Patent Application
20230414674
The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Sep. 8, 2023, is named 42256-613_201_SL.xml and is 15,688 bytes in size.
Follicular neogenesis is the generation of new hair follicles (HF) after birth. Although humans are born with a full complement of hair follicles, hair follicles can change in size and growth characteristics. For example, hair follicles can ultimately degenerate and disappear as in baldness or in permanent scarring (cicatricial) alopecias. Other common baldness and less common hair loss conditions, such as discoid lupus erythematosis, congenital hypotrichosis, lichen planopilaris, and other scarring alopecias are in need of regeneration of HF.
In certain aspects, disclosed herein is a method of growing a hair follicle comprising: (a) preparing human induced pluripotent stem cells (hiPSCs); (b) differentiating the hiPSCs into hair follicle bulge stem cells (HFBSCs); and (c) implanting the HFBSCs into skin of a subject.
In some embodiments, the hiPSCs have one, two, three, four, five, six, or more markers selected from the group consisting of CD200, ITGA6, ITGB1, OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81 and SSEA4. In some embodiments, the HFBSCs have one, two, three, four, five, six, or seven markers selected from the group consisting of CD200, ITGA6, ITGB1, KRT15, KRT18, KRT19, and P63. In some embodiments, the preparing in (a) comprises introducing, by electroporation, non-integrating episomal plasmid vectors encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28 and an shRNA for human p531. In some embodiments, the electroporation is via a Neon transfection system. In some embodiments, the differentiating in (b) comprises formation of embryoid bodies (EBs) in a floating culture. In some embodiments, the differentiating in (b) comprises plating the EBs onto coated plates. In some embodiments, the coated plates are collagen I coated plates. In some embodiments, the method further comprises, prior to (c), (b1) differentiating the hiPSCs into keratinocytes. In some embodiments, the differentiating in (b1) comprises employing all-trans retinoic acid (ATRA) and L-ascorbic acid (L-AA) to induce the hiPSC to form ectoderm and then the addition of bone morphogenic protein-4 (BMP-4) and epidermal growth factor (EGF). In some embodiments, the differentiating in (b1) is according to a sequential differentiation protocol. In some embodiments, the implanting in (c) comprises intradermal injection. In some embodiments, the implanting in (c) occurs at 15-19 days in vitro (DIV). In some embodiments, the implanting in (c) occurs 16-18 DIV. In some embodiments, the HFBSCs have not yet started expressing the keratinocyte associated molecules KRT5 and KRT14. In some embodiments, the method further comprises treating hair loss and/or a condition in a subject in need thereof, the condition is alopecia, ectodermal dysplasia, monilethrix, Netherton syndrome, Menkes disease, or hereditary epidermolysis bullosa. In some embodiments, the subject is a human subject.
In certain aspects, disclosed herein is a composition, comprising (a) hair follicle bulge stem cells (HFBSCs); and (b) media, wherein the HFBSCs express markers CD200, ITGA6, ITGB1, KRT15, KRT18, KRT19, and P63.
In some embodiments, the HFBSCs do not express the keratinocyte associated molecules KRT5 or KRT14. In some embodiments, the composition is made by a process comprising: (a) preparing human induced pluripotent stem cells (hiPSCs); and (b) differentiating the hiPSCs into the HFBSCs. In some embodiments, the hiPSCs have one, two, three, four, five, six, or more markers selected from the group consisting of CD200, ITGA6, ITGB1, OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81 and SSEA4. In some embodiments, the preparing hiPSCs in (a) comprises introducing, by electroporation, non-integrating episomal plasmid vectors encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28 and an shRNA for human p531. In some embodiments, the differentiating in (b) comprises formation of embryoid bodies (EBs) in a floating culture. In some embodiments, the differentiating in (b) comprises plating the EBs onto coated plates. In some embodiments, the coated plates are collagen I coated plates. In some embodiments, the HFBSCs is made by the method disclosed herein.
In certain aspects, disclosed herein is a hair follicle replacement method, comprising: (a) obtaining human pluripotent stem cells (hPSCs); (b) differentiating the hPSCs, thereby producing differentiated hPSCs toward becoming keratinocytes; (c) capturing and isolating at least a portion of the differentiated hPSCs, wherein the portion of the differentiated hPSCs expresses hair follicle bulge stem cell markers (HFBSCM); and (d) transplanting the portion of the differentiated hPSCs into a patient in need thereof.
