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Cited by 487Hair Growth and DisordersPluripotent Stem Cells ResearchSkin and Cellular Biology Research

Hair-bearing human skin generated entirely from pluripotent stem cells

Open access
Lee, Jiyoon, Rabbani, Cyrus C., Gao, Hongyu, Steinhart, Matthew R., Woodruff, Benjamin M., Pflum, Zachary E., Kim, Alexander, Heller, Stefan +3 more·2020·Nature
DOI 10.1038/s41586-020-2352-3PMC PMC7593871

Published version of a preprint

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Abstract

The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain 1,2. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met 3-9. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4-5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are 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 the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery.

AI snapshot

Written from the paper’s full text.

TL;DR

A stepwise human pluripotent stem cell organoid culture using TGFβ/FGF pathway modulation self-assembles hair-bearing skin with stratified epidermis, fat-rich dermis, pigmented hair follicles, sebaceous glands, sensory neurons, and Merkel cells. Single-cell RNA-sequencing and foetal comparison suggest second-trimester facial skin identity, and grafted organoids form planar hair-bearing skin on nude mice.

Key findings

  • Hair germs appeared at 70 ± 5 days for WA25 and 72 ± 4 days for DSP-GFP organoids; hair follicle production occurred in 87.4% of WA25, 87.2% of DSP-GFP, and 70.8% of WA01 organoids. Pigmented hair-bearing organoids formed in 53.5% of WA25 and 76.2% of DSP-GFP cultures (p=0.077).
  • scRNA-seq identified 23,329 cells at day-6 and 18,190 cells at day-29; day-6 populations were epithelial ~59%, neuro-glial ~8%, mesenchymal ~8%, and cycling ~25%, while day-29 was dominated by mesenchymal cells ~70%, with epithelial ~17%, neuro-glial ~7%, and cycling ~6%.
  • Organoids showed craniofacial identity, including first pharyngeal arch markers and lack of HOX gene expression, and were proposed as equivalent to human facial skin in the second trimester.
  • In xenografts, 55% of grafts (15 of 27, 3 experiments) grew 2–5 mm hairs, 22% had in-grown hairs, and 22% failed; no grafts showed ulceration or uncontrolled tumour-like growth.

Methods

  • hPSCs (WA25 hESC, DSP-GFP hiPSC, and WA01 validation) were aggregated at 3,500 cells per U-bottom well, differentiated with Matrigel, SB431542, FGF, and BMP-4, then LDN/FGF at day-3; organoids were matured in floating culture on an orbital shaker for up to ~150 days.
  • scRNA-seq used 10x Genomics Chromium, CellRanger, and Seurat; day-6 organoids (n=10) and day-29 organoids (n=5) were pooled, and day-48 WA25 organoids (n=6) had tail structures removed before dissociation.
  • Xenografts used day 113–145 hair-bearing WA25 organoids with tails removed, implanted into 1–2 mm incisions in back skin of 5–6-week-old female nude mice, then analysed by histology, immunostaining, and electron microscopy.

Limitations

  • The paper states that sweat glands were absent, that additional molecular and physiological profiling is needed to determine whether other somatosensory neuron subtypes and nerve endings can be induced, and that long-term (>1 year) observation is needed to determine terminal hair fate or growth cycling.
  • The paper notes that off-target cell lineages require additional control before clinical reconstitution, and that cell-line variability suggests further protocol optimization may be needed.
  • The text also makes evident that no statistical test was used to predetermine sample size, investigators were not blinded to treatment groups, and experiments were generally not randomized; xenograft failures and in-grown hairs were attributed partly to technical issues.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41586-020-2352-3

Cites

48 of 48 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

44 of 56 cited sources checked · 10 supported · 1 flagged · accuracy not yet measured

  • Interpretationown data
    § Abstract

    Within the neuro-glial group, PAX6+HES5+ neuroectoderm-like cells clustered near PLP1+SOX10+PAX3+ CNCC-like cells, suggesting that CNCC arise from a neuroectoderm intermediate.

    “Within the neuro-glial group, PAX6+HES5+ neuroectoderm-like cells clustered near PLP1+SOX10+PAX3+ CNCC-like cells (Fig. 2d), suggesting that CNCC arise from a neuroectoderm intermediate11.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] Tchieu, J. et al. A modular platform for differentiation of human PSCs into all major ectodermal lineages. Cell Stem Cell 21, 399–410 (2017). source

      In Results, “Expression analysis of purified cell types from the ectoderm” (page unavailable), the paper reports that neuroectoderm clustered near hESCs, whereas neural crest clustered near cranial placode—not neuroectoderm. It treats neuroectoderm and neural crest as distinct lineages and does not infer that cranial neural crest arises through a neuroectoderm intermediate. (LLM, conf=0.82)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation
    § Abstract

    Reconstruction of appendage-bearing skin in cultures and in bioengineered grafts remains an unmet biomedical challenge.

