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Cited by 132Pluripotent Stem Cells ResearchHair Growth and DisordersSkin and Cellular Biology Research

Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells

Open access
Lee, Jiyoon, van der Valk, Wouter H., Serdy, Sara A., Deakin, CiCi, Kim, Jin, Le, Anh Phuong, Koehler, Karl R.·2022·Nature Protocols
DOI 10.1038/s41596-022-00681-yPMC PMC10461778
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Abstract

Human skin uses millions of hairs and glands distributed across the body surface to function as an external barrier, thermoregulator and stimuli sensor. The large-scale generation of human skin with these appendages would be beneficial, but is challenging. Here, we describe a detailed protocol for generating hair-bearing skin tissue entirely from a homogeneous population of human pluripotent stem cells in a three-dimensional in vitro culture system. Defined culture conditions are used over a 2-week period to induce differentiation of pluripotent stem cells to surface ectoderm and cranial neural crest cells, which give rise to the epidermis and dermis, respectively, in each organoid unit. After 60 d of incubation, the skin organoids produce hair follicles. By day ~130, the skin organoids reach full complexity and contain stratified skin layers, pigmented hair follicles, sebaceous glands, Merkel cells and sensory neurons, recapitulating the cell composition and architecture of fetal skin tissue at week 18 of gestation. Skin organoids can be maintained in culture using this protocol for up to 150 d, enabling the organoids to be used to investigate basic skin biology, model disease and, further, reconstruct or regenerate skin tissue. Skin organoids are generated from human pluripotent stem cells in a three-dimensional in vitro culture system. The mature organoids contain stratified skin layers, pigmented hair follicles, sebaceous glands, Merkel cells and sensory neurons.

AI snapshot

Written from the paper’s full text.

TL;DR

This protocol describes generation of hair-bearing skin organoids entirely from human pluripotent stem cells in 3D culture, with defined signaling manipulations over 2 weeks and maturation up to 150 days. The organoids form stratified skin, pigmented hair follicles, sebaceous glands, Merkel cells, and sensory neurons, recapitulating features of fetal skin at week 18 of gestation.

Key findings

  • After 60 days of incubation, skin organoids produce hair follicles; by day ~130 they reach full complexity with stratified skin layers, pigmented hair follicles, sebaceous glands, Merkel cells, and sensory neurons, recapitulating fetal skin tissue at week-18 gestation.
  • Hair placodes and germs appear at days 55–75, hair pegs at days 75–90, and more elaborate hair follicles with sebaceous glands and a bulge region by days 110–130.
  • With 5 ng/mL BMP4 on day 0, the protocol anticipates >90% of skin organoids will consistently produce hairs; previously, an average of 87% generated hair follicles with 2.5 ng/mL BMP4.
  • Skin organoids can be maintained in culture for up to 150 days; beyond 150 days they display abnormal features such as a compacted core of dead squamous cells and fused hair follicles.

Methods

  • Step-by-step 3D protocol using human ESCs or iPSCs, including WA25, WA01, DSP-GFP, SOX2-GFP, HIST1H2BJ-GFP, LMNB1-GFP, TUBA1B-GFP, and mND2-0 lines; differentiation is initiated in 96-well U-bottom plates and transitioned to floating culture in OMM1%M on an orbital shaker at 65 rpm.
  • Defined signaling regimen: day 0 E6SFB (2% Matrigel, 10 μM SB, 4 ng/mL bFGF, 5 ng/mL BMP4), day 3 LDN/bFGF treatment (final 200 nM LDN, 50 ng/mL bFGF), then long-term maturation with medium changes.
  • Characterization includes morphological monitoring, cryosectioning and immunostaining, whole-mount immunostaining with qCe3D clearing, single-cell dissociation for scRNA-seq, and xenografting as previously demonstrated.

Limitations

  • Stated: the cystic structure limits shedding and accumulates cornified tissue in the core; after day 150, floating dead keratinocytes erupt to the surface, and hair shafts can become kinked or curled.
  • Stated: organoids lack sweat glands, blood vessels, arrector pili muscle (rarely observed), and immune cells; hair follicles do not contain a medulla layer and resemble lanugo hair rather than adult terminal hair.
  • Stated: off-target cell lineage induction occurs, with highly variable chondrocyte and myocyte populations; outcomes are cell line dependent, and BMP4 concentration/timing may require optimization.

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

Integrity signals

Registry

Crossref · 10.1038/s41596-022-00681-y

Cites

79 of 79 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

34 of 41 cited sources checked · 6 supported · 0 flagged · accuracy not yet measured

  • Findingcitation

    With a slight modification of the mouse IEO culture regimen (i.e., removing the WNT stimulus that is critical for otic induction), we could steer the cell aggregate toward primarily forming hairy skin tissue.