In some embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs). In some embodiments, the hPSCs are hiPSC-derived hair follicle bulge stem cells (hiPSC-HFBSC). In some embodiments, the hiPSCs are derived from cells of the patient. In some embodiments, the cells of the patient are selected from the group consisting of fibroblasts, renal epithelial cells, and blood cells. In some embodiments, the transplanting in (d) occurs at least 15 days after the differentiating in (b). In some embodiments, the transplanting in (d) occurs at least 16 days after the differentiating in (b). In some embodiments, the transplanting in (d) occurs at least 17 days after the differentiating in (b). In some embodiments, the transplanting in (d) occurs at least 18 days after the differentiating in (b). In some embodiments, the transplanting in (d) occurs at least 19 days after the differentiating in (b). In some embodiments, the portion of the differentiated hPSCs is at a stage before the portion of the differentiated hPSCs has experienced downregulation of key integrins and key surface glycoproteins and before the portion of the differentiated hPSCs has started expressing keratinocyte-associated molecules. In some embodiments, the key integrins comprise at least integrin α6 and/or integrin β1. In some embodiments, the key surface glycoproteins comprise at least CD200. In some embodiments, the stage is before the portion of the differentiated hPSCs has started expressing keratinocyte-associated molecules KRT5 and KRT14. In some embodiments, the stage is after the portion of the differentiated hPSCs has expressed at least one of KRT15, KRT18, and KRT19, and P63. In some embodiments, the transplanting in (d) comprises transplanting the portion of the differentiated hPSCs intradermally above the muscle coat. In some embodiments, the capturing and isolating in (c) comprises flow cytometry. In some embodiments, the capturing and isolating in (c) comprises capturing and isolating cells that co-express CD200 and integrin α6. In some embodiments, the capturing and isolating in (c) comprises capturing and isolating cells that co-express CD200 and integrin β1. In some embodiments, the patient in need thereof has alopecia or has lost hair follicles from an injury or burn.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
As disclosed herein, when using human induced pluripotent stem cells (hiPSCs) to achieve hair follicle (HF) replacement, one can emulate the earliest fundamental developmental processes of gastrulation, ectodermal lineage commitment, and dermo-genesis. Viewing hiPSCs as a model of the epiblast, mapping the dynamic up- and down-regulation of the developmental molecules that determine HF lineage can provide insights to ascertain the precise differentiation stage and molecular requirements for grafting HF-generating progenitors. To yield an integrin-dependent lineage like the HF in vivo, hiPSC derivatives may need to be co-expressed, just prior to transplantation, the following combination of markers: integrins a6 and b1 and the glycoprotein CD200 on their surface; and, intracellularly, the epithelial marker keratin 18 and the hair follicle bulge stem cell (HFBSC)-defining molecules transcription factor P63 and the keratins 15 and 19. If the degree of trichogenic responsiveness indicated by the presence of these molecules is not achieved (they peak on Days 11-18 of the protocol), HF generation may not be possible. Conversely, if differentiation of the cells is allowed to proceed beyond the transient intermediate progenitor state represented by the HFBSC, and instead cascades to their becoming keratin 14 keratin 5 CD200− keratinocytes (Day 25), HF generation is equally impossible. Day 16-18 of differentiation may be the preferred time for transplanting. At this time point, the hiPSCs have lost pluripotency, have attained optimal expression of HFBSC markers, have not yet experienced downregulation of key integrins and surface glycoproteins, have not yet started expressing keratinocyte-associated molecules, and have sufficient proliferative capacity to allow a well-populated graft. This panel of markers may be used for isolating (by cytometry) HF-generating derivatives away from cell types unsuited for this therapy as well as for identifying trichogenic drugs.
Regenerative Medicine seeks to use stem cells to replace cells that have undergone destruction or senescence and death. Among the most sought cell types are hair follicles (HFs) for patients with inherited or immunogenic alopecia, or alopecia following severe wounding (e.g., burns, trauma, or surgery), or androgenetic alopecia (Chueh, S. C., et al. Therapeutic strategy for hair regeneration: hair cycle activation, niche environment modulation, wound-induced follicle neogenesis, and stem cell engineering. Expert Opin Biol Ther. 2013; 13(3):377-391). There has long been a debate over what qualities a stem cell and its derivatives should possess to enable HF replacement (see, e.g., Veraitch, O., et al. Human induced pluripotent stem cell-derived ectodermal precursor cells contribute to hair follicle morphogenesis in vivo. J Invest Dermatol. 2013; 133(6):1479-1488; and Yang, R., et al. Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells. Nat Commun. 2014; 5:3071). Herein presented the developmental and molecular requirements that human pluripotent stem cells (hPSCs) and their derivatives must acquire to reconstitute HFs, whether using human embryonic stem cells (hESCs) or, more immunologically desirable, patient recipient-specific human induced pluripotent stem cells (hiPSCs). These insights into successful engraftment were gained by examining the dynamics of up- and down-regulation of the key molecules that ensure proper HF lineage commitment by hPSCs. hPSCs intended for HF replacement may need to co-express the following combination of markers: key components of the integrin signaling pathway-integrin a6 (ITGA6) (see, e.g., Li, A. et al. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA. 1998; 95(7):3902-3907; and Ma, D. R., et al. A review: the location, molecular characterisation and multipotency of hair follicle epidermal stem cells. Ann Acad Med Singapore. 2004; 33(6):784-788) and integrin 131 (ITGB1) (see, e.g., Bata-Csorgo, Z., et al. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes. J Clin Invest. 1995; 95(1):317-327; Jones, P. H. Epithelial stem cells. Bioessays. 1997; 19(8):683-690; and Jones, P. H. et al. Stem cell patterning and fate in human epidermis. Cell. 1995; 80(1):83-93; and Jones, P. H. et al. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell. 1993; 73(4):713-724) and, just prior to transplantation, the surface glycoprotein CD200 (see, e.g., Ohyama, M. Hair follicle bulge: a fascinating reservoir of epithelial stem cells. J Dermatol Sci. 2007; 46(2):81-89; and Ohyama, M. et al. Characterization and isolation of stem cell-enriched human hair follicle bulge cells. J Clin Invest. 2006; 116(1):249-260) together with keratin 18 (KRT18) (an epithelial marker) (Maurer, J. et al. Contrasting expression of keratins in mouse and human embryonic stem cells. PLoS One. 2008; 3(10):e3451) and the following hair follicle bulge stem cell (HFBSC) markers: transcription factor P63 (see, e.g., Ma, D. R. et al. (2004), and Pallegrini, G. et al. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA. 2001; 98(6):3156-3161), keratin 15 (KRT15) ((see, e.g., Ma, D. R. et al. (2004), and Lyle, S., et al. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. J Cell Sci. 1998; 111(Pt 21):3179-3188), and keratin 19 (KRT19) (see, e.g., Ma, D. R. et al. (2004); Commo, S. et al. The human hair follicle contains two distinct K19 positive compartments in the outer root sheath: a unifying hypothesis for stem cell reservoir?Differentiation. 2000; 66(4-5):157-164; and Michel, M. et al. Germain L. Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. J Cell Sci. 1996; 109(Pt 5):1017-1028) CD200, ITGA6, and ITGB1 on the surface of the hPSC likely interact with specific matrix receptors that not only mediate cell adhesion, survival, and proliferation (Rowland, T. J., Roles of integrins in human induced pluripotent stem cell growth on Matrigel and vitronectin. Stem Cells Dev. 2010; 19(8):1231-1240), but also entrance into a differentiation pathway that allows the emergence of HFBSCs in vitro and HFs in vivo. If the developmental stage and degree of responsiveness indicated by the presence of these markers is not achieved, HF replacement will not be possible. In other words, to yield optimal HFs in vivo, we make the developmental case for transplanting at Day 16 of differentiation—the point at which the hPSCs have lost almost all expression of pluripotency markers; have attained optimal expression levels of HFBSC markers but not yet progressed toward a keratinocyte fate; have not yet experienced downregulation of key integrins and surface CD200; and possess sufficient proliferative capacity to produce a well-populated graft. In fact, the necessity of these markers may be used to separate hiPSC derivatives that will yield HFs away from a heterogeneous population of derivatives that are disadvantageous for this therapeutic indication or perhaps even inimical (i.e., tumorigenic). The markers may also be used to identify trichogenic drugs.
