Journal Logo

Science of Aging, Part 2: Original Articles

The Rise of Induced Pluripotent Stem Cell Approach to Hair Restoration

Pinto, Antonella PhD; Terskikh, Alexey V. PhD

Author Information
Plastic and Reconstructive Surgery: December 2021 - Volume 148 - Issue 6S - p 39S-46S
doi: 10.1097/PRS.0000000000008785
  • Free

Abstract

Across time and cultures, hair conveys the image of health and prosperity or may indicate membership, affiliation, or a disease history. Although mainly considered a cosmetic rather than a medical condition, hair loss is a relevant social issue associated with diminished self-esteem, increased distress, and even depressive episode reflecting the important role that hair plays in many cultures around the world. Given the fundamental limitations of the current hair transplantation approaches (ie, limited amount of hairs genetically resistant to hair loss in the donor area) and the widely underestimated psychological consequences of hair loss, better options for hair restoration are urgently needed.

Hair loss, in particular androgenetic alopecia, which the most common form of hair loss in men, has genetic underpinnings1 that could be linked to the perturbations/imbalance in various signaling pathways2 hinting at a broader connection between hair loss and function of other tissues and organs. Recently, the remarkable complexity of hair follicle became quite apparent. Indeed, each follicle can be considered as a mini organ uniquely characterized by cycles in which many of the hair follicle structures are lost and then fully reconstituted with each cycle.3–5

Hair cycling capability is rooted in dedicated stem cell niches, which respond to cues from dermal papilla (DP) and the surrounding cells to become active or stay quiescent, during a highly coordinated series of bidirectional epithelial–mesenchymal interactions that control the growth activity of hair follicles since the very early stage of development.6,7 The first morphologically detectable step in hair follicle genesis is the formation of an epidermal placode, which recruits cells from the underlying dermis to form aggregates of mesenchymal cells, known as dermal condensates. These condensates mark the location of the new hair follicle and become the DP as it further compacts.8 During this growth period, epithelial cells in physical opposition to the DP begin to differentiate into concentric cylinders to form the central hair shaft (HS) that emerges from the skin surface. Melanocytes reside above the DP within the epithelial compartment and provide pigmentation to the HS.9 However, melanocyte stem cells are located in the bulge and maintained in close contact with resident bulge epithelial stem cells (EpSCs).10

Once morphogenesis is completed, follicles are prompted to enter the first hair cycle by an unknown stimulus presumed to emanate from the DP, whose continuous interaction with the epithelial compartment governs the transition between distinct hair cycle stages.8 The hair cycle consists of phases of growth (anagen), degeneration (catagen), and rest (telogen), coordinated by the balance between stimulators and inhibitors and repeated numerous times in adult life.6

Among many cell types present in hair follicle4,11,12 DP cells and epithelial cells constitute the functional core of hair follicle. Over half a century ago, Cohen13 and Oliver14 independently documented that transplantation of intact dermal papillae isolated from adult rats and guinea pigs into recipient skin induce de novo follicle development and hair growth. However, culturing DP cells in the dish diminishes their folliculogenic potential especially noticeable for human DP cells where attempts to improve culture conditions met with limited success.15,16 Notwithstanding, the 3D culture conditions provided significant improvement over 2D monolayers to propagate human DP cells in the dish.17 Such approach resulted in restoration of many genes expressed in naive DP cells freshly isolated from the human skin and enabled the formation of some hair follicle structures upon transplantation.17

Hair follicles have been successfully bioengineered by Tsuji and colleagues.18 In this approach, primary mouse back hair follicles were dissociated to isolate and amplify DP cells and epithelial cells, which were then aggregated around a nylon thread followed by transplantation of such construct.18 Alternatively, Cristiano and colleagues used a biomimetic approach combining 3D printed collagen matrix with human adult folliculogenic cells obtained from dissociated hair follicles to engineer human hair follicles.19

The iPSC-based approach provides a unique opportunity to apply a wealth of knowledge in the field of stem cell research to hair restoration. The first clinical trial of patient-derived iPSC therapy to replace and repair dying cells in retina has been recently launches in the United States by National Eye Institute, which is a part of the National Institutes of Health. This and other iPSC-based protocols currently examined by FDA, will pave the way for many iPSC-based therapies to be deployed in the near future.