    “Reconstruction of appendage-bearing skin in cultures and in bioengineered grafts remains an unmet biomedical challenge3–9.”
    Quote in the paperSubstantial claim7 of 7 sources checked
    • cites [3] Yang, R. et al. Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells. Nat. Commun. 5, 3071 (2014). source

      The Abstract and Introduction describe obtaining folliculogenic human epithelial stem cells for tissue engineering as a challenge. They do not specifically say that reconstructing appendage-bearing skin in culture remains unmet. The Results (Characterization of iPSCs-derived EpSCs in vivo; page unavailable) report hair follicles and epidermis forming in experimental grafts on mice, so the paper does not support an unqualified claim that reconstruction in grafts remains unmet. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [4] Abaci, H. E. et al. Tissue engineering of human hair follicles using a biomimetic developmental approach. Nat. Commun. 9, 5301 (2018). source
    • cites [5] Gledhill, K. et al. Melanin transfer in human 3D skin equivalents generated exclusively from induced pluripotent stem cells. PLoS ONE 10, e0136713 (2015). source

      The Introduction (page unavailable) says current cultured human 3D skin equivalents lack appendages and that adding complex structures such as appendages remains a hurdle. The paper does not address whether reconstructing appendage-bearing skin in bioengineered grafts remains a challenge. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [6] Itoh, M. et al. Generation of 3D skin equivalents fully reconstituted from human induced pluripotent stem cells (iPSCs). PLoS ONE 8, e77673 (2013). source
    • cites [7] Lei, M. et al. Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells. Proc. Natl Acad. Sci. USA 114, E7101–E7110 (2017). source

      The Limitations and Waning Immunity passages discuss pertussis outbreaks, vaccine exemptions, and vaccine durability, not skin reconstruction. Searches for appendage-bearing skin, tissue-engineered grafts, hair follicles, and skin culture found no passage addressing the claim. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [8] Heitman, N., Saxena, N. & Rendl, M. Advancing insights into stem cell niche complexities with next-generation technologies. Curr. Opin. Cell Biol. 55, 87–95 (2018). source

      The Modeling complexity section says flat cultures lack tissue complexity and reports that older mouse skin clusters lost the ability to induce hair follicles, although this could be rescued. It also describes murine skin organoids that make hair and sebaceous glands. These passages support limitations in cultured skin reconstruction, but the paper does not address bioengineered skin grafts or characterize their reconstruction as an unmet challenge. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [9] Koehler, K. R. et al. Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells. Nat. Biotechnol. 35, 583–589 (2017). source

      The Abstract (page unavailable) describes human inner ear organoids and briefly mentions keratinocyte-like cells, but does not address reconstruction of appendage-bearing skin or bioengineered skin grafts. Searches for skin appendages, grafts, and the claimed unmet challenge found no passage addressing that point. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Premisecitation
    § Abstract

    TGFβ inhibition promotes ectoderm induction from pluripotent stem cells.

    “TGFβ inhibition promotes ectoderm induction from pluripotent stem cells, whereas BMP activation promotes surface ectoderm (alternatively, non-neural ectoderm) induction and suppresses neuroectoderm induction9–12.”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [9] Koehler, K. R. et al. Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells. Nat. Biotechnol. 35, 583–589 (2017). source
    • cites [10] Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D. & Hashino, E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013). source

      The paper’s concluding passage (Abstract; page unavailable) says TGFβ inhibition acts with BMP activation in the induction of non-neural ectoderm in vitro. It does not establish that TGFβ inhibition alone promotes ectoderm induction from pluripotent stem cells, as the cited clause could imply. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [11] Tchieu, J. et al. A modular platform for differentiation of human PSCs into all major ectodermal lineages. Cell Stem Cell 21, 399–410 (2017). source
    • cites [12] Wilson, P. A. & Hemmati-Brivanlou, A. Induction of epidermis and inhibition of neural fate by Bmp-4. Nature 376, 331–333 (1995). source
  • Premisecitation
    § Abstract

    Growing functional skin appendages in culture systems containing epidermal and dermal cells has been persistently difficult.