Disclosed herein is a method of growing a hair follicle. The method comprises: (a) preparing human induced pluripotent stem cells (hiPSCs); (b) differentiating the hiPSCs into hair follicle bulge stem cells (HFBSCs); and (c) implanting the HFBSCs into skin of a subject.
In some embodiments, the hiPSCs have at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or more markers selected from the group consisting of CD200, ITGA6, ITGB1, OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and SSEA4. In some embodiments, the hiPSCs have 1, 2, 3, 4, 5, 6, 7, or 8 markers selected from the group consisting of CD200, ITGA6, ITGB1, OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and SSEA4. In some embodiments, the hiPSCs have all of the markers of CD200, ITGA6, ITGB1, OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and SSEA4.
In some embodiments, the HFBSCs have at least one, at least two, at least three, at least four, at least five, at least six, or more markers selected from the group consisting of CD200, ITGA6, ITGB1, KRT15, KRT18, KRT19, and P63. In some embodiments, the HFBSCs have one, two, three, four, five, or six markers selected from the group consisting of CD200, ITGA6, ITGB1, KRT15, KRT18, KRT19, and P63. In some embodiments, the HFBSCs have all markers of CD200, ITGA6, ITGB1, KRT15, KRT18, KRT19, and P63.
In some embodiments, the preparing in (a) comprises introducing, by electroporation, non-integrating episomal plasmid vectors encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28 and an shRNA for human p531. In some embodiments, the electroporation is via a Neon transfection system.
In some embodiments, the differentiating in (b) comprises formation of embryoid bodies (EBs) in a floating culture. In some embodiments, the differentiating in (b) comprises plating the EBs onto coated plates. In some embodiments, the coated plates are collagen I coated plates.
In some embodiments, the method further comprises, prior to (c), (b1) differentiating the hiPSCs into keratinocytes. In some embodiments, the differentiating in (b1) comprises employing all-trans retinoic acid (ATRA) and L-ascorbic acid (L-AA) to induce the hiPSC to form ectoderm and then the addition of bone morphogenic protein-4 (BMP-4) and epidermal growth factor (EGF). In some embodiments, the differentiating in (b1) is according to a sequential differentiation protocol.
In some embodiments, the implanting in (c) comprises intradermal injection. In some embodiments, the implanting in (c) occurs at 15-19 days in vitro (DIV). In some embodiments, the implanting in (c) occurs at 16-18 DIV. In some embodiments, the implanting in (c) occurs at 15 DIV. In some embodiments, the implanting in (c) occurs at 16 DIV. In some embodiments, the implanting in (c) occurs at 17 DIV. In some embodiments, the implanting in (c) occurs at 18 DIV. In some embodiments, the implanting in (c) occurs at 19 DIV.
In some embodiments, the HFBSCs have not yet started expressing the keratinocyte associated molecules KRT5 and KRT14.
In some embodiments, the method further comprises treating hair loss and/or a condition in a subject in need thereof. In some embodiments, the condition is alopecia, ectodermal dysplasia, monilethrix, Netherton syndrome, Menkes disease, or hereditary epidermolysis bullosa.
In some embodiments, the subject is a human subject.
In one aspect, the present disclosure provides a composition, comprising (a) hair follicle bulge stem cells (HFBSCs); and (b) media. In some embodiments, the HFBSCs express markers CD200, ITGA6, ITGB1, KRT15, KRT18, KRT19, and P63.
In some embodiments, the HFBSCs do not express the keratinocyte associated molecules KRT5 or KRT14. In some embodiments, the HFBSCs is made by any method disclosed herein in this application.
In some embodiments, the composition is made by a process comprising: (i) preparing human induced pluripotent stem cells (hiPSCs); and (ii) differentiating the hiPSCs into the HFBSCs.
In some embodiments, the hiPSCs have one, two, three, four, five, six, or more markers selected from the group consisting of CD200, ITGA6, ITGB1, OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81 and SSEA4. In some embodiments, the preparing hiPSCs in (i) comprises introducing, by electroporation, non-integrating episomal plasmid vectors encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28 and an shRNA for human p531.
In some embodiments, the differentiating in (ii) comprises formation of embryoid bodies (EBs) in a floating culture. In some embodiments, the differentiating in (ii) comprises plating the EBs onto coated plates. In some embodiments, the coated plates are collagen I coated plates.
In another aspect, the present disclosure provides a method for hair follicle replacement, the method comprising: (a) obtaining human pluripotent stem cells (hPSCs); (b) differentiating the hPSCs, thereby producing differentiated hPSCs toward becoming keratinocytes; (c) capturing and isolating at least a portion of the differentiated hPSCs, wherein the portion of the differentiated hPSCs expresses hair follicle bulge stem cell markers (HFBSCM); and (d) transplanting the portion of the differentiated hPSCs into a patient in need thereof.