Given remarkable amplification capacity of iPSC cells, this approach represents a virtually unlimited source of folliculogenic cells providing the differentiation protocols are efficient and robust. Similar to that observed for other iPSC-derived cells, it is likely that iPSC-derived dermal and epithelial cells represent immature fetal-like cellular states, which are more efficient at forming hair follicles upon transplantation compared to adult-derived cells. Such expectations are consistent with the bone fide developmental potential of fetal cells which are classically used for transplantation experiments.

Historically, EpSCs were among the first adult stem cells isolated from human skin and amplified in the dish giving rise to holoclones, meroclones, and paraclones with distinct clonogenic potential.20 Remarkably, in vitro amplified human cells EpSCs maintain their repopulation potential.21–23 Thirty years after this discovery, such in vitro amplified autologous engineered keratinocyte enabled the regeneration of the entire human epidermis.24 In addition, EpSCs capable of contributing to skin epidermis and hair follicles have been derived from human iPSC.25 Such human iPSC-derived EpSCs were recently used to repairing full-thickness skin defects in nude mice26 paving the way to the future applications in therapeutic applications.

The DP within hair follicles in different locations originate from two distinct developmental lineages, neural crest27 and mesenchyme.28 Within the beard and mustache hair in humans (whiskers in animals), DPCs are originated from neural crest. Curiously, the scalp hair in humans (which comprise mesenchyme originated DPCs) is distinct from beard and mustache hair both morphologically and with respect to the gene expression profile.29 The mechanism of DP cell development from dermal fibroblasts has been understood in great details the mechanism of mouse DP cell specification from dermal fibroblasts during development has been elucidated in a series of elegant papers7,30–32; the mechanism of specification of DPCs from neural crest has not been investigated. Based on these developmental origins, there should be at least two different routes of iPSCs differentiation into DP cells: through the neural crest intermediate and directly from mesenchymal lineage. More recently, a spontaneous formation of hair follicles within iPSC-derived skin-containing organoids has been described.33,34 Below we will examine and compare each of these approaches to hair regeneration.

GENERATION OF DP CELLS VIA MESENCHYMAL ROUTE; INDUCED DP-SUBSTITUTING CELLS

Most hair follicles covering animal and human body are encompassing DP cells derived from the mesenchymal germ layer. Recent work from Okano, Ohyama and colleagues explored the possibility to derive folliculogenic DP cells from iPSC via mesenchymal intermediate.35 Human bone marrow contains a small (0.1%) subset of self-renewing cells marked by high levels of LNGFR (CD271), THY-1(CD90), and VCAM-1 (CD106); these cells had robust multilineage differentiation potential36 raising intriguing possibility of directing these cells towards DP fate. Veraitch et al., took advantage of a novel protocol driving LNGFR(+) THY-1(+) cells from human iPSCs based on embryoid body formation and culturing cells in mesenchymal stem cells (MSC) serum-free medium containing PDGF, TGF-β, and FGF, reported to promote MSC.37 Indeed, iPSC-derived MSCs (iMSCs) were able to differentiate into osteoblast, adipocyte and chondrocyte, lineages described for human bone marrow LNGFR(+) THY-1(+) subset.35 To promote DP fates, Veraitch et al. employed treatment with retinoic acid and then to DP cell-activating culture (DPAC) medium containing WNT, BMP, and FGF factors which was developed to restored lost expression of DP genes in serially passaged hDP cells.15 This treatment resulted in downregulation of multipotency-related MSC signature (NANOG, ZSCAN10, FZD5, BMP7, and ZFP64) and upregulated hDP signature genes RGS2, BMP4, LEF1, BAMBI, DIO2, LPL, and SNCAIP715 producing cells termed induced DP-substituting cells (iDPSCs)35 (Fig. 1).