    “With a slight modification of the mouse IEO culture regimen (i.e., removing the WNT stimulus that is critical for otic induction), we could steer the cell aggregate toward primarily forming hairy skin tissue”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [29] 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, “Optimal treatment regimen for HF induction” (page unavailable), reports hair-follicle-bearing skin organoids under the SB/BMP-FGF/LDN regimen and says that adding CHIR, previously used to encourage inner-ear organoid induction, produced presumptive hair follicles alongside inner-ear organoids. This supports deriving hairy skin without that added stimulus, but the passage does not establish that WNT stimulation is critical for otic induction or quantify whether the resulting tissue was primarily hairy skin. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Findingcitation

    By days 110-130, the hair pegs formed more elaborate hair follicles containing sebaceous glands and a bulge region with Merkel cells and NFATC1+ hair follicle stem cell-like cells.

    “By days 110–130, the hair pegs formed more elaborate hair follicles containing sebaceous glands (sebum-producing glands) and a bulge region with Merkel cells (touch-sensing cells) and nuclear factor of activated T cells 1 (NFATC1)+ hair follicle stem cell-like cells”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [25] Lee, J. et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature 582, 399–404 (2020). source

      The organoid-development passage (indexed as Abstract; page unavailable) reports hair-follicle formation through day 120, and the Merkel-cell passage places Merkel-like cells near a presumed follicle bulge. The passage on sebaceous glands and NFATC1+ bulge stem cell-like cells, however, describes out-grown xenografted follicles, not follicles shown to have those features by days 110–130 in culture. Our copy does not establish that specific timing for the full combination of features. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    These findings marked the first demonstration that de novo hair could be generated entirely from mouse PSCs in culture.

    “These findings marked the first demonstration that de novo hair could be generated entirely from mouse PSCs in culture.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] Lee, J. et al. Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 22, 242–254 (2018). source
  • Interpretationown data

    Comparative analysis with fetal hair suggests that the skin organoids in vitro are reminiscent of lanugo hair, which sheds just before or after birth.

    “Our comparative analysis with fetal hair suggests that the skin organoids in vitro are reminiscent of lanugo hair, which sheds just before or after birth”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [25] Lee, J. et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature 582, 399–404 (2020). source

      The passage on organoid hair follicles (labelled Abstract in our copy; no page given) compares their appearance with foetal skin and suggests they produce lanugo- or vellus-like hair follicles. It does not establish that they are specifically lanugo hair, and our copy does not state that lanugo hair sheds just before or after birth. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Interpretationown data

    Based on scRNA-seq and immunostaining data, the end-stage skin organoids (~150 days of culture) represent the human skin tissues at the second-trimester stage of the developing fetus.

    “Based on our scRNA-seq and immunostaining data, the end-stage skin organoids (~150 days of culture) represent the human skin tissues at the second-trimester stage of the developing fetus”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [25] Lee, J. et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature 582, 399–404 (2020). source
  • Classified as probably not a substantial claim. 20
  • May combine two claims. 29

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

    Premisecitation

    The mouse is a standard model for investigating skin development and disease in vivo.

    “The mouse is a standard model for investigating skin development and disease in vivo”
    Quote in the paperNot a substantial claim4 of 6 sources checked
    • cites [12] Wu, X., Scott, L., Washenik, K. & Stenn, K. Full-thickness skin with mature hair follicles generated from tissue culture expanded human cells. Tissue Eng. 20, 3314–3321 (2014). source
    • cites [13] Klicznik, M. M. et al. A novel humanized mouse model to study the function of human cutaneous memory T cells in vivo in human skin. Sci. Rep. 10, 11164 (2020). source

      The Introduction says skin-humanized mice are used to study human inflammatory skin conditions in vivo, supporting the disease portion. It does not establish that mice are a standard model for investigating skin development; the model described here is specifically designed to study human cutaneous T cells. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [14] Agarwal, Y. et al. Development of humanized mouse and rat models with full-thickness human skin and autologous immune cells. Sci. Rep. 10, 14598 (2020). source

      The Introduction says in vivo models for studying skin-targeting insults and pathogens primarily involve mice and rats, and the Discussion calls rodent models primary platforms for investigating skin-associated diseases (page not provided). These passages support mice as a standard model for skin disease, but do not establish their standard use for investigating skin development. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [15] Salgado, G., Ng, Y. Z., Koh, L. F., Goh, C. S. M. & Common, J. E. Human reconstructed skin xenografts on mice to model skin physiology. Differentiation 98, 14–24 (2017). source
    • cites [16] Takagi, R. et al. Bioengineering a 3D integumentary organ system from iPS cells using an in vivo transplantation model. Sci. Adv. 2, e1500887 (2016). source