In some embodiments, the hPSCs are human induced pluripotent stem cells (hiPSCs).
In some embodiments, the hPSCs are hiPSC-derived hair follicle bulge stem cells (hiPSC-HFBSC). In some embodiments, the hiPSCs are derived from cells of the patient. In some embodiments, the hiPSCs are derived from cells of another subject.
In some embodiments, the cells of the patient are selected from the group consisting of fibroblasts, renal epithelial cells, and blood cells.
In some embodiments, the transplanting in (d) occurs at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or more days after the differentiating in (b).
In some embodiments, the portion of the differentiated hPSCs is at a stage before the portion of the differentiated hPSCs has experienced downregulation of key integrins and key surface glycoproteins and before the portion of the differentiated hPSCs has started expressing keratinocyte-associated molecules.
In some embodiments, the key integrins comprise at least integrin α6 and/or integrin β1.
In some embodiments, the key surface glycoproteins comprise at least CD200.
In some embodiments, the stage is before the portion of the differentiated hPSCs has started expressing keratinocyte-associated molecules KRT5 and KRT14.
In some embodiments, the stage is after the portion of the differentiated hPSCs has expressed at least one of KRT15, KRT18, and KRT19, and P63.
In some embodiments, the transplanting in (d) comprises transplanting the portion of the differentiated hPSCs intradermally above the muscle coat.
In some embodiments, the capturing and isolating in (c) comprises flow cytometry.
In some embodiments, the capturing and isolating in (c) comprises capturing and isolating cells that co-express CD200 and integrin α6.
In some embodiments, the capturing and isolating in (c) comprises capturing and isolating cells that co-express CD200 and integrin β1.
In some embodiments, the patient in need thereof has alopecia or has lost hair follicles from an injury or burn.
Abbreviations used:
ATRA, all-trans retinoic acid; BMP, bone morphogenic protein; DAPI, 4, 6-Diamidino-2-phenylindole; DIV, days in vitro; EB, embryoid body; EGF, epidermal growth factor; FAD, a mixture of Ham's F12 and Dulbecco's Modified Eagle's Medium; FC, flow cytometry; hESCs, Human embryonic stem cells; HF, hair follicle; HFBSC, hair follicle bulge stem cell; hiPSC, human induced pluripotent stem cells; hiPSC-HFBSC, hiPSC-derived hair follicle bulge stem cells; hPSCs, human pluripotent stem cells; HSCA, human specific cytoplasmic antigen; HSNA, human specific nuclear antigen; ICC, immunocytochemistry; ITGA6, integrin a6; ITGB1, integrin 131; KRT, keratin; L-AA, L-ascorbic acid; LEF1, lymphoid enhancer-binding factor 1; MDC, mouse dermal cells; MSX2, msh homeobox 2; PHLDA1, Pleckstrin Homology Like Domain Family A Member 1; TDAG51, T-Cell Death-Associated Gene 51; TRPS1, trichorhinophalangeal syndrome type I.
Experimental Section
Material & Methods
Human Pluripotent Stem Cells (hPSCs)
Two types of hPSCs were used. For hESCs, the “gold standard” for hPSCs, the H9 line (Wicell) was employed. The hiPSCs were generated from normal human skin fibroblasts (obtained from de-identified donors) by using a Neon transfection system to introduce, by electroporation, non-integrating episomal plasmid vectors encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28 and an shRNA for human p53 (see, e.g., Okita K., et al. A more efficient method to generate integration-free human iPS cells. Nat Methods. 2011; 8(5):409-412).
Characterization of hiPSCs
As previously described (see, e.g., Imaizumi Y. et al. Mitochondrial dysfunction associated with increased oxidative stress and alpha-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue. Mol Brain. 2012; 5:35; Ohta, S. et al. Generation of human melanocytes from induced pluripotent stem cells. PLoS One. 2011; 6(1):e16182; and Takahashi K., et al. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc. 2007; 2(12):3081-3089), hiPSCs colonies were characterized in vitro by both immune-cytochemistry (ICC) and flow cytometry (FC) for the presence of the following standard pluripotency markers using monoclonal antibodies against OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81 & SSEA4. Pluripotency of the hiPSCs was further confirmed by proving their ability to form teratomas when injected into the flanks of immuno-incompetent (NSG) mice (Jackson Laboratory). The teratomas were examined histologically for the presence of cells representative of the 3 fundamental primitive germ layers (ectoderm, mesoderm, and endoderm), hence confirming pluripotency as disclosed herein.
Differentiation of hiPSCs into HFBSCs
As a continuation of previous work (Ibrahim M. R, et al. Deriving keratinocyte progenitor cells and keratinocytes from human-induced pluripotent stem cells. Curr Protoc Stem Cell Biol. 2020; 54(1):e119), the overall strategy was to differentiate hiPSCs toward becoming keratinocytes but, just prior to that end-point, capture and isolate an intermediate transient progenitor cell population in vitro that expressed “HFBSCs” markers and could generated HFs. As schematized in
More detailed explanation of the protocol is shown in
In Vitro Characterization of hiPSC-Derived HFBSCs (“hiPSC-HFBSCs”)
ICC was performed using primary monoclonal antibodies against KRT18 (an epithelial marker); ITGA6, ITGB1, P63, KRT15, and KRT19 (HFBSCs markers); and KRT14 (a terminally differentiated keratinocyte marker). The temporal expression of CD200, ITGA6, and ITGB1 were monitored in differentiating hiPSCs using FC analysis. Relative gene expression of OCT4, P63, KRT15, KRT19, KRT8, KRT18, KRT5, and KRT14 in hiPSC-derived cells at days in vitro (DIV) 0, 11, 18, and 25 was assessed using qPCR analysis as described herein.