F1
Fig. 1.:
iDPSCs exhibit functional DP properties in vivo. Co-grafting of hKCs and hDPCs (A) or iDPSCs (B) covered with FBs gave rise to cystic structures with focal aggregates (arrows), which contained fine HF-like structures (arrowheads), suggesting DP properties of iDPSCs. C. a representative regenerated structure using a combination of iDPSCs+hKC+FBs. hDPCs or iDPSCs were stained red with CellBrite Orange Cytoplasmic Membrane Dye. (Veraitch, et al., (2017). Induction of hair follicle dermal papilla cell properties in human induced pluripotent stem cell-derived multipotent LNGFR(+)THY-1(+) mesenchymal cells. Scientific reports, 7, 42777. https://doi.org/10.1038/srep42777. Reprinted with permission from CC-BY 4.0.)

Most importantly, iDPSCs have contributed to formation of hair-like structures in vivo when co-transplanted with primary human keratinocytes and human fibroblasts.35 These proof-of-principle experiments have established a feasibility of human DP cells derivation from iPS cells via mesenchymal route recapitulating the developmental pathway of DP cells present in the majority of the 5 million of hair follicles in the body. Further optimization will be required to improve the efficiency of iDPSCs for hair follicle induction possibly eliminating the need of co-transplanting human fibroblasts.

GENERATION OF DP CELLS FROM THE NEURAL CREST CELLS

The first protocol for derivation of human embryonic stem (ES) cells into neural crest cells (NC) was developed by Studer and colleagues based on monolayer cultures.38 In parallel, we developed a simplified protocol based on small clusters of human ES cell colonies cultured in suspension.39 Because human ES cells do not produce much BMP2/440 such conversion of ES into NC in suspension does not require BMP inhibitors.39 The neural spheres produced within 5–6 days of such treatment attached to plastic and produce large numbers of emigrating cells devoted of SOX2 and expressing SOX10 and other neural crest markers.39 Subsequently, we have characterized molecular signature of these NC cells and serendipitously uncovered many features of cranial NC derived by human ES conversion in suspension.40 Indeed, the suspension protocol for deriving NC was subsequently used to generate and characterize multiple human cranial cell types.41–44

Around the same time, Okano and Sommer groups conducted lineage tracing using transgenic mice encoding Wnt1 promoter-driven Cre crossed to floxed-EGFP; both groups observed NC cells and NC-derived cells express EGFP. In the adult mice, all DP cells in the whisker follicles expressed EGFP supporting their NC and in particular cranial NC origin.45,46

The above findings prompted us to consider generating DP versus NC intermediate. We reasoned that DP cells bear similarities with most common dermal cells such as fibroblasts47 and also express mesenchymal stem cell (MSC) markers.48 Therefore, we enriched differentiating hESC-derived NCs cultures for mesenchymal cells using preferential adherence to tissue culture plastic, a known property of MSC.49 We observed about 20% of hESC-NC cultures routinely adhered to plastic. Consistent with the fact that cranial NC give rise to multiple mesenchymal lineages,45 only ~50% of differentiated cells contained markers of DP cells such versican and alkaline phosphatase.50 We transplanted hESC-DP cells in Nude in combination with E18.5 epidermal cells from C57BL/6 mice using classical patch assay51 and observed induction of hair follicles, which occasionally penetrated the skin (Fig. 2). The dark coloration of HSs was most likely due to the presence of melanocyte progenitors within epidermal preparations from newborn black hair-bearing C57BL/6 mice. The efficiency of hair follicle induction by hESC-DP was comparable to that of control mouse dermal cells from newborn animals. To ensure that such growth is functionally driven by human cells (as opposed to mouse cells in the presence of human cells nearby), we have genetically engineered hESC to express EGFP and repeated the experiments. Using confocal microscopy, we have documented that hESC-DP cells have incorporated into the DP (in fact the majority of DP cells expressed EGFP). Mechanistically, we have documented that derivation of folliculogenic hESC-DP cells required BMP signaling,50 consistent with its role in mouse DP cells where it is required for their hair follicle-inductive properties.30

