Patent Application
20220333082
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.
Bioengineering definition: Application of engineering and biological principles for purposefully defining cellular behaviour and or cellular disposition within an engineered synthetic tissue and or organ. Furthermore, the designed modification of genetic information to code altered cellular functions with beneficial impact on biologically derived materials and/or cellular behaviour. In addition, the use of engineering and biological principles to create novel tissue and/or organs and or biologically derived materials.
The use of vertebrata adult progenitors or adult stem cells has enabled the derivation of a variety of cell types within said adult progenitor tissue. In addition, vertebrata embryonic stem cells which count with a higher differentiation potential enable deriving all adult cell types in said vertebrata species, particularly adult stem cells and terminally differentiated cells. Furthermore, the reprogramming process from differentiated cells towards higher pluripotency states (primed or naive) provides an inexhaustible cell source.
The reprogramming process from differentiated cells towards cells of higher pluripotency state may be achieved through the exogenous synthetic expression of transcriptional factors and or epigenetic modifiers that reinforce the core transcriptional network of said pluripotent state. The simplicity of this process has placed reprogrammed stem cells as a convenient cell source for bioengineering and regeneration.
The term “pluripotent stem cell”, as used herein, encompasses “induced pluripotent stem cell”, or “iPSC”, a type of pluripotent stem cell derived from a differentiated non-pluripotent cell. Methods for the generation of iPSC or iPS cells are well-known in the art (Zhou et al., Stem Cells 27 (11): 2667-74 (2009); Huangfu et al, Nature Biotechnol. 26 (7): 795 (2008); Woltjen et al., Nature 458 (7239): 766-770 (2009); Zhou et al., Cell Stem Cell 8:381-384 (2009).
To generate iPSC, it is preferable to deliver one or more “reprogramming factors” to non-pluripotent cells using episomal vectors (e.g. Sendai virus, adenovirus). Unlike lentiviral and retroviral vectors, episomal vectors do not integrate into the host's cell genome and will be lost, which results in iPSC with “zero footprint”. This is preferable as no permanent viral genetic material will be present in the final iPSC.
As is appreciated by those of skill in the art, iPSC can be generated using a number of reprogramming factors, such as OCT4, KLF4, SOX2, c-MYC, NANOG, LIN28. These reprogramming factor genes are provided on episomal vectors such as are known in the art and commercially available. For example, CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit (Thermo Fisher Scientific, catalog number A34546) or Episomal iPSC Reprogramming Vectors (Thermo Fisher Scientific, catalog number A14703).
In addition, zero footprint human iPS cell lines are also commercially available, e.g. Human Episomal iPSC Line (Thermo Fisher Scientific, catalog number A18945)
As is known in the art, a source of non-pluripotent cells for iPSC generation could be bone marrow and blood cells, fibroblasts, keratinocytes, epithelial cells of kidney and bladder etc.
The term “dermal papilla stem cell”, as used herein, encompasses stem cells that are isolated from the hair follicle dermal compartments, such as dermal papilla and dermal sheath. As will be appreciated by those in the art, the dermal papilla and dermal sheath contain cells that are capable to induce new hair growth when transplanted alone or in combination with skin epithelial cells (Jahoda et al, Nature. 311(5986):560-2 (1984); Jahoda et al, Exp Dermatol. 10(4):229-37 (2001); McElwee et al, J Invest Dermatol. 121(6):1267-75 (2003). However, none of the current approaches for using dermal papilla stem cell were able to produce hair follicles with clinically and cosmetically satisfying results.
The terms “dermal papilla”, “dermal papilla stem cells” and “dermal sheath cup cells’ are used indistinguishably form each other and the use of one includes the use of the other.
Methods for the isolation of dermal papilla stem cell from autologous and allogeneic hair follicles are well-known in the art (Gledhill et al, Methods Mol Biol. 989:285-92 (2013); Topouzi et al. Exp Dermatol. 26(6):491-496 (2017).