Co-Culture of hiPSC-HFBSCs & Mouse Dermal Cells (MDCs) in Vitro
hiPSC-HFBSCs were co-cultured with freshly isolated MDCs using 2 systems: (1) a transwell system, which allows 2 types of cells in monolayer to communicate with each other, but only via diffusible factors because they are separated by a porous membrane that allows the transit of only molecules but not cells; and (2) a 3-dimensional (3D) aggregate of the 2 cell types which allows cell-cell contact. Co-culturing was performed for 1 week, from DIV 11 to DIV 18. For the transwell co-cultures, 2.5×105 hiPSC-HFBSCs were seeded onto a collagen coated surface in the bottom well while an equal number of MDCs were seeded onto permeable transwell inserts in the top well (Corning, Corning, NY). For the 3D co-culture system, equal numbers of hiPSC-HFBSCs and MDCs (2.5×105) were mixed together in a Matrigel supported aggregate (1:1 mixture of Matrigel and “modified FAD medium”) supplemented with BMP-4 (25 ng/mL), ATRA (1 mM), and EGF (20 ng/mL). qPCR analysis of hair differentiation markers was performed on the co-cultured hiPSC-HFBSCs at DIV 18. The HF-associated genes assayed included KRT75, msh homeobox 2 (MSX2), lymphoid enhancer-binding factor 1 (LEF1), and trichorhinophalangeal syndrome type I (TRPS1) (see, e.g., Fantauzzo, K. A. et al. Dynamic expression of the zinc-finger transcription factor Trps1 during hair follicle morphogenesis and cycling. Gene Expr Patterns. 2008; 8(2):51-57; Gu, L. H. and Coulombe, P. A. Keratin expression provides novel insight into the morphogenesis and function of the companion layer in hair follicles. J Invest Dermatol. 2007; 127(5):1061-; 1073; Kobielak, A. and Fuchs, E. Links between alpha-catenin, NF-kappaB, and squamous cell carcinoma in skin. Proc Natl Acad Sci USA. 2006; 103(7):2322-2327; Kobielak, A. and Fuchs, F. The new keratin nomenclature. J Invest Dermatol. 2006; 126(11):2366-2368; and Rendl, M. et al. Molecular dissection of mesenchymal-epithelial interactions in the hair follicle. PLoS Biol. 2005; 3(11):e331).
Transplantation and Characterization of hiPSC-HFBSCs In Vivo
Patch grafting assays were performed as described previously (see, e.g., Kobayashi, T. et al. Canine follicle stem cell candidates reside in the bulge and share characteristic features with human bulge cells. J Invest Dermatol. 2010; 130(8):1988-1995; and Zheng, Y. et al. Organogenesis from dissociated cells: generation of mature cycling hair follicles from skin-derived cells. J Invest Derma-tol. 2005; 124(5):867-876). hiPSC-HFBSCs were combined on DIV 16 with freshly isolated MDCs from the backs of black C57BL/6 mice. Equal numbers of HFBSCs and MDCs (2.5×106 each) were combined in 100 mL phosphate buffered saline (PBS) and injected intradermally into anesthetized (general) SCID mice above the muscle coat, where the cells could remain tightly packed and in contact with each other with little dispersion. Intradermal injection, in contrast to subcutaneous injection, not only prevented dispersion of the cells (cell aggregation and cell-cell interaction proving critical for organogenesis) but also provided a more appropriate microenvironment for the transplanted cells, the dermis being the natural milieu for growing hair.
Three-to-six weeks after implantation, the resulting growths were dissected, and processed for histological and immunohistochemical evaluation as described herein. The protocol was approved by the Sanford Burnham Prebys Institutional Animal Care and Use Committee.
Statistical Analysis
One-Way ANOVA was used to calculate the P values. A P-value of <0.05 was considered significant.
Reprogramming of Normal Human Skin Fibroblasts into Normal hiPSCs Using Episomal DNA Cocktail
To generate hiPSCs, normal human skin fibroblasts obtained from de-identified donors were electroporated using a Neon transfection system to introduce non-integrating episomal plasmid vectors encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28 and an shRNA for human p53 (Okita et al. (2011))
A. Thawing & Maintaining Human Skin Fibroblast Cells
B. Electroporation of Human Fibroblasts Using Neon Transfection System
C. Amplification of the Transfected Cells
D. Re-Seeding the Transfected Cells on Feeders
E. Reprogramming
F. Colony Pickup
Differentiation of hiPSCs into HFBSCs and Keratinocytes:
The goal of these procedures was to differentiate hiPSCs into keratinocytes and to capture the intermediate transient multipotent stem cell population in vitro called the “HFBSC”. To differentiate hiPSCs into keratinocytes, we used a sequential differentiation protocol that employs ATRA and L-AA to induce hiPSC to form ectoderm, which was then differentiated into KPCs and keratinocytes. In this protocol ectoderm is precluded from continuing to become neuroectoderm via BMP4 suppression. The protocol is divided into 2 parts; formation of EBs in floating culture and plating of the obtained EBs on collagen I coated plates.
A. Reagents
“DKSM medium” is a commercially-available serum-free medium optimized for the isolation and expansion of human keratinocytes without the need for bovine pituitary extract (BPE) supplementation or the use of fibroblast feeder layers. The abbreviation DKSM stands for “Defined Keratinocyte Serum Free Medium” and was obtained from Thermo Fisher Scientific (Cat #10744019). In this formulation, the growth-promoting activity of BPE has been emulated instead by defined trophic factors that can maintain cell morphology, physiological markers, and growth of the cultured keratinocytes. DKSM has a low calcium concentration (<0.1 mM) which maintains keratinocytes in an undifferentiated proliferative stage.