    “Culture systems containing epidermal and dermal cells have been used to model human skin outside of the body for research or clinical uses for over 40 years7,8,10; however, growing functional skin appendages in these systems has been persistently difficult.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [7] Lei, M. et al. Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells. Proc. Natl Acad. Sci. USA 114, E7101–E7110 (2017). source

      The Discussion and Limitations sections address pertussis vaccination, outbreaks, and nonmedical vaccine exemptions, not skin culture systems. Searches for functional skin appendages, epidermal and dermal cells, and related terms found no passage addressing the claim. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [8] Heitman, N., Saxena, N. & Rendl, M. Advancing insights into stem cell niche complexities with next-generation technologies. Curr. Opin. Cell Biol. 55, 87–95 (2018). source

      In “Modeling complexity: 2D co-cultures and 3D tissue-recapitulating organoids” (page unavailable), the review says 3D epidermal–dermal clusters from old mouse skin lost the ability to induce hair follicles, although that ability could be rescued. This supports a difficulty in a particular culture system, but does not establish that growing functional skin appendages has been persistently difficult across the skin culture systems described in the claim; the review also describes murine organoids capable of making hair and sebaceous glands. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [10] Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D. & Hashino, E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013). source

      The paper’s Abstract describes generating inner-ear sensory epithelia and mechanosensitive hair cells from mouse embryonic stem cells. Searches for skin appendages and epidermal–dermal culture systems found no passage addressing whether functional skin appendages are difficult to grow in those systems. The paper’s inner-ear hair cells are not skin appendages. (LLM, conf=0.77)

      Experimental — accuracy not yet measured
  • Findingown data
    § Abstract

    The epithelial group contained three epidermal sub-clusters: CXCL14+ basal, KRT1+ intermediate, and KRT4+ peridermal keratinocytes.

    “The epithelial group contained three epidermal sub-clusters: CXCL14+ basal, KRT1+ intermediate, and KRT4+ peridermal keratinocytes (Fig. 2e, Extended Data Fig. 5)22.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [22] Joost, S. et al. Single-cell transcriptomics reveals that differentiation and spatial signatures shape epidermal and hair follicle heterogeneity. Cell Syst. 3, 221–237 (2016). source

      The Results sections on unbiased clustering and subclustering describe murine epidermal and hair-follicle populations, including basal cells marked by Krt14 and Krt5. The searched passages do not describe an epithelial group divided into CXCL14+ basal, KRT1+ intermediate, and KRT4+ peridermal sub-clusters. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 15
  • May combine two claims. 26
  • The quote only partly states this claim. 2

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Abstract

    Culture systems containing epidermal and dermal cells have been used to model human skin outside of the body for research or clinical uses for over 40 years.

    “Culture systems containing epidermal and dermal cells have been used to model human skin outside of the body for research or clinical uses for over 40 years7,8,10; however, growing functional skin appendages in these systems has been persistently difficult.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [7] Lei, M. et al. Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells. Proc. Natl Acad. Sci. USA 114, E7101–E7110 (2017). source

      The Discussion addresses pertussis vaccination and community immunity, not skin culture systems. Searches for epidermal and dermal cell cultures, skin models, and the claimed 40-year history found no relevant passage in our copy. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [8] Heitman, N., Saxena, N. & Rendl, M. Advancing insights into stem cell niche complexities with next-generation technologies. Curr. Opin. Cell Biol. 55, 87–95 (2018). source

      The “Modeling complexity” section (page unavailable) describes cultured epidermal and dermal clusters as a skin model, supporting the general point that these cells have been used in culture. Its example uses mouse cells, however, and our copy does not establish use to model human skin for over 40 years or for clinical purposes. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [10] Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D. & Hashino, E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013). source

      The paper’s Abstract describes a 3D culture model of inner-ear development and notes an epithelium that mimics embryonic epidermis. Our copy does not address culture systems combining epidermal and dermal cells to model human skin, their research or clinical uses, or a 40-year history (searched for skin culture and its history). (LLM, conf=0.77)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation
    § Abstract

    Organoid HFs are periodically spaced throughout the epidermis, suggesting that appendage patterning mechanisms are preserved in organoids.

    “Moreover, organoid HFs are periodically spaced throughout the epidermis suggesting that appendage patterning mechanisms are preserved in organoids18.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Chuong, C.-M., Yeh, C.-Y., Jiang, T. X. & Widelitz, R. Module-based complexity formation: periodic patterning in feathers and hairs. Wiley Interdiscip. Rev. Dev. Biol. 2, 97–112 (2013). source

      Section 4, “Engineering of the Periodic Patterning Process” (page unavailable), reports evenly spaced hairs in reconstituted skin grown on a matrix on a mouse. This supports periodic spacing as a feature of hair formation, but the paper does not report hair follicles in organoids or establish that appendage-patterning mechanisms are preserved in organoids. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation
    § Abstract

    BMP activation promotes surface ectoderm induction and suppresses neuroectoderm induction.