      The Methods section describes mouse skin samples and transplantation into mice, and the Results section uses natural mouse skin to assess a bioengineered skin system in vivo. This supports the use of mice to investigate skin development, but the paper does not establish that mice are a standard model for investigating skin disease. (LLM, conf=0.80)

      Experimental — accuracy not yet measured
    • cites [17] Zheng, Y. et al. Organogenesis from dissociated cells: generation of mature cycling hair follicles from skin-derived cells. J. Invest. Dermatol. 124, 867–876 (2005). source

      The Results (page unavailable) describe an in vivo mouse assay in which injected skin-derived cells formed hair follicles, supporting the use of mice to investigate an aspect of skin development. The cited paper does not establish that mice are a standard model for skin research generally, or report an investigation of skin disease. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The hPSC-derived skin organoids can recapitulate many features of full-thickness human fetal skin and be xenografted on nude mice.

    “This protocol provides step-by-step guidance to form human pluripotent stem cell (hPSC)-derived skin organoids, which we have previously shown can recapitulate many features of full-thickness human fetal skin and be xenografted on nude mice”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [25] Lee, J. et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature 582, 399–404 (2020). source

  • May combine two claims.

    Premisecitation

    A clear demonstration that appendage-bearing skin could be recreated in culture remained elusive until our publication where we demonstrated how to generate hair-bearing skin from mouse PSCs.

    “Despite some advances, a clear demonstration that appendage-bearing skin could be recreated in culture remained elusive, until our publication where we demonstrated how to generate hair-bearing skin from mouse PSCs”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [29] Lee, J. et al. Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 22, 242–254 (2018). source

      The Discussion states that this study generated hair-follicle-bearing skin in vitro from mouse PSCs under serum-free conditions. But the claim that appendage-bearing skin had not previously been recreated in culture is too broad: the Discussion also describes earlier work in which mouse embryonic skin cells formed structures with protruding hair follicles in a hanging drop. The previously elusive advance was a defined in vitro method using PSCs. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    A critical distinction between human and mouse IEO inductions is that TBXT+ mesodermal lineage cells appear to be missing in the human IEO system, perhaps due to the higher dosage of a TGF-β pathway inhibitor used in the human protocol.

    “A critical distinction between our human and mouse IEO inductions is that TBXT+ mesodermal lineage cells appear to be missing in the human IEO system, perhaps due to the higher dosage of a TGF-β pathway inhibitor used in the human protocol”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [33] 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 paper’s discussion of non-neural conversion (page not provided) says that, unlike its mouse culture, the human culture did not induce Brachyury (TBXT)+ mesendoderm cells. The Online Methods report 10 μM of the TGF-β inhibitor SB-431542 in the human protocol, but the retrieved passages neither establish that this dose was higher than in the mouse protocol nor suggest that the dose explains the difference. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Human skin equivalents have notable drawbacks, such as a lack of crucial skin tissue structures (e.g., hair follicles and sebaceous glands) and limited culture durations (e.g., 2-3 weeks).

    “However, human skin equivalents have notable drawbacks, such as a lack of crucial skin tissue structures (e.g., hair follicles and sebaceous glands) and limited culture durations (e.g., 2–3 weeks)”
    Quote in the paperSubstantial claim4 of 4 sources checked
    • cites [19] Itoh, M. et al. Generation of 3D skin equivalents fully reconstituted from human induced pluripotent stem cells (iPSCs). PLoS ONE 8, e77673 (2013). source

      The Discussion describes the paper’s in vitro 3D skin equivalent as lacking skin appendages, supporting the structural drawback. The Methods report harvesting the equivalent after approximately 18–20 days of preparation, but do not say that its culture duration is limited to 2–3 weeks. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [20] Yang, R. et al. Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells. Nat. Commun. 5, 3071–3071 (2014). source

      The Results section (page unavailable) reports that the authors’ 3D skin equivalents formed a multilayered epidermis and that they could not identify human sebaceous glands in their skin reconstitution assays. It does not establish a general absence of hair follicles in human skin equivalents or a 2–3-week culture-duration limit; the reported two-and-a-half-week interval concerns an in vivo assay, not culture duration. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [21] 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 says human skin equivalents lack appendages, supporting the point about missing structures such as hair follicles and sebaceous glands. The Methods describe harvesting this study’s constructs 14 days after raising them to the air–liquid interface, but do not establish a general culture-duration limit of 2–3 weeks. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [22] Abaci, H. E. et al. Tissue engineering of human hair follicles using a biomimetic developmental approach. Nat. Commun. 9, 5301 (2018). source

      The Introduction (page not available) says currently available human skin constructs have poor long-term viability and lack appendages such as hair follicles. The Results report no sebocyte differentiation in the authors’ constructs and describe culturing them for three weeks, but do not establish that human skin equivalents generally have a 2–3-week culture limit. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    The in vitro hair follicle developmental timeline corresponds well to that of human fetal hair follicle development in vivo: the hair placode, germ, peg, and follicle differentiation stages occur, mostly, between 80 and 170 days (12-25 gestational weeks), where the full anagen stage hair follicles develop by day 126 (18 gestational weeks).