B. Media
Day 0:
Embryoid Body (EB) Formation
On the 2nd day (Day 1):
On Day 2:
From Day 3 to Day 7:
From Day 8 to Day 11:
From Day 12 to Day 25
To passage hiPSC-HFBSCs:
Immunocytochemistry (ICC)
Materials:
Prepare the Blocking Buffer (BB):
Procedure:
Immunofluorescence Staining of Cells for Flow Cytometry (FC)
Materials:
Prepare the Flow Buffer:
Procedure:
Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
RNA Isolation: (RNeasy Mini Kit, Qiagen, Cat. #74104)
Synthesize first-strand cDNA (SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR, Invitrogen, Catalog #11752-050)
Real-time PCR (SensiFAST™ SYBR® No-ROX Kit, Bioline, BIO-98005) Reaction mix composition: Prepare a PCR master mix. The volumes given in Table 5 are based on a standard 20 μL final reaction mix and can be scaled accordingly.
The conditions for 3-step cycling are presented in Table 6.
Patch Assay
Requirements:
Procedure: Sterilize the Animal (Betadine and Ethanol 70%)
Second Day (Day of Injection):
Immunohistochemistry (IHC)
Using Shandon Apparatus Technique
Materials Required:
Procedures:
Results
One aspect of the present disclosure is to exploit insights from the dynamic up- and down-regulation of the key developmental molecules that determine HF lineage commitment by hPSCs. The insights can ascertain the precise differentiation stage and molecular requirements, as indicated by surrogate biomarkers for successful HF replacement in vivo based on the conversion of hiPSCs into engraftable hair generating cells. The results can produce a strategy that entailed differentiating hiPSCs toward becoming keratinocytes in vitro but, just prior to that end-point, capturing and isolating an intermediate transient progenitor cell population, which expressed HFBSC-associated markers and could generate HFs in vivo before going on to become (which may be less desirable) non-HF-producing keratinocytes (See
For more details, see Ibrahim, M. R., et al. (2021). The Developmental & Molecular Requirements for Ensuring that Human Pluripotent Stem Cell-Derived Hair Follicle Bulge Stem Cells Have Acquired Competence for Hair Follicle Generation Following Transplantation. Cell transplantation, 30, 9636897211014820, the content of which is entirely incorporated herein by reference.
Generation and Characterization of hiPSCs
hiPSCs were generated de novo by electroporating into de-identified normal human skin fibroblasts, non-integrating episomal plasmid vectors encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28 and an shRNA for human p53 (Okita, K. (2011)). The hiPSC clones exhibited appropriate hPSC morphology and were isolated about 21-45 days post-transfection. Fourteen distinct clones were generated following 2 independent rounds of transfections. Of these 14 clones, 2 unrelated clones were selected at random for the studies described here.
Given that hESCs are the gold standard for hPSCs, hESCs were also studied through all subsequent steps in parallel with the hiPSCs for validation. Like the hESCs, the new hiPSCs expressed the following panel of pluripotency immunomarkers as determined by ICC and FC: the nuclear transcription factors NANOG, SOX2, and OCT4; the surface markers TRA-1-60, TRA-1-81, and SSEA4. Pluripotency of the hiPSC clones was further confirmed by demonstrating their ability to form teratomas containing cells representative of all 3 fundamental primitive germ layers in immunocompromised NSG mice as shown in
Generation, Characterization, & Differentiation of hiPSCs into HFBSCs
With the conversion of hiPSCs into floating EBs (
As shown in
Co-expression of CD200, ITGA6, and ITGB1 along with SOX2 on hESCs as well as on hiPSCs, in
By DIV 5, the EBs acquired a cystic morphology, at which time they were plated (
Expression Dynamics of the Molecules Determining hiPSC-HFBSC Fate
The hPSC-derived cells obtained by the differentiation protocol can be divided into 3 stages: hPSCs (Stage #1) becoming HFBSCs (Stage #2), and then, if no additional cues are presented (including cues from an in vivo environment following transplantation), progression to becoming mature keratinocytes (Stage #3).
Emergence of the expression of KRT8, KRT18, P63, KRT15, and KRT19 indicated differentiation of the hPSCs toward HFBSCs (see
The dynamic changes in these various lineage-determining molecules are detailed based on ICC, FC, and q-PCR.
By ICC and FC, expression of the pluripotency markers that defined the hPSCs decreased (See
With regard to the surface glycoprotein CD200, its peak expression appeared obligatory for HFBSC generation. CD200 was uniformly expressed by undifferentiated hPSC and continued to be expressed during ectodermal differentiation and differentiation of those ectodermal cells into HFBSCs until DIV 18. CD200 expression persisted even as that of the pluripotency markers disappeared during the earliest differentiation stages. However, starting at DIV 18 and reaching a nadir at DIV 25 (FIG. Panels A and B), CD200 began to downregulate (about 40% of cells have lost CD200 expression by DIV 25) concomitant with increased expression of KRT14 (
Hence, downregulation of CD200 with concomitant upregulation of KRT14 appeared to separate the HFBSC stage (Stage #2) from the keratinocyte stage (Stage #3). This “border zone” may hold translational significance if Stage #2 cells (hiPSC-HFBSCs) but not Stage 3 cells (KRT14+ keratinocytes), could engraft to yield HFs in vivo.