As is appreciated by those of skill in the art, dermal papilla cells can also be generated by in vitro differentiation of iPSC (Gnedeva et al, PLoS One. 10(1):e0116892 (2015); Veraitch et al, Sci Rep. 7:42777 (2017).
In addition, dermal papilla primary cells and cell lines are commercially available, e. g. Human Follicle Dermal Papilla Cells (HFDPC) (PromoCell, catalogue number C-12071).
Differentiation of stem cells can be achieved through the use of culture conditions with defined combinations of nutrients, co-factors, growth factors and or small molecules. Differentiation protocols can rely on external signals or intrinsic signals, and the differentiation propensity of adult cell types varies from one cell type to another and is dependent on its hierarchy within the pluripotency level and linage commitment level.
Differentiation of stem cells towards tissue specific cells types is widely and efficiently applicable, the derivation of specific cell types can be biased by means of autocrine or paracrine signals. Paracrine signals of adjacent differentiated cells act on stem cells as lineage specific attractors, cellular identity attractors and mimicking developmental organizing centres.
A variety of differentiation protocols to derive some components of vertebrata hair follicle in vivo have been proposed (Blanpain et al., Cell 118, 635-648 (2004); Patent application CN107164310A). Similarly, there are methods for the in vitro differentiation of hair follicles from single populations of mouse pluripotent stem cells (Lee et al., Cell 22, 242-254 (2017)). However, none of them have successfully developed in vitro human hair follicles from two populations of cells, such as human pluripotent stem cells and an attractor population of human dermal papilla stem cells. Furthermore, the current protocols rely on using cell culture media supplemented with animal-derived xenogeneic growth factors (e.g. fetal bovine serum), rendering the bioengineered hair follicles unsuitable for transplantation to humans. In addition, none of the existing protocols have successfully developed transplantable bioengineered sheets containing human hair follicles.
A variety of methods for tissue fabrication for multiple tissue types have been proposed (Patent application US20170130192A1; U.S. Pat. No. 6,096,347A; Patent JP5893786B2; Patent application WO2017077985A1). However, none of them successfully developed in vitro hair follicles, or tissue sheets containing hair follicles, or integrate biodegradable material scaffolds for aiding the generation of asymmetry in the structure of said tissue sheets or in the shafts of said hair follicles.
The lack of bioengineered hair follicles and hair follicle-containing sheets with defined position of hair shafts, results in the use of natural sources for cosmetic surgeries, such us autologously donated human hair samples. However, the transplantation of autologous remaining hair follicles from a non-balding area of the scalp to a balding area possesses several limitations such as limited number of available donor hair follicles, reduced hair density and scarring at the donor area. On the other hand, using autogenous stem cells to derive the bioengineered hair follicles provides an unlimited source for autologous transplantation, which eliminates the aforementioned risks.
However, the ultimate goal of hair regenerative therapy is using allogeneic hair follicles and hair follicle-containing sheets for hair restoration. Compared to autologous cells, allogeneic pluripotent stem cells and dermal papilla stem cells are easier from a manufacturing standpoint and allow the generation of well-screened, standardized, high-quality “off-the-shelf” allogeneic hair follicles. However, due to cells' antigenicity, allogeneic hair follicles would elucidate a strong recipient's immune response and would be rejected. To circumvent the problem of rejection, reduction or elimination of cells' antigencity and generation of hypoimmunogenic universally-acceptable hair follicles will be required. Currently, there are no methods for creating hypoimmunogenic dermal papilla stem cells and hypoimmunogenic universally-acceptable hair follicles.
By “hypoimmunogenic pluripotent stem cell”, “hypoimmunogenic dermal papilla stem cell” and “hypoimmunogenic hair follicle” herein are meant a pluripotent stem cell, dermal papilla stem cell and hair follicle that do not induce an immunological response sufficient enough for allogeneic transplant rejection.