    “TGFβ inhibition promotes ectoderm induction from pluripotent stem cells, whereas BMP activation promotes surface ectoderm (alternatively, non-neural ectoderm) induction and suppresses neuroectoderm induction9–12.”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [9] Koehler, K. R. et al. Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells. Nat. Biotechnol. 35, 583–589 (2017). source
    • cites [10] Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D. & Hashino, E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013). source
    • cites [11] Tchieu, J. et al. A modular platform for differentiation of human PSCs into all major ectodermal lineages. Cell Stem Cell 21, 399–410 (2017). source
    • cites [12] Wilson, P. A. & Hemmati-Brivanlou, A. Induction of epidermis and inhibition of neural fate by Bmp-4. Nature 376, 331–333 (1995). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Abstract

    Dermal fibroblasts have two embryonic origins: mesoderm cells (body and scalp skin) and cranial neural crest cells (CNCC; facial skin).

    “Dermal fibroblasts have two embryonic origins: mesoderm cells (body and scalp skin) and cranial neural crest cells (CNCC; facial skin)13,14.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [13] Minoux, M. & Rijli, F. M. Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development. Development 137, 2605–2621 (2010). source
    • cites [14] Driskell, R. R. et al. Distinct fibroblast lineages determine dermal architecture in skin development and repair. Nature 504, 277–281 (2013). source

      The paper’s Abstract describes two dermal fibroblast lineages that form the upper and lower dermis, not two embryonic origins. Searches for mesoderm, cranial neural crest, and the claimed body/scalp versus facial-skin distinction found no passage addressing those origins. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data
    § Abstract

    All cell sub-types were present in WA25 and DSP-GFP organoids, but in different proportions.

    “All cell sub-types were present in WA25 and DSP-GFP organoids, but in different proportions (Fig. 2b, c), suggesting further protocol optimization may be needed to reduce variability19.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [19] Velasco, S. et al. Individual brain organoids reproducibly form cell diversity of the human cerebral cortex. Nature 570, 523–527 (2019). source

      The paper’s Methods (Statistics and Reproducibility) lists the stem cell lines studied, but not WA25 or DSP-GFP. The paper reports consistent overall cell-type proportions across its own lines, so its findings cannot verify which subtypes were present in WA25 and DSP-GFP organoids or whether their proportions differed (searched: WA25, DSP-GFP, cell-type proportions). (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationown data
    § Abstract

    Presumptive dermal cells expressed FGF7 (Keratinocyte Growth Factor), which may be a key driver of epidermal stratification in this system.

    “Presumptive dermal cells expressed FGF7 (Keratinocyte Growth Factor), which may be a key driver of epidermal stratification in this system (Extended Data Fig. 6f)28,29.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [28] Zhu, X.-J. et al. BMP-FGF signaling axis mediates Wnt-induced epidermal stratification in developing mammalian skin. PLoS Genet. 10, e1004687 (2014). source
    • cites [29] Richardson, G. D. et al. KGF and EGF signalling block hair follicle induction and promote interfollicular epidermal fate in developing mouse skin. Development 136, 2153–2164 (2009). source

  • Classified as probably not a substantial claim.

    Premisecitation
    § Abstract

    Appendage-bearing skin organoids can be generated via directed differentiation of epidermal and dermal cells from mouse pluripotent stem cells.

    “We recently reported that appendage-bearing skin organoids can be generated via directed differentiation of epidermal and dermal cells from mouse pluripotent stem cells (PSCs)3.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [3] Yang, R. et al. Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells. Nat. Commun. 5, 3071 (2014). source

      The Results (Characterization of iPSCs-derived EpSCs in vivo) report hair-follicle formation after combining pluripotent-stem-cell-derived epithelial cells with dermal cells. But the epithelial cells came from human iPSCs, while the dermal cells were freshly isolated from neonatal mice—not differentiated from mouse PSCs. The cited study therefore does not support the claim that both cell types were directed-differentiated from mouse PSCs. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation
    § Abstract

    Neural clusters contained cells expressing SOX2 (neural progenitors), DCX (CNS neurons) or PRPH (PNS neurons).