Before testing the above rationale/hypothesis directly via transplantation studies, one may need to determine whether Stage 2 cells had the competence to express hair differentiation markers when exposed to proper inductive cues (
The “bulge activation hypothesis” holds that signals from the dermal papillae (DP) (the mesenchymal component of the HF) stimulate resting HFBSCs to generate transient amplifying cells which can then form HFs and hair shafts in vivo (Catsarelis, G. et al. Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell. 1990; 61(7):1329-1337). Accordingly, an experiment was conducted to co-culture hiPSC-HFBSCs with freshly isolated MDCs for 1 week using two different paradigms (
Characterization In Vivo of hiPSC-HFBSCs Following Transplantation
In search for the optimal molecular profile for inducing hiPSCs to yield HF-competent HFBSCs, sustained co-expression of CD200, ITGA6 and ITGB1 together with emergence of other HFBSC-associated gene products such as P63, KRT15, KRT19, and KRT18 (i.e., Stage 2 cells) can be a solution. Towards that end, hiPSC-HFBSCs were translated into SCID mice at DIV 16. DIV 16 represented the time point at which there was maximal expression of CD200 (before its down-regulation, as shown by FC) as well as of KRT15, KRT19, and KRT18 (as shown by RT-PCR). Transplantation of the hiPSC-HFBSCs was accomplished by co-injection of HFBSCs and MDCs intradermally above the muscle coat, where the cells could remain tightly packed and in contact with each other with little dispersion. The hiPSC-derived HFBSCs yielded HF in vivo (
Positive immunoperoxidase staining (brown) of representative reconstituted HFs with an antibody against TDAG51 (a known HF stem cell marker) are shown in
Three to six weeks following transplantation, histological analysis showed that the injected Stage 2 cells aggregated to form small cystic spheres in the host dermis. Human-like multilayered epidermis was also formed in the grafts. The cysts consisted of both basal keratinocytes and stratified epidermis. Pilosebaceous units growing outward from the cyst were evident (
Human origin of the epithelial cells in the HFs and epidermis was confirmed by immunopositivity for the human-specific nuclear antigen (HSNA) which was detected in about 60-70% of growing HFs cells. Human derivation of the cells was further confirmed by the positive immunoreactivity for a human-specific cytoplasmic antigen (HSCA) (
A cell-based treatment for alopecia has a challenge to ascertain that newly generated HFs contain a mechanism for cycling. The donor-derived HFs bore a “bulge region” in vivo rendering them capable of cycling and renewal. Indeed, KRT15 expression in the bulge region of the chimeric HFs and in the basal layer of the epidermis (
Assessing the Necessary & Sufficient Elements for HF Generation
An important step in confirming the necessity and causality of the putative suite of developmental determinants was to first eliminate and then re-add each factor systematically and demonstrate first an inability and then a reacquisition of the ability to yield engraftable HF-generating HFBSCs. However, all of the seven molecules (markers) required for complete HF generation were also fundamental to the earliest stages of normal development. Neither of the seven genes can be knock-out, and certainly not all seven, (even conditionally) in the hPSCs without disrupting normal development and confounding interpretation of the experiments. Furthermore, they could be knocked out even in the starting fibroblasts prior to being reprogrammed into hiPSCs because each is obligatory for the reprogramming process itself as well as for subsequent expansion, self-renewal, and acquisition of pluripotency (see, e.g., Sellheyer, K. et al. PHLDA1 (TDAG51) is a follicular stem cell marker and differentiates between morphoeic basal cell carcinoma and desmoplastic trichoepithelioma. Br J Dermatol. 2011; 164(1):141-147; and Sellbeyer, K. et al. Follicular stem cell marker PHLDA1 (TDAG5 1) is superior to cytokeratin-20 in differentiating between trichoepithelioma and basal cell carcinoma in small biopsy specimens. J Cutan Pathol. 2011; 38(7):542-550). Given these limitations, a series of studies that would serve the same purpose as a “knock-out” but without perturbing the system and obfuscating interpretation through ambiguity, were performed, as detailed below.
The expression profile of each of the markers that hPSC-derived HFBSC ultimately achieve is mapped. Test transplants of cells were done at each epoch along the developmental trajectory from “epiblast” (i.e., undifferentiated hPSC) to “ectoderm” to “HFBSC” to “keratinocyte”. Each period had a different constellation of markers among the seven. The question is which constellation yielded HFs in vivo.
Similar to what has been observed above, P63, KRT15, KRT18, and KRT19 start to be expressed at DIV 11, peak at DIV18, and decrease by DIV 25. CD200, while expressed earlier than DIV 11 (when the cells are still in their pluripotent state), ebb by DIV 25, the point at which KRT5 and KRT14 expression becomes ascendant, heralding the emergence of keratinocytes. ITGA6 and ITGB1 are expressed starting at DIV 0 through DIV 25; if these integrins are not expressed, HF also fails to develop. Transplantation of cells prior to DIV 11 or after DIV 25 fails to yield HF. Therefore, we could conclude that HF generation requires the expression of CD200, ITGA6, ITGB1, KRT15, KRT18, KRT19, and P63 and that a constellation lacking one or more of these molecules was insufficient to yield HFs.
Therefore, transplantation at DIV 16-18 may be favored, at which time the cells have lost expression of pluripotency markers, continue to express integrins ITGA6 and ITGB1, have attained optimal expression levels of the HFBSC markers KRT15 and KRT19 (in conjunction with P63), express the epithelial marker KRT18, have not yet experienced downregulation of the surface glycoprotein CD200, have not yet started expressing the keratinocyte-associated molecules KRT5 and KRT14, and have attained optimal proliferative capacity to allow a well-populated graft. The optimal site for transplantation was intradermal and above the muscle coat to prevent cell dispersion. Transplantation of MDCs alone failed to yield HFs in vivo.
Analysis
To find out how best to produce and select pluripotent stem cell derivatives for reliable and efficient therapeutic HF replacement, a developmental approach to this question can be taken by generating HFs as they might emerge if starting in the epiblast (a stage emulated by hESCs and, for clinical use, patient-specific hiPSCs) and progressing iteratively through gastrulation and on to dermal organogenesis.