As will be appreciated by those in the art, there were several strategies proposed for generation of non-immunogenic iPSC and iPSC-derived differentiated cells (Xia et al, PNAS 116 (21) 10441-10446 (2019); Deuse et al, Nature Biotechnology 37, pages 252-258 (2019); patent application WO2018132783A1, Patent application WO2016183041A3). However, none of them successfully developed in vitro bioengineered hypoimmunogenic hair follicles, or hypoimmunogenic tissue sheets containing hair follicles,
The present application describes a method for deriving hair follicles and sheets containing hair follicles using autologous sources, and can be implemented in good manufacturing procedures pipelines and xeno-free conditions. Furthermore, using as source biodegradable grids containing and carrying said hair follicles, facilitates the downstream surgical process by augmenting the hair follicle count used and handled per surgical step.
The present invention also describes a method for deriving hair follicles and sheets containing hair follicles using allogeneic sources that are genetically-modified to avoid host immune response. This invention provides genetically-modified induced pluripotent stem cells (iPSC)_and dermal papilla stem cells (DPSC) that lack major immune antigens and produce signals preventing immune attack. Therefore, bioengineered allogeneic hair follicles formed from those iPSC and DPSC do not trigger immune responses and avoid immune rejection, which allows them to be used as universal donor hair follicles for allogeneic transplantation.
As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better clarify the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.
The words “grid”, “mesh”, “scaffold” and “frame” are used indistinguishably form each other and the use of one includes the use of the others. In addition, the words “cavities”, “chambers” and “pockets” are used indistinguishably form each other and the use of one includes the use of the others.
Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all groups used in the appended claims.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art. Generally, nomenclatures used in connection with techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.
The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e. g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001); Ausubel et al., Current Protocols in Molecular Biology, J, Greene Publishing Associates (1992, and Supplements to 2002); Handbook of Biochemistry: Section A Proteins, Vol 11976 CRC Press; Handbook of Biochemistry: Section A Proteins, Vol II 1976 CRC Press. The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.
A cell when used herein may preferably be a mammalian cell. A mammalian cell may preferably be a cell from an human, antelope, antilopini, beaver, buffalo, caracal, cat, cheetah, chinchilla, cow, deer, eland, elephant, ermine, faux, fisher, fox, genet, giraffe, goat, golden jackal, hedgehog, horse, leopard, lynx, lion, marten, mink, monkey, ape, nutria, otter, rabbit, rhinoceros, sable, serval, sheep, shrew, stoat, swine, wolf, australian brushtail possum, mouse, rat, Camelidae, their subspecies and Pantholopinae such as: Dromedary Camel (Camelus dromedarius), Bactrian Camel (Camelus bactrianus) and it's wildtype, Llama (Llama glama), Alpaca (Vicugna pacos), Vicuña (Vicugna vicugna), Guanaco (Lama guanicoe) and Tibetan Antilope (Pantholops hodgsonii) with human preferred. A preferred mammalian cell is a stem cell or induced pluripotent stem cell or dermal papilla stem cell. The mammalian cells may be obtained from a biopsy. The mammalian cell may be from a cell line, e.g. a deposited cell line or a commonly available cell line.
Some of the objects of the present disclosure are as listed herein below.
It is an object of the present disclosure to provide for methods to derive hair follicles as a source for plastic surgery.
It is an object of the present disclosure to provide for methods to derive bioengineered hair follicles containing sheets as a source for plastic surgery.
It is an object of the present disclosure to provide for methods to derive bioengineered hair follicles containing sheet with the exclusive disposition of hair filaments towards one side or facet of said bioengineered hair follicles containing sheet.
It is another object of the present disclosure to provide for a method to derive bioengineered hair follicles avoiding the need for allogeneic hair follicle donation.
It is another object of the present disclosure to provide for autologous bioengineered hair follicles to avoid the risk for tissue immune rejection.
It is yet another object of the present disclosure to provide bioengineered hypoimmunogenic hair follicles from universal donor dermal papilla stem cells and induced pluripotent stem cells.