F2
Fig. 2.:
Folliculogenic capacity of human ES cells-derived DP cells. A. Stereo image of the whole mounts of mouse keratinocytes transplanted in combination with hESC-DPCs. B. Occasional hair shafts from A. grown through the skin. (C, D) GFP-positive DPs of newly formed hairs (GFP/bright field, confocal microscopy) are positive for Versican and Alkaline Phosphatase (AP). E. Quantification hairs induced by keratinocytes transplanted alone or in combination with mDC, hDP or hESC-DP. F. Dynamics of hair inductive capability of ESC-DP cells with time of differentiation from hESC-NC (day 0) shown as number of hairs formed per transplantation (trend visualized by the red line) or hESC differentiated in presence of serum (blue diamond) in comparison with keratinocytes alone (visualized by the dashed line). All data are represented as mean ±SEM and were analyzed with one-way ANOVA (Kruskal-Wallis test, Dunn’s Multiple Comparisons). *, P < 0.05; **, P < 0,001. Scale bars 1 mm. Gnedeva, K. et al. (2015). Derivation of hair-inducing cell from human pluripotent stem cells. PloS One, 10(1), e0116892. https://doi.org/10.1371/journal.pone.0116892. Reprinted with permission from CCBY 3.0 US.

Following these proof-of-principle studies, we set to overcome two major limitations of the original experiments with respect to clinical applications: first, the use of ES cells as a source of pluripotent cells and second, the use of fetal bovine serum which is a poorly defined reagent. We have revised and improved our original differentiation protocol to generate DP cells from human iPSC via NC-intermediate using a defined combination of growth factors that were selected to recapitulate the physiological steps of hair follicle development. Specifically, we employed Wnt10b, R-spondin, FGF20, and BMP6, which were previously implicated in mouse hair development. Indeed, Wnt signaling is the key pathway for hair follicle morphogenesis52 and R-spondin is a potent physiological enhancer of Wnt signaling.53,54 In addition, FGF20 governs the formation of primary and secondary dermal condensations in developing hair follicles,55 whereas BMP6 provides important signals to DP cells and is required for their hair follicle-inducing properties.30

It has been observed that cultured rodent DP cells spontaneously aggregate when delivered inside dermis forming a papillae-like clumps that apparently produced significant amount of extracellular matrix.56 In contrast, such spontaneous aggregation has not been observed in cultured human DP cells injected into skin.17 The aggregation of human iPSC-DPCs in spheroid-like structures resulted in upregulation of several genes (eg, syndecan-1 and integrin alpha-9) compared to a monolayer culture. We employed single-cell sequencing to compare gene signatures of DP cells grown as monolayers and upon aggregation. As expected, we observed that human iPSC-DPCs are distinct from freshly isolated human DP cells; both populations dramatically alter their expression profile upon aggregation into spheres. Depside remaining two distinct populations, both iPSC-DPCs and freshly prepared human DP cells, upregulates many common gene characteristics of 3D environment compared to monolayer grown human DP cells.15,17 We have demonstrated that human iPSC-DPs are capable of inducing hair follicles upon transplantation into Nude mice when combined with mouse or human EpSCs (Pinto et al., manuscript in submission).

SPONTANEOUS GENERATION OF HAIR FOLLICLES WITHIN SKIN ORGANOIDS

A spontaneous generation of hair follicles within skin organoids has been reported by the Koehler and colleagues.33,34 This differentiation strategy produced uniform epithelial cysts, which after an incubation period of 4–5 months, gave rise to stratified epidermis, fat-rich dermis, and pigmented hair follicles (Fig. 3). Clearly visible from day 18, such organoids gradually became bipolar, with the epidermal cyst partitioned to one pole (that the authors called the head) and an opaque cell mass at the opposite pole (referred to as the tail). The skin organoids reached a hair-bearing stage only after 70 days in culture when hair-germ-like buds began extending radially outward from the surface of organoids. After more than 100 days in culture, skin organoids were comparable to 18-week human fetal skin viewed from the dermal side with melanocytes distributed evenly throughout the epithelium and concentrated in the matrix region of hair follicles. Using immunostaining and electron microscopy, the organoid hair follicles showed most of the unique cellular layers of hair follicles, except the medulla layer, which is characteristic of adult terminal hair follicles, suggesting that the generated HFs are similar to those of vellus hairs akin to that of the skin of the cheek and outer ear.