    “Neural clusters contained cells expressing SOX2 (neural progenitors), DCX (CNS neurons) or PRPH (PNS neurons) (Extended Data Fig. 5)23.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [23] Zeisel, A. et al. Molecular architecture of the mouse nervous system. Cell 174, 999–1014 (2018). source

      The Results sections on postnatal neurogenesis and the peripheral nervous system describe progenitor and neuronal clusters, but the passages retrieved do not identify them by SOX2, DCX, or PRPH expression. Searches for all three gene names, individually and together, found no passage in our copy establishing the claimed marker assignments; no page numbers were available. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation
    § Abstract

    NFATC1 expression was predominantly cytoplasmic, consistent with follicular bulges in foetal skin; adult bulge cells displayed nuclear NFATC1 expression, a sign of maturity.

    “The expression pattern of NFATC1 was predominantly cytoplasmic, which was consistent with follicular bulges in foetal skin; by contrast, adult bulge cells displayed nuclear NFATC1 expression, a sign of maturity (Extended Data Fig. 10)2,36.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [2] Lee, J. et al. Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 22, 242–254 (2018). source

      The Results section, “Skin organoid-derived HFs contain bulge-like regions” (page not available), reports predominantly cytosolic NFATc1 in organoid bulge cells and likens those cells to late-embryonic stem cells. It also identifies nuclear NFATc1 as a marker of both nascent and adult hair-follicle stem cells and suggests that its rarity may indicate limited maturation. It does not directly show the stated fetal-versus-adult expression contrast or establish nuclear localization as specific to adult maturity. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [36] Horsley, V., Aliprantis, A. O., Polak, L., Glimcher, L. H. & Fuchs, E. NFATc1 balances quiescence and proliferation of skin stem cells. Cell 132, 299–310 (2008). source

  • May combine two claims.

    Interpretationown data
    § Abstract

    Organoid neurons were reminiscent of NC-derived neurons found in cranial or dorsal root ganglia, with medium-large soma NEFH+PRPH+ neurons and smaller soma NEFH−PRPH+ neurons.

    “The organoid neurons were reminiscent of NC-derived neurons found in cranial or dorsal root ganglia, with medium-large soma Neurofilament-Heavy Chain (NEFH)+ Peripherin (PRPH)+ neurons and smaller soma NEFH−PRPH+ neurons (Fig. 3o, Supplementary Video 7)23,31.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [23] Zeisel, A. et al. Molecular architecture of the mouse nervous system. Cell 174, 999–1014 (2018). source

      The Results section, “Peripheral Nervous System” (page unavailable), identifies dorsal-root-ganglion sensory neurons and a neurofilament branch, supporting part of the comparison. It does not describe the claimed NEFH/PRPH combinations or their association with medium-large versus smaller somata, nor does it establish the comparison with cranial ganglion neurons. The organoid observations are presented as the citing authors’ own results, not as findings of this source. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [31] Gatto, G., Smith, K. M., Ross, S. E. & Goulding, M. Neuronal diversity in the somatosensory system: bridging the gap between cell type and function. Curr. Opin. Neurobiol. 56, 167–174 (2019). source

      The “Sensory afferent heterogeneity” section describes neural-crest-derived trigeminal and dorsal root ganglion sensory neurons as having similar molecularly defined types. It mentions neurofilament as a historical sensory-afferent marker, but does not describe the claimed NEFH/PRPH combinations or their association with soma size. The organoid observations are the citing authors’ own results, not findings reported by this source. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data
    § Abstract

    There was a lack of HOX gene expression, which would indicate more caudal PAs.

    “Furthermore, we noted a lack of HOX gene expression, which would indicate more caudal PAs (Extended Data Fig. 5)26.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Minoux, M. et al. Mouse Hoxa2 mutations provide a model for microtia and auricle duplication. Development 140, 4386–4397 (2013). source

  • May combine two claims.

    Findingown data
    § Abstract

    Immunostaining revealed that epidermal germ cells were LHX2+PCAD+EDAR+, and dermal condensates were SOX2+PDGFRα+P75+.

    “As in nascent mouse HFs, immunostaining revealed that epidermal germ cells were LHX2+PCAD+EDAR+, and dermal condensates were SOX2+PDGFRα+P75+ (Fig. 1i–l, Extended Data Fig. 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Abstract

    Hair-bearing skin organoid generation was not restricted to these cell lines or the Koehler laboratory.

    “Hair-bearing skin organoid generation was not restricted to these cell lines or the Koehler laboratory.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Abstract

    The neuro-glial group contained clusters of Schwann cell precursor cell (SCP)-like cells (PLP1+MPZ+), as well as melanocytes (PMEL+MITF+).