A dermal papilla stem cell derived from a mammal, wherein said mammal cell is selected from human, antelope, antilopini, beaver, buffalo, caracal, cat, cheetah, chinchilla, cow, deer, eland, elephant, ermine, faux, fisher, fox, genet, giraffe, goat, golden jackal, hedgehog, horse, leopard, lynx, lion, marten, mink, monkey, ape, nutria, otter, rabbit, rhinoceros, sable, serval, sheep, shrew, stoat, swine, wolf, australian brushtail possum, mouse, rat, Camelidae, their subspecies and Pantholopinae such as: Dromedary Camel (Camelus dromedarius), Bactrian Camel (Camelus bactrianus) and it's wildtype, Llama (Llama glama), Alpaca (Vicugna pacos), Vicuña (Vicugna vicugna), Guanaco (Lama guanicoe) and Tibetan Antilope (Pantholops hodgsonii) with human preferred.
The mammal dermal papilla stem cell of item 1, wherein said mammal cell markers is selected from Sox2-positive, Sox9-positive, Nestin-positive, P63-positive, CD133-positive, AP-positive, alpha-SMA-positive or combinations of the above.
A pluripotent stem cell derived from a mammal, wherein said mammal cell is selected from human, antelope, antilopini, beaver, buffalo, caracal, cat, cheetah, chinchilla, cow, deer, eland, elephant, ermine, faux, fisher, fox, genet, giraffe, goat, golden jackal, hedgehog, horse, leopard, lynx, lion, marten, mink, monkey, ape, nutria, otter, rabbit, rhinoceros, sable, serval, sheep, shrew, stoat, swine, wolf, australian brushtail possum, mouse, rat, Camelidae, their subspecies and Pantholopinae such as: Dromedary Camel (Camelus dromedarius), Bactrian Camel (Camelus bactrianus) and it's wildtype, Llama (Llama glama), Alpaca (Vicugna pacos), Vicuña (Vicugna vicugna), Guanaco (Lama guanicoe) and Tibetan Antilope (Pantholops hodgsonii) with human preferred.
The mammal pluripotent stem cell of item 3, wherein said pluripotent stem cell type is selected from embryonic stem cell, primed stem cell or induced pluripotent stem cell, with induced pluripotent stem cell preferred.
A nucleic acid molecule, wherein said nucleic acid molecule comprises at least one nucleotide sequence encoding at least one fitness gene, selection marker indicating homologous or heterologous recombination when integrated in the genome of a vertebrata cell, wherein the selection markers when being expressed confers chemical resistance or is optically discriminable, e.g. in FACS or any fluorescence guided capture, and wherein the nucleotide sequence encoding a selection marker indicating homologous or heterologous recombination in a vertebrata cell is flanked 5′ and or 3′ by nucleotide sequences that are homologous to nucleotide sequences of a nucleic acid sequence present in the vertebrata cell.
The nucleic acid molecule of item 5, wherein said nucleic acid molecule comprises a chemical resistance selection marker selected from neomycin resistance, hygromycin resistance, HPRT1, puromycin resistance, puromycin N-acetyl-transferase, blasticidin resistance, G418 resistance, phleomycin resistance, nourseothricin resistance or chloramphenicol resistance.
The nucleic acid molecule of item 5, wherein said optical discriminability is different emission wavelength. The nucleic acid molecule of item 5, wherein said selection marker indicating homologous or heterologous recombination is a fluorescent protein.
The nucleic acid molecule of item 7, wherein said fluorescent protein is selected from Sirius, SBFP2, Azurite, EBFP2, mKalamal, mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl, mTFP1, MiCy, iLOV, AcGFPI, sfGFP, mEmerald, EGFP, mAzamiGreen, cfSGFP2, ZsGreen, mWasabi, SGFP2, Clover, mClover2, EYFP, mTopaz, mVenus, SYFP2, mCitrine, YPet, ZsYellowl, mPapayal, mKO, mOrange, mOrange2, mK02, TurboRFP, mRuby2, eqFP611, DsRed2, mApple, mStrawberry, FusionRed, mRFP1, mCherry, mCherry2, dTOMATO, tdTOMATO, tagBFP, photoactivatable or photoswitchable fluorescent protein.