    “The in vitro hair follicle developmental timeline corresponds well to that of human fetal hair follicle development in vivo: the hair placode, germ, peg, and follicle differentiation stages occur, mostly, between 80 and 170 days (12–25 gestational weeks), where the full anagen stage hair follicles develop by day 126 (18 gestational weeks; Fig. 1)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [37] Duverger, O. & Morasso, M. I. To grow or not to grow: hair morphogenesis and human genetic hair disorders. Semin. Cell Dev. Biol. 25, 22–33 (2014). source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation

    Skin organoids form a sphere-like cyst where hair shafts sprout inward toward the fluid-filled core, with hair bulbs protruding outward from the surface of the cysts.

    “Skin organoids form a sphere-like cyst where hair shafts sprout inward toward the fluid-filled core, with hair bulbs protruding outward from the surface of the cysts”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [25] Lee, J. et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature 582, 399–404 (2020). source

      The paper describes a spherical, cyst-like skin organoid (Abstract, page unavailable) and reports that hair germ-like buds extend radially outward from its surface (Induction of human skin organoids, page unavailable). Our copy does not establish the claimed inward growth of hair shafts toward a fluid-filled core or specifically identify the outward structures as hair bulbs. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [29] Lee, J. et al. Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 22, 242–254 (2018). source

      The Discussion describes the organoids as cysts with radially oriented hair follicles, and the Results describe an inside-out epidermal mantle and protruding bulb-like structures. However, our copy does not state that hair shafts sprout inward toward a fluid-filled core. The Results also say follicle orientation varies with culture conditions: follicles wrap around floating organoids but grow outward in Matrigel droplets. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    After 1 month of transplantation, the skin organoid cysts had unfurled and integrated into the host skin, forming about 1-2 mm sized planar skin with outward-growing hairs.

    “After 1 month of transplantation, we found that the skin organoid cysts had unfurled and integrated into the host skin, forming about 1–2 mm sized planar skin with outward-growing hairs.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [25] Lee, J. et al. Hair-bearing human skin generated entirely from pluripotent stem cells. Nature 582, 399–404 (2020). source

      The “Organoids form planar skin in vivo” passage (page unavailable) reports that cystic organoids unfurled and integrated with host skin, with outward-growing hairs in 55% of grafts. It describes 1–2 mm incisions, not a measured size of the resulting planar skin. The Methods (page unavailable) say the mice were observed for about 7 or 14 weeks after surgery, so the claimed one-month outcome is not established. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation

    The precursor cells of basal keratinocytes, squamous cells, and melanocytes are present within the skin organoid, setting the stage for researchers to investigate mechanisms of cancer initiation.

    “The precursor cells of basal keratinocytes, squamous cells, and melanocytes are present within the skin organoid, setting the stage for researchers to investigate mechanisms of cancer initiation”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [56] Yum, M. K. et al. Tracing oncogene-driven remodelling of the intestinal stem cell niche. Nature 594, 442–447 (2021). source

      The Methods section describes intestinal epithelial organoids made from mouse small-intestinal crypts, not skin organoids. Searches for skin organoids, keratinocyte and melanocyte precursors found no passage addressing the claimed cell types or their use to investigate cancer initiation. The Extended Data mention imaging skin tissue, but not a skin organoid. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Skin organoids can be readily generated from patient-derived cells and used to model genetic disorders, such as epidermolysis bullosa.

    “Furthermore, skin organoids can be readily generated from patient-derived cells and used to model genetic disorders, such as epidermolysis bullosa”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [59] Wenzel, D. et al. Genetically corrected iPSCs as cell therapy for recessive dystrophic epidermolysis bullosa. Sci. Transl. Med. 6, 264ra165–264ra165 (2014). source
    • cites [60] Sebastiano, V. et al. Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa. Sci. Transl. Med. 6, 264ra163–264ra163 (2014). source

      The Abstract and Methods describe generating stratified epidermis in organotypic cultures from RDEB patient-derived, gene-corrected cells. They do not describe skin organoids used to model the disorder, and the Results note variability between cell lines rather than establishing that generation is readily achieved. Page numbers are unavailable in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [61] Tolar, J. et al. Induced pluripotent stem cells from individuals with recessive dystrophic epidermolysis bullosa. J. Invest. Dermatol. 131, 848–856 (2011). source

