henhaobio
Sign in

NatureDevelopmental BiologyPeer-reviewed journal article

Hair-bearing human skin generated entirely from pluripotent stem cells

Jiyoon LeeiD, Cyrus C. RabbaniiD, Hongyu GaoiD, Matthew R. SteinhartiD, Benjamin M. WoodruffiD, Zachary E. Pflum, Alexander KimiD, Stefan HelleriD, Yunlong LiuiD, Taha Z. Shipchandler, Karl R. KoehleriD

PDFDOIPreprint
In-depth full-text digest available · Jump to digest ↓

Abstract

The skin is important for regulating bodily fluid retention and temperature, guarding against external stresses, and mediating touch and pain sensation. The skin is also susceptible to damage from burns, diseases, or genetic defects, which affect nearly one billion people worldwide1,2. For the advancement of skin regenerative therapies, it remains challenging to construct new skin with hair follicles and nerves in tissue cultures and in bioengineered skin grafts3-8. Here, we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use step-wise modulation of the TGF{beta} and FGF signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During 4-5 months incubation, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis, and pigmented hair follicles equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking human touch circuitry. Single-cell RNA sequencing and direct comparison to foetal specimens suggest that skin organoids are equivalent to human facial skin in the second-trimester of development. Moreover, we show that skin organoids produce planar hair-bearing skin when grafted on nude mice. Together, our results demonstrate the self-assembly of nearly complete skin tissue in vitro that can be used to reconstitute skin in vivo. We anticipate that our skin organoid model will be foundational to future studies of human skin development, disease modelling, or reconstructive surgery.

In-depth summary

Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 1 (8 Jul 2019)

Stepwise modulation of TGFβ, BMP, and FGF signaling in human pluripotent stem cell aggregates generates innervated, pigmented hair-bearing skin organoids that form planar human skin when grafted onto nude mice.

Conventional skin cultures and bioengineered grafts lack appendages such as hair follicles, sebaceous glands, and sensory nerve circuitry. Co-inducing cranial surface ectoderm and neural crest cells within a 3D aggregate recapitulates embryonic epidermal-mesenchymal interactions to self-assemble appendage-bearing human facial skin in vitro.

Key results

  1. ShownTimed TGFβ, BMP, and FGF modulation co-induces cranial surface ectoderm and pharyngeal arch 1 neural crest derivatives that self-organize into stratified skin cysts.

    Treating 3,500-cell hPSC aggregates with Matrigel, SB431542, FGF2, and BMP4 on day 0 and LDN-193189 and FGF2 on day 3 yielded epithelial cysts covered by PDGFRα+ mesenchymal and SOX10+P75+ neuro-glial cells; scRNA-seq of 18,190 day-29 cells (9,268 WA25; 9,013 DSP-GFP) showed 70-71% mesenchymal, 15-18% epidermal, and 4-10% neuro-glial cells with PRRX1+DLX1/4/5+PITX1+ facial identity. · Abstract; Optimized hPSC differentiation; Figure legends

  2. ShownSkin organoids reproducibly develop dozens of periodic hair follicles across multiple human embryonic and induced pluripotent stem cell lines and laboratories.

    Hair germs emerged at 70 ± 5 days in WA25 (n = 80) and 72 ± 4 days in DSP-GFP (n = 80) organoids, forming hair follicles in 87.4 ± 3.5% of WA25 (n = 212, 9 experiments), 87.2 ± 4.1% of DSP-GFP (n = 212, 9 experiments), and 70.8% (92/130) of WA01 organoids in an independent lab, averaging 64 (range 9-285) and 48 (range 7-128) follicles per WA25 and DSP-GFP organoid. · Abstract; Cell culture quantitative analysis: Frequency of…; Number of HFs produced; Protocol validation (Heller Lab)

  3. ShownLate-stage organoids differentiate melanocytes, adipocytes, cartilage, and sensory-like neuron networks targeting Merkel cells in hair follicle bulges.