The nucleic acid molecule of item 5, wherein said fitness gene is selected from telomerase, Ras, Abl, Akap13, Araf, Tim, Atf, Axl, Bcl, Braf, Brca, Brip, Cbl, Csf1r, 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.
The nucleic acid molecule of items 9, wherein said nucleotide sequence encoding a fitness gene comprises a promoter driving expression of said selection markers.
The nucleic acid molecule of item 10, wherein said promoter is constitutive or inducible.
The nucleic acid molecule of any one of the preceding items, wherein homologous or heterologous recombination is induced by random integration, TALENs, ZFNs, meganucleases, or CRISPR nuclease.
The vertebrata dermal papilla stem cell of item 1, wherein said dermal papilla stem cell is engineered to contain at least one nucleic acid molecule of item 5, at least one chemical selection marker of item 6 and/or at least one optically active selection marker of item 7 and 8, at least one fitness gene of item 9.
The vertebrata pluripotent stem cell of item 3, wherein said pluripotent stem cell is engineered to contain at least one nucleic acid molecule of item 5, at least one chemical selection marker of item 6 and/or at least one optically active selection marker of item 7 and 8, at least one fitness gene of item 9.
A grid of blended biodegradable substrates, wherein said substrates blend components is selected from polyglycolic acid, polyacetic acid, epsilon-caprolactones, polydioxanones, lactides, hydrogels, or combinations of the above.
A extracellular matrix blend, wherein said matrix blend components is selected from laminin, fibronectin, collagen, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, elastin, integrin, cadherin, selectin, connexins, claudins, occludins, and chemically modified extracellular matrix proteins, or combinations of the above.
Three-dimensional cell aggregate composed of cellular mixtures of item 1 and item 3, and extracellular matrix item 16.
A media blend to facilitate cell aggregate formation, wherein said media 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.
A media blend to facilitate the patterning and paracrine signalling between cells item 1 and item 3, wherein said media 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[1,5-a]pyrimidin-3-yl]-quinoline, dorsomorphin, L-alanyl-glutamine, item 6, or combinations of the above.
A media blend to facilitate the maturation of cellular aggregates item 7, patterning and paracrine signalling between cells item 1 and item 3, and maturation of cellular aggregates item 7 into hair follicles, wherein said media 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-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, item 6, or combinations of the above.
A polymer surface, wherein said surface is selected from poly-acrylates, poly-ornithine, poly-olefines, hydrogels, poly-amino acids, and poly-peptides.
A bioengineered hair follicle, wherein said bioengineered hair follicle is composed of the derivatives of cells item 1 and item 3, and extracellular matrix item 16, cultured successively on media item 18, item 19, and item 20, and produced from matured derivatives of cell aggregate item 17.
A loaded biodegradable grid, wherein said loaded biodegradable grid is composed of the biodegradable grid item 15 containing at least one cellular aggregate item 17.
A bioengineered hair follicles containing sheet, wherein said bioengineered hair follicles containing sheet is composed of the loaded biodegradable grid item 23, cultured successively on media item 18, item 19, and item 20, and wherein successive culture on media item 18, item 19 and item 20 results in the conversion of cellular aggregates item 17 into hair follicles item 22.
Hypoimmunogenic dermal papilla stem cell of item 1, wherein said hypoimmunogenic cell comprises of one or more of the following: 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-Ig, IL-35, HLA-E, HLA-G, C1-inhibitor and combinations thereof.
Hypoimmunogenic cell of item 25, wherein said hypoimmunogenic cell is created by using genome editing technologies including CRISPR/Cas, TALEN, CRISPR/Cas, Zinc finger (ZNF), viral, plasmid, interfering RNA, with CRISPR/Cas preferred.