      The Results section reports that patient-derived RDEB iPS cells formed skin-like structures within teratomas, and the Discussion describes their potential for modeling RDEB. The paper does not report readily generating skin organoids; its skin-like structures formed in vivo after injection into mice. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [62] Itoh, M., Kiuru, M., Cairo, M. S. & Christiano, A. M. Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells. Proc. Natl Acad. Sci. USA. 108, 8797–8802 (2011). source

      The Results and Discussion (pages not provided) show that the authors generated 3D skin equivalents from cells derived from patients with recessive dystrophic epidermolysis bullosa and used them as in vitro disease models. The paper calls these constructs skin equivalents, not organoids, and describes a multistep construction protocol; it does not establish that skin organoids can be readily generated. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    To successfully generate skin, co-development of epidermis and dermis is required, and precise alterations of major signaling pathways (TGF-β, BMP4, and bFGF) are critical.

    “To successfully generate skin, co-development of epidermis and dermis is required, and precise alterations of major signaling pathways (TGF-β, BMP4, and bFGF) are critical”
    Quote in the paperSubstantial claim5 of 6 sources checked
    • cites [20] Yang, R. et al. Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells. Nat. Commun. 5, 3071–3071 (2014). source

      The Methods (Patch assay for skin reconstitution; page unavailable) combine epithelial cells with neonatal mouse dermal cells, and the Results (Generation of iPSCs-derived EpSCs; page unavailable) say that the timing of EGF was critical in a protocol using BMP4. The paper does not establish that epidermis and dermis must co-develop to generate skin generally, or identify precise alterations of TGF-β and bFGF as critical; its differentiation protocol emphasizes EGF, retinoic acid, and BMP signaling. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [29] Lee, J. et al. Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 22, 242–254 (2018). source

      The Introduction says epidermal–dermal interplay is required for terminal skin development and appendage formation, and the Results describe co-development of both layers in mouse skin organoids. The Results also identify a TGFβ, BMP4 and FGF-2 treatment regimen as optimal for hair-follicle-bearing organoids, but report that some follicles formed without FGF treatment. The paper therefore supports the importance of these pathways, not the stronger claim that precise alterations of all three are required to generate skin. Page numbers are unavailable in our copy. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [30] 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 timed changes to TGFβ, BMP, and FGF signaling to generate inner-ear precursors, and reports epidermis-like epithelium in some aggregates. It does not say that these changes are critical for generating skin or that epidermis and dermis must co-develop; the cited result concerns inner-ear development. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [33] 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 paper reports using FGF-2, BMP4, and a TGF-β inhibitor to direct non-neural epithelium toward inner ear organoids, and says BMP concentration may need fine-tuning between cell lines (Abstract; page unavailable). It also reports keratinocyte-like cells, but does not establish that epidermis and dermis must co-develop to generate skin or that these signaling changes are critical for skin generation. (LLM, conf=0.79)

      Experimental — accuracy not yet measured
    • cites [36] Wilson, P. A. & Hemmati-Brivanlou, A. Induction of epidermis and inhibition of neural fate by Bmp-4. Nature 376, 331–333 (1995). source
    • cites [72] Tchieu, J. et al. A modular platform for differentiation of human PSCs into all major ectodermal lineages. Cell Stem Cell 21, 399–410.e7 (2017). source

      The Results section ('BMP signaling through TFAP2A is necessary to generate non-CNS fates'; page unavailable) reports dose-dependent BMP4 induction, FGF-pathway inhibition, and differentiation into epidermal cells. The Method Details describe a keratinocyte protocol using BMP4 and signaling inhibitors. These passages support signaling control of epidermal differentiation, but they do not establish that epidermis and dermis must co-develop to generate skin, or that the specified TGF-β, BMP4, and bFGF alterations are critical for that outcome. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    The epidermal organoids formed an epidermis and a dermis, which generated hair follicles resembling bona fide mouse hair follicles.

    “However, we were surprised to find that the epidermal organoids formed an epidermis and a dermis, which generated hair follicles resembling bona fide mouse hair follicles.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After days 55-75 of differentiation, we observed the initial formation of hair placodes and germs, with further differentiation into hair pegs occurring during days 75-90 of differentiation.

    “After days 55–75 of differentiation, we observed the initial formation of hair placodes and germs, with further differentiation into hair pegs occurring during days 75–90 of differentiation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    By day ~130, the skin organoids reach full complexity and contain stratified skin layers, pigmented hair follicles, sebaceous glands, Merkel cells, and sensory neurons.