F3
Fig. 3.:
Structure of hair follicles in the skin organoids. A, Schematic of a typical skin organoid. B, C, Dark-field images of day-140 skin organoids, showing adipose and HF bulb (B), and human fetal forehead skin at 18 weeks (C). Scale bars, 250 μm B; 100 μm C. Reprinted by permission from Springer Nature Customer Service Center GmbH: Spinger Nature, Nature, Hairbearing human skin generated entirely from pluripotent stem cells. Lee, J. et al., 2020.

CONCLUDING REMARKS

The advantages of iPSC-based approach stem from a unique nature of pluripotent cells. First, iPSCs have a unique in vitro amplification capacity of these cells while preserving genomic and epigenetic integrity, pluripotency, and differentiation potential.57 Such robust amplification enables generation of large numbers of iPCSs and, subsequently, differentiated cells that are required for therapeutic applications. Second, iPSCs have a potential to generate all cellular fates required to regenerate hair follicles making this an attractive one-stop-shop for several cell lineage required to build hair follicle. The generation and characterization of patient-specific iPSC cells have been continuously improving by many investigators and institutions58 culminating in a robust GMP manufacturing process,57 which could be cost-effective through the use of automation and robotics.59 The iPSC-based approach to hair regeneration still needs to overcome number of challenges, which are common to other regenerative medicine protocols based on pluripotent stem cells. One of the key problems is the development of robust and reproducible differentiation protocols, which generate homogeneous population at an appropriate maturation stage. At present, it seems that the generation of DP cells through the neural crest intermediate has the lowest barrier to enter the clinical trials in the next few years.

Current iPSC-based approaches are geared toward autologous transplantation. Although it ensures a perfect immunological match, the autologous approach is extremely expensive, limiting the number of potential patients. Several strategies are anticipated to enable the allogenic iPSC-based approach in the near future, for instance generating “universal” iPSCs line through HLA editing.60 Alternatively, one can envisage the development of very large iPSC banks to provide the best matching of the donor and recipient HLA types.61 Allogenic approach will inevitably bring down the cost of iPSC-based hair restoration, eventually to the level that is affordable for all who can benefit from the treatment.