Hypoimmunogenic pluripotent stem cell of item 3, wherein said hypoimmunogenic cell comprises of one or more of the following: 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-Ig, IL-35, HLA-E, HLA-G, C1-inhibitor and combinations thereof.
Hypoimmunogenic pluripotent stem cell of item 27, wherein said hypoimmunogenic cell is created by using genome editing technologies including CRISPR/Cas, TALEN, CRISPR/Cas, Zinc finger (ZNF), viral, plasmid, interfering RNA, with CRISPR/Cas preferred.
An in vitro method for manufacturing and deriving bioengineered hair follicles item 22 and bioengineered hair follicles containing sheet item 24:
(a) Subjecting population of dermal papillae stem cell of item 1 to liquid merging with stem cell item 3 on media item 18, and aggregate them and bring together through centrifugal force, and culture them between one hour and two days. The ratio of cells of item 1 and item 3 is adjusted within 1:1 to 1:1×10e5. The combined count of cells item 1 and item 3 per cellular aggregate ranges between 100 and 1×10e5. Cell populations are alternatively aggregated inside the spaces of biodegradable grid item 15.
(b) Coating of the three-dimensional aggregate in (a) and liquid coat it with extracellular matrix item 16 to generate the cellular aggregate item 17. Cell aggregates are alternatively coated inside the spaces of biodegradable grid item 15 yielding item 23.
(c) Subjecting the cellular aggregate item 17 to culture on media item 19 between one hour and eight days. Cell aggregates item 17 are alternatively deposited into the spaces of biodegradable grid item 15 yielding item 23.
(d) Subjecting the cellular aggregate item 17 in (c), or item 23 in (c), and patterned with media item 19 to media item 20 between seven and eighty days.
(e) Item 17 in (d) results in a bioengineered hair follicle item 22, and item 23 in (d) results in a bioengineered hair follicle containing sheet item 24.
(f) Collection of the resulting suspension hair follicles item 22 in (e), or resulting biodegradable hair follicles containing sheet item 24 in (e).
A method for deriving autologous bioengineered hair follicles or autologous bioengineered hair follicles containing sheets.
(g) Obtain dermal papillae stem cells item 1 from target individual, and obtain induced pluripotent stem cells item 3 from target individual.
(h) Derive bioengineered hair follicles or bioengineered hair follicles containing sheets from target individual cells in (g), using method item 29.
A method for deriving allogeneic hypoimmunogeneic bioengineered hair follicles or allogenic hypoimmunogeneic bioengineered hair follicles containing sheets.
One or more cavities integrated and contained in the biodegradable grid item 15, wherein said cavities includes a wide opening towards one side of the grid and a narrow opening towards the opposite side of the grid.
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.
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.
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
a—item 17 three dimensional cell aggregate
b—item 22 bioengineered hair follicle
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
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
Bottom: GFP-positive (no direct involvement of DPSC)
Middle: GFP; Bottom: merge (Fixation o/n 20180629 in 4% PFA, 1×PBS)
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 (
Day 8: hOMM cultivation only, media change 50% every other day (500 ul OMM volume).
Day 12 20% DPSC (DPSC not visible anymore, organoids form similar structures, theoretically too early):
Similar to previous approaches
Day 35 (20180502):
Cond. 1, 90% iPSC, 6:
Day 74 (20180610), organoid No. 16:
Day 78 (20180614), 90%, Cond. 1, Organoid No. 13,
Day 82, Cond. 1, 50%, No. 16
Day 82, 90%, Cond. 1, No. 13
Day 82, 90%, Cond. 2, No. 13
Day 92, 90%, Cond. 1, No. 13
Day 92, 95%, Cond. 3, No. 32
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.
The protocol is flexible allowing the de novo formation in different BMP4 conditions.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/068813 | 7/3/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62872379 | Jul 2019 | US |