    “By day ~130, the skin organoids reach full complexity and contain stratified skin layers, pigmented hair follicles, sebaceous glands, Merkel cells, and sensory neurons”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Qualitatively, hair-forming skin organoids arose more consistently and reproducibly with constant agitation; yet, instances of organoids forming hair follicles were also observed in a static culture.

    “Qualitatively, we found that hair-forming skin organoids arose more consistently and reproducibly with constant agitation; yet, instances of organoids forming hair follicles were also observed in a static culture.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Using mouse PSCs, these signaling manipulations led to multi-lineage organoids containing both ectoderm (on-target; characterized by Tfap2a and E-cadherin expression) and mesoderm (off-target; characterized by Tbxt expression) cell types.

    “Using mouse PSCs, these signaling manipulations led to multi-lineage organoids containing both ectoderm (on-target; characterized by Tfap2a and E-cadherin expression) and mesoderm (off-target; characterized by Tbxt expression) cell types.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    We also observed adipocytes, sensory neurons, Schwann cells, and melanocytes developing in a proper spatial organization relative to native human skin.

    “We also observed adipocytes (fat cells), sensory neurons, Schwann cells, and melanocytes (pigment cells) developing in a proper spatial organization relative to native human skin.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The surface ectoderm layers, induced early in the culture, formed both IEOs and epidermal organoids, mimicking the formation of the skin that covers the outer ear.

    “Not surprisingly, we found that the surface ectoderm layers, induced early in the culture, formed both IEOs and epidermal organoids, mimicking the formation of the skin that covers the outer ear.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Rudimentary mechanosensory complex-like structures form at the hair follicle bulge-nerve interface, and on rare occasions free nerve endings are observed in the organoid epidermis.

    “Regarding nerve endings, we have characterized rudimentary mechanosensory complex-like structures forming at the hair follicle bulge-nerve interface and, on rare occasions, we observe free nerve endings in the organoid epidermis.”
    Quote in the paperSubstantial claim

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

    Findingnone given

    Some hair follicles will have pigmentation such as that seen in 18-week fetal skin.

    “Some hair follicles will have pigmentation such as that seen in 18-week fetal skin (Fig. 3g-i).”
    Quote in the paperNot a substantial claim

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

    Findingnone given

    Approximately 1.6 × 105 cells can be dissociated per skin organoid at day 6.

    “~day 6 (1.6 × 105 cells)”
    Quote in the paperNot a substantial claim

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

    Findingown data

    After ~30 days of differentiation, the epithelial cyst should be composed of stratified epidermis with basal, intermediate, and periderm layers.

    “After ~30 days of differentiation, the epithelial cyst should be composed of the stratified epidermis with basal, intermediate, and periderm layers (Fig. 3b, c).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The epithelial cyst will become covered by a layer of mesenchymal and neuroglial cells over the week following day 8.

    “The epithelial cyst will become covered by a layer of mesenchymal and neuroglial cells over the next week.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    Visually, under a microscope, floating cells appear to occupy ~50-55% of the area of a well when they adhere on the plate.

    “Visually, under the microscope, floating cells appear to occupy ~50–55% of the area of a well when they adhere on the plate.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The WA25 human ESC (hESC) line has a higher basal level of endogenous BMP4 compared with wild type C (WTC)-background cell lines and requires less exogenous recombinant BMP4 to induce surface ectoderm.

    “For example, we have found that the WA25 human ESC (hESC) line has a higher basal level of endogenous BMP4 compared with wild type C (WTC)-background cell lines and requires less exogenous recombinant BMP4 to induce surface ectoderm (Fig. 6).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    Although the neural crest (NC) lineage in skin organoids produces important skin lineage cells (i.e., melanocytes for pigmentation and neurons for touch/pain sensing), it also produces highly variable chondrocyte and myocyte populations.

    “Although the neural crest (NC) lineage in skin organoids produces important skin lineage cells (i.e., melanocytes for pigmentation and neurons for touch/pain sensing), it also produces highly variable chondrocyte and myocyte populations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    The organoids currently lack sweat glands, blood vessels, arrector pili muscle (rarely observed), and immune cells.

    “For example, the organoids currently lack sweat glands, blood vessels, arrector pili muscle (rarely observed), and immune cells”
    Quote in the paperSubstantial claim

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

    Findingown data

    Approximately 3 × 105 cells can be dissociated per skin organoid at day 130.

    “~day 130 (3 × 105 cells)”
    Quote in the paperNot a substantial claim

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

    Findingnone given

    Approximately 2 × 105 cells can be dissociated per skin organoid at day 50 without the tail portion.

    “~day 50 (2 × 105 cells without tail portion)”
    Quote in the paperNot a substantial claim

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

    Findingnone given

    The average size of a day 30 skin organoid is 2 mm.