REFERENCES

1. Hagenaars SP, Hill WD, Harris SE, et al. Genetic prediction of male pattern baldness. PLoS Genet. 2017;13:e1006594.
2. Heilmann-Heimbach S, Hochfeld LM, Paus R, et al. Hunting the genes in male-pattern alopecia: how important are they, how close are we and what will they tell us? Exp Dermatol. 2016;25:251–257.
3. Paus R, Cotsarelis G. The biology of hair follicles. N Engl J Med. 1999;341:491–497.
4. Sasaki GH. Review of human hair follicle biology: dynamics of niches and stem cell regulation for possible therapeutic hair stimulation for plastic surgeons. Aesthetic Plast Surg. 2019;43:253–266.
5. Castro AR, Logarinho E. Tissue engineering strategies for human hair follicle regeneration: How far from a hairy goal? Stem Cells Transl Med. 2020;9:342–350.
6. Müller-Röver S, Handjiski B, van der Veen C, et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol. 2001;117:3–15.
7. Rendl M, Lewis L, Fuchs E. Molecular dissection of mesenchymal-epithelial interactions in the hair follicle. PLoS Biol. 2005;3:e331.
8. Sennett R, Rendl M. Mesenchymal-epithelial interactions during hair follicle morphogenesis and cycling. Semin Cell Dev Biol. 2012;23:917–927.
9. Slominski A, Wortsman J, Plonka PM, et al. Hair follicle pigmentation. J Invest Dermatol. 2005;124:13–21.
10. Tanimura S, Tadokoro Y, Inomata K, et al. Hair follicle stem cells provide a functional niche for melanocyte stem cells. Cell Stem Cell. 2011;8:177–187.
11. Schneider MR, Schmidt-Ullrich R, Paus R. The hair follicle as a dynamic miniorgan. Curr Biol. 2009;19:R132–R142.
12. Chen CL, Huang WY, Wang EHC, et al. Functional complexity of hair follicle stem cell niche and therapeutic targeting of niche dysfunction for hair regeneration. J Biomed Sci. 2020;27:43.
13. Cohen J. The transplantation of individual rat and guineapig whisker papillae. J Embryol Exp Morphol. 1961;9:117–127.
14. Oliver RF. The induction of hair follicle formation in the adult hooded rat by vibrissa dermal papillae. J Embryol Exp Morphol. 1970;23:219–236.
15. Ohyama M, Kobayashi T, Sasaki T, et al. Restoration of the intrinsic properties of human dermal papilla in vitro. J Cell Sci. 2012;125(Pt 17):4114–4125.
16. Qiao J, Zawadzka A, Philips E, et al. Hair follicle neogenesis induced by cultured human scalp dermal papilla cells. Regen Med. 2009;4:667–676.
17. Higgins CA, Chen JC, Cerise JE, et al. Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth. Proc Natl Acad Sci U S A. 2013;110:19679–19688.
18. Toyoshima KE, Asakawa K, Ishibashi N, et al. Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches. Nat Commun. 2012;3:784.
19. Abaci HE, Coffman A, Doucet Y, et al. Tissue engineering of human hair follicles using a biomimetic developmental approach. Nat Commun. 2018;9:5301.
20. Barrandon Y, Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci U S A. 1987;84:2302–2306.
21. Morgan JR, Barrandon Y, Green H, et al. Expression of an exogenous growth hormone gene by transplantable human epidermal cells. Science. 1987;237:1476–1479.
22. Barrandon Y, Green H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-alpha and epidermal growth factor. Cell. 1987;50:1131–1137.
23. Barrandon Y, Li V, Green H. New techniques for the grafting of cultured human epidermal cells onto athymic animals. J Invest Dermatol. 1988;91:315–318.
24. Hirsch T, Rothoeft T, Teig N, et al. Regeneration of the entire human epidermis using transgenic stem cells. Nature. 2017;551:327–332.
25. Yang R, Zheng Y, Burrows M, et al. Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells. Nat Commun. 2014;5:3071.
26. Zhou H, Wang L, Zhang C, et al. Feasibility of repairing full-thickness skin defects by iPSC-derived epithelial stem cells seeded on a human acellular amniotic membrane. Stem Cell Res Ther. 2019;10:155.
27. Wong CE, Paratore C, Dours-Zimmermann MT, et al. Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin. J Cell Biol. 2006;175:1005–1015.
28. Paus R, Müller-Röver S, Van Der Veen C, et al. A comprehensive guide for the recognition and classification of distinct stages of hair follicle morphogenesis. J Invest Dermatol. 1999;113:523–532.
29. Rutberg SE, Kolpak ML, Gourley JA, et al. Differences in expression of specific biomarkers distinguish human beard from scalp dermal papilla cells. J Invest Dermatol. 2006;126:2583–2595.
30. Rendl M, Polak L, Fuchs E. BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties. Genes Dev. 2008;22:543–557.
31. Mok KW, Saxena N, Heitman N, et al. Dermal condensate niche fate specification occurs prior to formation and is placode progenitor dependent. Dev Cell. 2019;48:32–48.e5.