    “The neuro-glial group contained clusters of Schwann cell precursor cell (SCP)-like cells (PLP1+MPZ+), as well as melanocytes (PMEL+MITF+) (Fig. 2h, Extended Data Fig. 5).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Abstract

    On average, hair germs appeared at 70 ± 5 days for WA25 and 72 ± 4 days for DSP-GFP organoids.

    “On average, hair germs appeared at 70 ± 5 days for WA25 and 72 ± 4 days for DSP-GFP organoids.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data
    § METHODS

    WA25 skin organoids produced an average of 48 hair follicles per organoid (min 9, max 285) across 80 specimens collected from 9 independent experiments between days 75-147.

    “WA25: average 48 hair follicles per organoid, min=9, max=285”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Abstract

    The other grafts had in-grown hairs (22%) or failed (22%) due to technical issues.

    “The other grafts had in-grown hairs (22%) or failed (22%) due to technical issues (Fig. 4c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Abstract

    In pigmented skin organoids, PMEL+ melanocytes were evenly distributed throughout the epithelium and concentrated in the matrix region of HFs.

    “In pigmented skin organoids, we found premelanosome protein (PMEL)+ melanocytes evenly distributed throughout the epithelium and concentrated in the matrix region of HFs (Fig. 3d, e, Supplementary Video 3).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Abstract

    Late-stage organoids developed hyaline cartilage in the tail region.

    “Another interesting feature of late-stage organoids was hyaline cartilage, which developed in the tail region (Fig. 3a, b, Extended Data Fig. 8).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Abstract

    For out-grown xenografts, histology confirmed that the organoid epidermis had integrated with the host epidermis, and the hair shafts were oriented perpendicular to the skin surface.

    “For out-grown xenografts, histology confirmed that the organoid epidermis had integrated with the host epidermis, and the hair shafts were oriented perpendicular to the skin surface, reminiscent of bona-fide human skin tissue (Fig. 4f, g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Abstract

    Xenograft sebaceous glands had multiple lobes similar to adult sebaceous glands and were located superficial to the HF bulge region.

    “Morphologically, xenograft sebaceous glands had multiple lobes similar to adult sebaceous glands and were located superficial to the HF bulge region (Fig. 4i, Extended Data Fig. 9).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § METHODS

    The difference in pigmented hair-bearing organoid frequency between WA25 and DSP-GFP cultures was not statistically significant by an unpaired Student's t-test with Welch's correction (p=0.077).

    “Statistical significance was determined in Figure 3g using an unpaired Student’s t-test with Welch’s correction to account for unequal standard deviations and sample sizes, p=0.077.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data
    § METHODS

    DSP-GFP skin organoids produced an average of 48 hair follicles per organoid (min 7, max 128) across 80 specimens collected from 9 independent experiments between days 75-147.

    “DSP-GFP: average 48 hair follicles per organoid, min=7, max=128”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Abstract

    Nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo.

    “Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § METHODS

    In general, experiments were not randomized.

    “In general, experiments were not randomized; however, organoid specimens used for histology and single-cell RNA-sequencing analysis were selected at random and not screened for quality or specific morphological characteristics.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § METHODS

    The skin organoid culture method was optimized and replicated by three investigators (K.R.K., J.L., and M.S.) using the WA25 and DSP-GFP cell lines.

    “The culture method was optimized and replicated by three investigators (K.R.K., J.L., and M.S.) using the WA25 and DSP-GFP cell lines.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Abstract

    Skin organoids represent craniofacial skin (superficial tissue) and cartilage (deep tissue) developing inside-out.

    “These data reinforce a tissue architecture model whereby skin organoids represent craniofacial skin (superficial tissue) and cartilage (deep tissue) developing inside-out.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductreasoning
    § METHODS

    S.H. and B.M.W. were blinded to the data already generated for this study with WA25 and DSP-GFP PSCs and were unfamiliar with the expected morphological changes during skin organogenesis.

    “S.H. and B.M.W. were blinded to the data already generated for this study with WA25 and DSP-GFP PSCs; thus, they were unfamiliar with the expected morphological changes during skin organogenesis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § METHODS

    No statistical test was used to predetermine the sample size.

    “No statistical test was used to predetermine sample size, the investigators were not blinded to the treatment groups.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given
    § METHODS

    In vivo experiments were performed after approval from the Indiana University Animal Care and Use Committee (IACUC) and complied with federal regulations.

    “In vivo experiments were performed after an approval from the Indiana University Animal Care and Use Committee (IACUC) and complied with federal regulations.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given
    § METHODS

    Organoid specimens used for histology and single-cell RNA-sequencing analysis were selected at random and not screened for quality or specific morphological characteristics.