    “The average size of a day 30 skin organoid is 2 mm.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    If the differentiation experiment is performed correctly, a translucent epithelial cyst should form by day 8 of differentiation.

    “If the differentiation experiment is performed correctly, a translucent epithelial cyst should form by day 8 of differentiation.”
    Quote in the paperNot a substantial claim

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

    Findingnone given

    Total time to observe hair germ formation is ~60 days.

    “Total time to observe hair germ formation: ~60 days”
    Quote in the paperNot a substantial claim

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

    Findingown data

    The static condition, without constant medium agitation on an orbital shaker, leads to increased variability and decreased reproducibility of organoid formation within an experiment.

    “Based on the qualitative observations in the Koehler laboratory, however, the static condition, without constant medium agitation on an orbital shaker, leads to increased variability and decreased reproducibility of organoid formation within an experiment.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Between days 55 and 75 of differentiation, depending on the cell lines, development of hair placodes, germs, and pegs will be visible under an inverted microscope at magnifications as low as 40X or 100X.

    “Between days 55 and 75 of differentiation depending on the cell lines, development of hair placodes, germs, and pegs will be visible under an inverted microscope at magnifications as low as 40X (4X microscope objective × 10X eyepiece) or 100X (10X microscope objective × 10X eyepiece; Fig. 5 and see Fig. 3c-e for immunostained images).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Findingnone given

    If the experiment is not optimal, the cell aggregates will be overpopulated with CNC cells and become opaque so that no sign of the epithelial cyst is visible.

    “If the experiment is not optimal, the cell aggregates will be overpopulated with CNC cells and become opaque so that no sign of the epithelial cyst is visible.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    By day 8, the epithelium forms as a surface lining of the sphere-like aggregate, and radial traces of migrating cells appear from the dark core to the epithelium.

    “By day 8, the epithelium forms as a surface lining of the sphere-like aggregate, and radial traces of migrating cells appear from the dark core to the epithelium.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    Hair follicle shafts appear to have difficulty rupturing the core epidermis and become abnormal with kinked or curled morphologies.

    “In the same context, hair follicle shafts appear to have difficulty rupturing the core epidermis and become abnormal with kinked or curled morphologies”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    The neuronal and glial components of skin organoids appeared to be CNC-derived.

    “The neuronal and glial components of skin organoids appeared to be CNC-derived.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Approximately 2 × 105 cells can be dissociated per skin organoid at day 30.

    “~day 30 (2 × 105 cells)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    Detached cell clumps will be passaged in tiny clusters of cells, averaging six cells per cell cluster.

    “The detached cell clumps will be passaged in tiny clusters of cells (average six cells per cell cluster).”
    Quote in the paperNot a substantial claim

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

    Findingnone given

    Total time to generate mature skin equipped with appendages is ~120 days onward.

    “Total time to generate mature skin equipped with appendages: ~120 days onward”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The skin organoids can be used to investigate basic skin biology, model disease, and further, the reconstruction or regeneration of skin tissue.

    “enabling the organoids to be used to investigate basic skin biology, model disease, and further, the reconstruction or regeneration of skin tissue.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The protocol can be performed in any laboratory setup for aseptic cell culture and by anyone trained in pluripotent stem cell (PSC) culture and cell differentiation techniques.

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

    Interpretationown data

    Even though 5 ng/mL BMP4 is used as a standardized concentration in this protocol, the optimal concentration must be determined cell line by cell line.

    “Even though 5 ng/mL BMP4 is used as a standardized concentration in this protocol, the optimal concentration must be determined cell line by cell line as we emphasized in the Fig. 6, ‘Experimental design’,”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The model may be suitable for studying hair follicle growth cycle initiation and perhaps the transition from anagen to catagen, but it may be limiting for studies of telogen-stage (resting phase) follicles.

    “Consequently, while the model may be suitable for studying hair follicle growth cycle initiation and perhaps the transition from anagen to catagen, it may be limiting for studies of telogen-stage (resting phase) follicles.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    In skin organoids, the cystic structure limits shedding and accumulates cornified tissue in the core.

    “In skin organoids, however, the cystic structure limits shedding and accumulates cornified tissue in the core.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

    Interpretationnone given

    The skin organoid system should be a suitable model for studying the development of human skin and its accessory tissues, as well as skin-related diseases.

    “The skin organoid system should be a suitable model for studying the development of human skin and its accessory tissues, as well as skin-related diseases.”
    Quote in the paperNot a substantial claim
46 of 46

  • The paper relies on this without support.

    The protocol can be performed in any laboratory setup for aseptic cell culture and by anyone trained in pluripotent stem cell (PSC) culture and cell differentiation techniques.