    By 100-140 days, PMEL+ melanocytes produced pigmented hairs in 53.5% of WA25 (n = 137, 8 experiments) and 76.2% of DSP-GFP (n = 91, 5 experiments) organoids, while 100% of examined organoids (>30 across 7 experiments) formed TUJ1+NEFH+ axons with S100β+ glia innervating KRT20+ Merkel cells in follicle bulge regions, matching day-132 human fetal forehead skin. · Abstract; Frequency of HF pigmentation; Figure legends

  4. ShownXenografted cystic skin organoids unfurl and integrate into planar hair-bearing human skin with mature sebaceous glands and bulge stem cell niches in nude mice.

    Implanting tail-dissected day-140 WA25 organoids into 1-2 mm dorsal incisions on NU/J nude mice yielded outward hair growth in 55% of grafts (15/27 across 3 experiments; 22% ingrown, 22% failed), with rete ridge-like epidermal junctions, host CD49F+ vasculature, multi-lobed SCD1+ sebaceous glands, and KRT15+LHX2+NFATC1+ bulge stem cell-like cells. · Abstract; Xenograft Experiments; Analysis of HFs growth in xenografts; Figure legends

Methods, data and reproducibility

Aggregated 3,500 dissociated WA25, DSP-GFP, or WA01 hPSCs per well, induced surface ectoderm on day 0 (2% Matrigel, 10 μM SB431542, 4 ng/ml FGF2, 2.5 ng/ml BMP4) and cranial neural crest on day 3 (200 ng/ml LDN-193189, 50 μg/ml FGF2), and matured cysts in shaker culture up to 150 days. Profiled organoids via 10x scRNA-seq (Seurat 2 CCA), immunostaining, electron microscopy, and xenografting into NU/J nude mice.

  • 10x Genomics single-cell RNA-seq of day-29 WA25 hESC and DSP-GFP hiPSC skin organoids and day-48 tail-removed WA25 skin organoids · human · n = 18,190 day-29 cells (9,268 WA25; 9,013 DSP-GFP) and 5 day-48 WA25 organoids
  • Long-term skin organoid differentiation cultures from WA25 hESCs, DSP-GFP hiPSCs, and WA01 (H1) hESCs · human · n = 212 WA25 (9 runs), 212 DSP-GFP (9 runs), and 130 WA01 (1 run) organoids
  • De-identified second-trimester human fetal facial skin (day-132 forehead and cheek) and adult facial skin reference tissues · human
  • Day-140 WA25 skin organoid xenografts implanted into 5-6 week-old female immunodeficient nude (NU/J) mice · human, mouse · n = 27 xenografts across 3 surgeries
Methods & tools
CellRanger (version 2.1.0), STAR, Seurat 2, Imaris 8, ImageJ, GraphPad Prism 8, ggplot2, ggrepel, SPRING
Reagents & environment
10x Genomics CellRanger 2.1.0 with bcl2fastq and STAR; R with Seurat 2, ggplot2, and ggrepel; Imaris 8 (Bitplane); ImageJ (Cell Counter plugin); GraphPad Prism 8.
Experimental conditions
Three-dimensional confocal/multiphoton reconstructions were processed on workstations at the Indiana Centre for Biological Microscopy.
Reproducibility gaps
Data are stated as available from the corresponding author upon request, and GEO accessions for raw/processed scRNA-seq files and Supplementary Data 1-4 HTML files were deferred to the peer-reviewed version.
Limitations (4)
  • Skin organoids lack mesoderm-derived endothelial and immune cell populations such as vasculature, Langerhans cells, and macrophages prior to in vivo grafting. · stated by the authors
  • Because organoids form inverted cysts with the epidermis facing inward, dead squamous cells accumulate in the core and hair follicle morphology degrades after roughly 150 days in culture. · stated by the authors
  • Electrophysiological profiling is still required to confirm functional mechanosensation in the neuron-Merkel cell complexes, and >1 year tracking is needed to assess whether grafted follicles cycle. · stated by the authors
  • Transmission electron microscopy of day-140 organoid hair follicles did not capture clear images of the hair shaft medullary layer. · stated by the authors

Read this if you develop stem-cell organoids, model human craniofacial skin and hair follicle morphogenesis, or study cutaneous sensory innervation and skin regeneration.

Citations
491
FWCI
66.84
Field percentile
top 1%

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISPeking '20Stabell '23Le '25Moriwaki '23Gopee '23Kageyama '22
72% semantic matchCell Biology
P. Peking, L. Krisch, M. Wolf+9 more·bioRxiv·Sep 10, 2020