32. Zhang Y, Tomann P, Andl T, et al. Reciprocal requirements for EDA/EDAR/NF-kappaB and Wnt/beta-catenin signaling pathways in hair follicle induction. Dev Cell. 2009;17:49–61.
33. Lee J, Böscke R, Tang PC, et al. Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 2018;22:242–254.
34. Lee J, Rabbani CC, Gao H, et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature. 2020;582:399–404.
35. Veraitch O, Mabuchi Y, Matsuzaki Y, et al. Induction of hair follicle dermal papilla cell properties in human induced pluripotent stem cell-derived multipotent LNGFR(+)THY-1(+) mesenchymal cells. Sci Rep. 2017;7:42777.
36. Mabuchi Y, Morikawa S, Harada S, et al. LNGFR(+)THY-1(+)VCAM-1(hi+) cells reveal functionally distinct subpopulations in mesenchymal stem cells. Stem Cell Reports. 2013;1:152–165.
37. Ng F, Boucher S, Koh S, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood. 2008;112:295–307.
38. Lee G, Kim H, Elkabetz Y, et al. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat Biotechnol. 2007;25:1468–1475.
39. Curchoe CL, Maurer J, McKeown SJ, et al. Early acquisition of neural crest competence during hESCs neuralization. PLoS One. 2010;5:e13890.
40. Bajpai R, Coppola G, Kaul M, et al. Molecular stages of rapid and uniform neuralization of human embryonic stem cells. Cell Death Differ. 2009;16:807–825.
41. Rada-Iglesias A, Prescott SL, Wysocka J. Human genetic variation within neural crest enhancers: molecular and phenotypic implications. Philos Trans R Soc Lond B Biol Sci. 2013;368:20120360.
42. Rada-Iglesias A, Bajpai R, Prescott S, et al. Epigenomic annotation of enhancers predicts transcriptional regulators of human neural crest. Cell Stem Cell. 2012;11:633–648.
43. Bajpai R, Chen DA, Rada-Iglesias A, et al. CHD7 cooperates with PBAF to control multipotent neural crest formation. Nature. 2010;463:958–962.
44. Bayless NL, Greenberg RS, Swigut T, et al. Zika virus infection induces cranial neural crest cells to produce cytokines at levels detrimental for neurogenesis. Cell Host Microbe. 2016;20:423–428.
45. Shakhova O, Sommer L. Neural crest-derived stem cells. In: The Stem Cell Research Community. StemBook. Cambridge: epub, University of Zurich; 2008. Available at: http://www.zora.uzh.ch.
46. Nagoshi N, Shibata S, Kubota Y, et al. Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad. Cell Stem Cell. 2008;2:392–403.
47. Driskell RR, Clavel C, Rendl M, et al. Hair follicle dermal papilla cells at a glance. J Cell Sci. 2011;124(Pt 8):1179–1182.
48. Hoogduijn MJ, Gorjup E, Genever PG. Comparative characterization of hair follicle dermal stem cells and bone marrow mesenchymal stem cells. Stem Cells Dev. 2006;15:49–60.
49. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–147.
50. Gnedeva K, Vorotelyak E, Cimadamore F, et al. Derivation of hair-inducing cell from human pluripotent stem cells. PLoS One. 2015;10:e0116892.
51. Zheng Y, Du X, Wang W, et al. Organogenesis from dissociated cells: generation of mature cycling hair follicles from skin-derived cells. J Invest Dermatol. 2005;124:867–876.
52. Rabbani P, Takeo M, Chou W, et al. Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration. Cell. 2011;145:941–955.
53. Schuijers J, Junker JP, Mokry M, et al. Ascl2 acts as an R-spondin/Wnt-responsive switch to control stemness in intestinal crypts. Cell Stem Cell. 2015;16:158–170.
54. Hagner A, Shin W, Sinha S, et al. Transcriptional profiling of the adult hair follicle mesenchyme reveals R-spondin as a novel regulator of dermal progenitor function. iScience. 2020;23:101019.
55. Huh SH, Närhi K, Lindfors PH, et al. Fgf20 governs formation of primary and secondary dermal condensations in developing hair follicles. Genes Dev. 2013;27:450–458.
56. Jahoda CA, Oliver RF. Vibrissa dermal papilla cell aggregative behaviour in vivo and in vitro. J Embryol Exp Morphol. 1984;79:211–224.
57. Shafa M, Walsh T, Panchalingam KM, et al. Long-term stability and differentiation potential of cryopreserved cGMP-compliant human induced pluripotent stem cells. Int J Mol Sci. 2019;21:E108.
58. Chen Y, Tristan CA, Chen L, et al. A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Nat Methods. 2021;18:528–541.
59. Konagaya S, Ando T, Yamauchi T, et al. Long-term maintenance of human induced pluripotent stem cells by automated cell culture system. Sci Rep. 2015;5:16647.
60. Koga K, Wang B, Kaneko S. Current status and future perspectives of HLA-edited induced pluripotent stem cells. Inflamm Regen. 2020;40:23.
61. Lee S, Huh JY, Turner DM, et al. Repurposing the cord blood bank for haplobanking of HLA-homozygous iPSCs and their usefulness to multiple populations. Stem Cells. 2018;36:1552–1566.
Copyright © 2021 by the American Society of Plastic Surgeons