    “This protocol can be performed in any laboratory setup for aseptic cell culture and by anyone trained in pluripotent stem cell (PSC) culture and cell differentiation techniques.”

  • The paper relies on this without support.

    The activity of each small molecule and protein may vary depending on vendor, lot, and in-lab handling.

    “Lastly, regarding the reagents and equipment for skin organoid culture, the activity of each small molecule and protein may vary depending on vendor, lot, and in-lab handling.”

  • The paper relies on this without support.

    Diverse coating materials used on differentiation culture plates lead to a higher variability and cell death in cultures.

    “Diverse coating materials used on differentiation culture plates also lead to a higher variability and cell death in cultures.”

  • The paper relies on this without support.

    Some cells fail to survive after the freezing-thawing process.

    “As some cells fail to survive after the freezing-thawing process, we recommend seeding at a higher rather than a lower cell seeding density relative to the recommended density when the viability of the cells in the frozen cryovial is unclear.”

  • The paper relies on this without support.

    Y must be removed for normal cell proliferation.

    “Y must be removed for normal cell proliferation.”

  • The paper relies on this without support.

    Passaging newly thawed cells at least twice before differentiation ensures cells are healthier and better adapted to their growing environment.

    “We recommend that newly thawed cells undergo the passaging process at least twice (i.e., this and next section) before being used for differentiation experiments. This ensures cells are healthier and better adapted to their growing environment.”

  • The paper relies on this without support.

    Over-incubating cells with the enzyme at 37 °C causes cell clumps to dissociate to single cells.

    “Therefore, do not over-incubate the cells with the enzyme at 37 °C to avoid cell clumps dissociating to single cells.”

  • The paper relies on this without support.

    Diluting accutase at least to a 1:4 ratio inactivates its enzymatic activity.

    “Addition of medium (dilution of enzyme at least to a 1:4 ratio) inactivates the enzymatic activity of accutase.”

  • The paper relies on this without support.

    Keeping cell cluster sizes bigger (average ten cells per cluster) than during normal passaging increases the viability of cells after freezing and thawing.

    “Keep cell cluster sizes bigger (average ten cells per cluster) than during the normal passaging process to increase the viability of cells after freezing and thawing.”

  • The paper relies on this without support.

    Media in the edge wells evaporate during the differentiation process, altering the final volume, leading to inconsistent and variable outcomes.

    “Media in the edge wells evaporate during the differentiation process, altering the final volume, leading to inconsistent and variable outcomes.”
10 of 10

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?

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.

  • Day 3 treatment has two different stated durations

    Minor

    The procedure labels the day 3 LDN and bFGF treatment as taking 20 minutes, while the timing summary assigns the same steps 30 minutes.

  • Day 6 medium volume exceeds the specified reservoir capacity

    Minor

    The instruction calls for pouring 11 mL into a reservoir specified as holding 10 mL.

2 of 2

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Authors
Lee, J., van der Valk, W. H., Serdy, S. A., Deakin, C., Kim, J., Le, A. P., & Koehler, K. R.
Affiliations
Boston Children's Hospital (US)
Corresponding author
  • Lee, Jiyoon
  • Koehler, Karl R.
ORCID
Venue
Nature Protocols
Publisher
Springer Science and Business Media LLC
Published
2022-03-23
Volume
17
Issue
5
Pages
1266-1305
ISSN
  • 1750-2799 (ISSN-L)
  • 1754-2189
DOI
10.1038/s41596-022-00681-y
PMC
PMC10461778
OpenAlex
W4220659425
Semantic Scholar
5db3155a1243e5476d31187fdfc2c55ccda5ae9b
Cited by
132
References
79
Citations by year
2026: 34 · 2025: 43 · 2024: 31 · 2023: 19 · 2022: 5
FWCI
6.76
Type
journal article
Language
en
Update notices
correction (2023-08-23)
Open access
Licence
Locations
  • Nature Protocols (publishedVersion)
  • PubMed (publishedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (acceptedVersion)
  • PubMed Central (submittedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
  • Leiden Repository (Leiden University) (submittedVersion)
Funders
  • U.S. Department of Health & Human Services | NIH | National Institute on Deafness and Other Communication Disorders (R01DC017461)
  • U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR075018)
  • U.S. Department of Health & Human Services | National Institutes of Health (UL1TR001108)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Health Sciences › Medicine › Urology › Hair Growth and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Skin and Cellular Biology Research
Keywords
skin organoids, hair follicle, human pluripotent stem cells, three-dimensional organoid culture, cranial neural crest cells, sebaceous glands, Merkel cells, sensory neurons, dermis, fetal skin, skin regeneration, disease modeling
MeSH terms
Cell Differentiation, Hair, Humans, Organoids, Skin, Hair Follicle, Pluripotent Stem Cells

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 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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