The dynamic up- and down-regulation of specific molecules that serve as landmarks for each stage of this developmental process can be mapped. From the mapped dynamic, one can find the precise point at which the appropriate molecular and developmental requirements were attained by the cell (as indicated by surrogate biomarkers) to yield optimal HF generation in vivo if transplanted. What emerged was a 3-stage profile that entailed differentiating hiPSCs (Stage #1) toward becoming keratinocytes in vitro (Stage 3) but, just prior to that endpoint, capturing and isolating an intermediate transient progenitor cell population (Stage #2), which are bona fide HFBSCs, not only based on their co-expression of HFBSC-associated molecules but, most importantly, by their ability to generate HFs in vivo upon transplantation. Cells that cascaded beyond “HFBSC Stage #2” into “keratinocyte Stage #3” lost that ability to generate HFs upon grafting. In short, to ensure that hiPSC-derived HFBSC have acquired the competence for HF generation, they must come to express, at the time of transplantation, CD200, ITGA, and ITGB1 on their surface, and KRT18, P63, KRT15, and KRT196, but not KRT5 or KRT14 (keratinocyte markers), intracellularly. Expression of the HFBSCs markers P63, KRT15, and KRT19 as well as the HFBSC-associated epithelial marker KRT18 increases significantly from DIV 11 until DIV 18 (marking the transition from hiPSC Stage #1 to HFBSC Stage #2. Although CD200 is expressed starting in Stage #1, it begins to wane at DIV 18, reaching its nadir at DIV 25. Keratinocyte markers KRT5 and KRT14 are low through Stages #1 and #2, but dominate by DIV 25. DIV 18 appears to represent a developmental border between Stage #2 (CD200 engraftable HF-yielding HFBSCs) and Stage #3 (unengraftable mature non-HF yielding KRT5/14 keratinocytes). Therefore, to yield HFs, transplantation, we learned, should take place after DIV 11 but no later than DIV 18; we have chosen DIV 16-18 as our optimal transplant time to insure that all pluripotency markers have downregulated and the HFBSC-associated molecules are at their peaks.
The expression of CD200, ITGA6 and ITGB1 on hiPSCs and hESCs is a sine qua non for the differentiation of hPSCs into HFBSCs is unexpected. The ICC and FC data not only clearly confirm their expression on Stage #1 cells, in association with other known pluripotency markers such as NANOG, SOX2, TRA-1-60, TRA-1-81, and SSEA4, but suggest that in the absence of such integrin-related molecules and glycoproteins, hPSCs cannot proceed to becoming HFBSC Stage #2 cells with competence for yielding HFs. There are two supporting evidences. First, the 3D co-cultures show that induction of the molecules mediating HF generation required cell-cell contact between receptive HFBSCs and trichogenic mesenchymal cells, an interaction mediated by these integrins. Second, downregulation of CD200 coincided with progression of Stage #2 HFBSCs toward a Stage #3 keratinocyte fate incapable of HF generation, while transplantation of the Stage #2 cells at the peak of their CD200 expression in conjunction with expression of the integrins consistently yielded HFs in vivo.
Beyond our empiric data, the expression of these surface glycoproteins makes biologic sense for creating hPSC-derived HFs. Integrins are transmembrane glycoproteins composed of an x subunit and a 13 subunit that are linked via non-covalent bonds. The x6 subunit associates with the 131 subunit or the 134 subunit to form x6131 and x6134 integrin heterodimers. x6131 is expressed on a variety of cell types and functions as a cellular receptor for matrix laminin42. ITGA6 is the x6 subunit (also known as CD49f); ITGB1 is the 131 subunit. x6131 integrins along with x6134 integrins confer functional characteristics to stem cells; x6131 may be the dominant integrin. More than 30 different stem cell types have been found to express ITGA6 on their membranes. During the reprogramming of fibroblasts into hiPSCs, ITGA6 is upregulated and focal adhesion kinase (FAK) is inactivated (via dephosphorylation). During differentiation of the hiPSCs, the converse takes place: ITGA6 levels diminish and FAK is activated (phosphorylated at residue Y397). Activation of ITGB1 also leads to FAK phosphorylation and reduction of NANOG, OCT4, and SOX2. Knockdown of ITGA6 can mimic ITGB1 activation and reduce or eliminate normal hESC and hiPSC colony development, self-renewal, and pluripotency. Hence, while the presence of these two arms of the integrin system are necessary for hPSC maintenance and differentiation, they may play dynamic opposing roles; obviously an equilibrium must be struck between them for first reprogramming and then development to proceed—in this case, all the way to becoming HF-competent HFBSCs. CD200 is a glycoprotein widely expressed on cancer stem cells (breast, colon, hematopoietic) and may be required to maintain growth, self-renew, metastasis, and to evade the immune system38. It is also a marker of HFBSCs (as well as limbal stem cells). This combination of surface molecules likely interacts with specific matrix receptors during culture which, in turn, influences the hiPSCs' adhesion, survival, proliferation, and entrance into particular differentiation “programs”. The expression of these molecules on hiPSCs appeared to ensure that the cells had acquired the molecular competence for becoming HFs in vivo. In fact, the necessity of these surface markers may be used for cytometry to extract hiPSC derivatives in Stage #2 that can yield HFs from a heterogeneous population that might contain cells disadvantageous for this therapeutic indication. For regulatory purposes, it might be a way to isolate therapeutic Stage #2 cells from undifferentiated and potentially tumorigenic hPSCs. These same markers, which are avatars for a desirable developmental trajectory, may be used to identify drugs that might enhance the efficiency of HF generation.
Based on the developmental dynamics mapped above, the transplantation at DIV 16-18 is favored. At DIV 16-18 the cells have lost expression of pluripotency markers, continue to express integrins ITGA6 and ITGB1, have attained optimal expression levels of the HFBSC-associated markers KRT15, KRT18, and KRT19 (as well as P63), have not yet experienced downregulation of CD200, have not yet started expressing the keratinocyte-associated molecules KRT5 and KRT14, and have attained optimal proliferative capacity to allow a well-populated graft. Transplantation should be done intradermally above the muscle coat, where the cells can remain tightly packed and in contact with each other with little dispersion. The guidance provided by the present study may bring us closer to replacing HFs lost in non-cicatricial and cicatricial alopecia through the use of a patient's own hiPSC-derived follicular stem cells.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims the benefit of U.S. Provisional Application No. 63/347,501, filed May 31, 2022, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63347501 | May 2022 | US |
