerol Sigma M6145 11.5M 450 μM 8 μl Normocin Invivogen Ant-nr-1 50 mg/ml 100 μg/ml 400 μl Note: This formulation is for 200 mL of medium which should be used for <2 weeks. We sterile filtered the medium before adding Insulin and other factors. A variation of this medium was previously used to generate cerebellar and anterior pituitary organoids.sup.6,7.

Human Organoid Maturation Medium

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TABLE-US-00004 Stock Final Volume Component Supplier Cat. No. Concentration Concentration (50 ml) Adv DMEM/F12 Gibco 12491-015 — 49% (v/v) 24.5 ml Neurobasal Gibco 21103-049 — 49% (v/v) 24.5 ml N2 supplement Gibco 17502-048 100x 0.5x   250 μl B27 -Vitamin A Gibco 12587-010  50x 1x 500 μl GlutaMAX Gibco 35050-079 100x 1x 500 μl Mercaptoethanol Gibco 21985-015 55 mM 0.1 mM 91 μl Normocin Invivogen Ant-nr-1 50 mg/ml 100 μg/ml 100 μl Note: This formulation is for 50 mL of medium, which should be used for <2 weeks. This medium is a custom-made hybrid of two media previously used to generate cerebral and gastric organoids.sup.8,9. B27 without Vitamin A was used to limit the influence of endogenously produced retinoic acid.