    “In general, experiments were not randomized; however, organoid specimens used for histology and single-cell RNA-sequencing analysis were selected at random and not screened for quality or specific morphological characteristics.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data
    § METHODS

    The final dissociated cell suspension used for single-cell RNA-sequencing had a concentration of ~1,000 cells/μl and >90% viability.

    “Final cell concentration was ~1,000 cells/μl and >90% viability.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Abstract

    After ~150 days in culture, a build-up of squamous cells in the core of skin organoids and abnormal HF morphologies were observed, suggesting that this timepoint may be the upper limit for skin organoid culture.

    “After ~150 days in culture, we observed a build-up of squamous cells in the core of skin organoids (Extended Data Fig. 9b) and abnormal HF morphologies, suggesting that this timepoint may be the upper limit for skin organoid culture.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § METHODS

    The investigators were not blinded to the treatment groups.

    “No statistical test was used to predetermine sample size, the investigators were not blinded to the treatment groups.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § METHODS

    All representative skin organoid images shown in the manuscript represent results from nine independent experiments of WA25 and DSP-GFP cultures and one WA01 culture.

    “All representative skin organoid images shown in this manuscript represent results from nine independent experiments of WA25 and DSP-GFP cultures and one WA01 culture (Figure 1c–f, i–l and Figure 3b, d–f”, h, i, k–m, o–r).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given
    § METHODS

    Unless stated otherwise, images are representative of specimens obtained from at least 3 separate experiments, and all immunostaining images are representative of a minimum of 3 times of the same staining.

    “Unless stated otherwise, images are representative of specimens obtained from at least 3 separate experiments.”
    • Also stated in: § METHODS · plain
      “All immunostaining images shown in this manuscript are representative of minimum 3 times of the same staining.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Abstract

    Skin organoid HFs reach a level of maturity roughly equivalent to second trimester foetal hair and have the cellular components for further maturation.

    “Together, these findings suggest that skin organoid HFs reach a level of maturity roughly equivalent to second trimester foetal hair and have the cellular components for further maturation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationnone given
    § Discussion

    The study establishes a novel model to investigate the cellular dynamics of developing human skin and its appendages-including sweat glands, which were absent from the skin organoids reported here.

    “Our study establishes a novel model to investigate the cellular dynamics of developing human skin and its appendages—including sweat glands, which were absent from the skin organoids reported here.”
    Quote in the paperSubstantial claim
38 of 38

  • The authors state this as an assumption.

    The site of axon targeting in organoid HFs is the bulge region.

    “which we presume to be the bulge region of the HFs”
1 of 1

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • SkOs often display inverted polarity, with the epidermis positioned internally and the dermis externally, and frequently undergo ectopic cartilage differentiation.

    “They often display inverted polarity, in which the epidermis is positioned internally and the dermis externally, and frequently undergo ectopic cartilage differentiation 14,15 .”

    From Vascularized human skin assembloids model compartmental skin organization and immune responsiveness in vitro (2026) · cites this paper as [14]

    The passage on organoid induction (page unavailable) describes an inner epithelial layer surrounded by outer mesenchymal cells, supporting the polarity description. The passage on late-stage organoids reports cartilage developing in the tail region, but does not establish how frequently it occurs or describe it as ectopic; it instead relates cartilage to the organoids’ craniofacial tissue architecture. (LLM, conf=0.83)

    Experimental — accuracy not yet measured

  • Skin organoids were equivalent to embryonic skin in development and mimicked normal hair folliculogenesis.

    “The skin organoids were shown to be equivalent to the embryonic skin in development and mimicked normal hair folliculogenesis.”

    From Reprogramming of three-dimensional microenvironments for in vitro hair follicle induction (2022) · cites this paper as [13]

    The Abstract says comparisons with foetal specimens suggest that the organoids resemble human facial skin in the second trimester, and reports that their hair follicles undergo morphogenesis similar to mammalian hair follicles. The claim captures the follicle finding but presents the qualified developmental comparison as a demonstrated equivalence to embryonic skin generally. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • There has been a complete absence of an endogenously differentiating vascularized, hair-bearing human skin equivalent that contains immune cell populations.

    “While many attempts to generate complex 3D skin equivalents have achieved vascularization, hair follicle generation, and immune cell co‐cultures, there has been a complete absence of endogenously differentiating a vascularized, hair‐bearing human skin equivalent that contains immune cell populations.[ 21 , 22 , 23 , 24 , 25 , 26 , 27 ]”

    From Coordinated Development of Immune Cell Populations in Vascularized Skin Organoids from Human Induced Pluripotent Stem Cells (2025) · cites this paper as [26]

    The paper reports hair-bearing human skin organoids but says it did not detect progenitors of Langerhans cells, which are resident skin immune cells (section labelled Abstract, page unavailable). It also reports that vasculature grew into the organoids only after grafting into mice. These findings support a gap in this model, but the paper does not establish the claimed complete absence of any endogenously differentiating human skin equivalent combining all three features. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • hiPSC-derived skin organoids have hair follicles, pigmentation, dermal stratification, appendage patterning, and innervation.

    “Recent advances in human induced pluripotent stem cell (hiPSC) technology have enabled the generation of skin organoids (SKO) with hair follicles, pigmentation, dermal stratification, appendage patterning, and innervation.[ 25 , 26 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]”

    From Coordinated Development of Immune Cell Populations in Vascularized Skin Organoids from Human Induced Pluripotent Stem Cells (2025) · cites this paper as [26]

    The paper reports hair follicles, pigmentation, periodically spaced appendages, and innervation in skin organoids; it also reports hair-bearing organoids from a hiPSC line (Abstract; no page available). But it describes a stratified epidermis and a fat-rich dermis, not dermal stratification. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Hair follicles in VSKOs are SOX2+, K14+, K15+, and K17+, as seen in the current SKO model.

    “Figure 7c also shows that the hair follicles in VSKOs are SRY‐box transcription factor 2 (SOX2)+, K14+, cytokeratin 15 (K15)+, and K17+, as seen in the current SKO model.[ 25 , 26 ]”

    From Coordinated Development of Immune Cell Populations in Vascularized Skin Organoids from Human Induced Pluripotent Stem Cells (2025) · cites this paper as [26]

    The paper reports SOX2+ dermal papilla clusters in organoid hair follicles (Abstract, Fig. 1i–l) and KRT15+ stem-cell-like cells in the presumptive bulge of xenografted follicles (Abstract, Fig. 4j). Our copy does not establish K14+ or K17+ hair follicles, nor does this paper examine the citing authors’ VSKOs. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • WA25 hair-follicle average differs between Results and Methods

    Major

    The Results report an average of 64 hair follicles per WA25 organoid, whereas the Statistics and Reproducibility section reports 48 for the same cell line and Figure 1h.

1 of 1

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Authors
Lee, J., Rabbani, C. C., Gao, H., Steinhart, M. R., Woodruff, B. M., Pflum, Z. E., Kim, A., Heller, S., Liu, Y., Shipchandler, T. Z., & Koehler, K. R.
Affiliations
  • Boston Children's Hospital (US)
  • Johns Hopkins University (US)
  • Indiana University School of Medicine
  • Stanford Medicine (US)
Corresponding author
Koehler, Karl R.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2020-06-03
Volume
582
Issue
7812
Pages
399-404
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-020-2352-3
PMC
PMC7593871
OpenAlex
W3032142305
Semantic Scholar
ca18b7b0af03573e144b07317b08925d4f05acf8
Cited by
487
References
44
Citations by year
2026: 82 · 2025: 85 · 2024: 85 · 2023: 75 · 2022: 80 · 2021: 68 · 2020: 12
FWCI
58.89
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
  • IUScholarWorks (Indiana University) (submittedVersion)
Funders
  • Indiana Clinical and Translational Sciences Institute (UL1TR001108, RR020128, TR001108)
  • University of Washington (5R24HD000836)
  • National Institutes of Health (1c06rr020128-01, 5R24 HD000836, UL-1-TR001108, TR001108, R01DC017461, R03DC015624, R01AR075018)
  • National Institute on Deafness and Other Communication Disorders (R01 DC017461, R03DC015624)
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01 AR075018)
  • National Institute of Child Health and Human Development
  • National Center for Research Resources (C06-RR020128)
  • National Center for Advancing Translational Sciences (UL1TR-001108)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (R24 HD000836, 5R24HD000836)
Topics
  • Health Sciences › Medicine › Urology › Hair Growth and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Skin and Cellular Biology Research
Keywords
skin organoids, pluripotent stem cells, hair follicle, sebaceous glands, TGF-beta signaling, FGF signaling, neural crest cells, Merkel cells, sensory neurons, Schwann cells, epidermis, dermis, skin grafting, skin development, disease modeling, reconstructive surgery
MeSH terms
RNA-Seq, Animals, Ectoderm, Female, Hair, Hair Color, Head, Humans, Mice, Nude, Organoids, Skin, Skin Transplantation, Hair Follicle, Pluripotent Stem Cells, Mice, Single-Cell Analysis, Heterografts

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-22.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-22.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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