Reprogramming of three-dimensional microenvironments for in vitro hair follicle induction
Open accessPublished version of a preprint
Abstract
During embryonic development, reciprocal interactions between epidermal and mesenchymal layers trigger hair follicle morphogenesis. This study revealed that microenvironmental reprogramming via control over these interactions enabled hair follicle induction in vitro. A key approach is to modulate spatial distributions of epithelial and mesenchymal cells in their spontaneous organization. The de novo hair follicles with typical morphological features emerged in aggregates of the two cell types, termed hair follicloids, and hair shafts sprouted with near 100% efficiency in vitro. The hair shaft length reached ~3 mm in culture. Typical trichogenic signaling pathways were up-regulated in hair follicloids. Owing to replication of hair follicle morphogenesis in vitro, melanosome production and transportation were also monitored in the hair bulb region. This in vitro hair follicle model might be valuable for better understanding hair follicle induction, evaluating hair growth and inhibition of hair growth by drugs, and modeling gray hairs in a well-defined environment.
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Written from the paper’s full text.
TL;DR
Low-concentration Matrigel (2% v/v) reprogrammed mouse embryonic epithelial and mesenchymal cells into core-shell aggregates, enabling in vitro hair follicle induction with ~100% sprouting efficiency and hair shafts reaching ~3 mm. The resulting hair follicloids recapitulated trichogenic signaling, pigmentation, and transplantation-based hair cycling, but lacked in vitro hair cycles and required rodent cells.
Key findings
- Matrigel supplementation at 2% (v/v) before aggregation changed cell organization to a core-shell structure and increased hair follicle sprouting from <1% (1 of 300 HFGs) to ~100% (~300 of 300 HFGs); FGF2 alone only improved sprouting to 6 of 300 HFGs (0.003 to 0.02 per HFG). Hair shafts reached ~3 mm at 23 days of culture.
- Hair follicloids showed hair follicle and surrounding-tissue markers including oil red O, α-SMA, CD34, Sox9, K5, Ki67, versican, gp100, TRP1, and c-Kit, and generated hair shafts with cuticles, medulla, and melanin granules; transplanted hair organoids showed hair regeneration and hair cycles for at least 10 months.
- ECM/adhesion PCR array showed 39 of 80 genes up-regulated and 1 down-regulated with Matrigel; up-regulated >1.5-fold genes included Fbln1, Fn1, Lama3, Lamb3, Lamc1, Col1a1, Col4a1, Col4a2, Col5a1, Vcan, Tgfbi, Itgb1, and MMPs. NF-κB and PI3K/Akt inhibitors completely suppressed sprouting, and Wnt10b-positive cells plus XAV-939 inhibition affected HFG-like structure formation and sprouting.
- α-MSH treatment produced dark-black hair shafts, increased total melanin, and increased TYRP1, TYRP2, TYR, and MITF expression at 6 days; Nexinhib20, a Rab27a antagonist, produced gray hair shafts, decreased total melanin, and decreased pigmentation gene expression.
Methods
- Mouse E18 dorsal skin epithelial and mesenchymal cells were dissociated, seeded in non-cell-adhesive round-bottom 96-well plates with 2% (v/v) Matrigel, cooled at 4°C for at least 30 min, then incubated at 37°C; optimized conditions used a 1:1 cell ratio and 20 × 10³ total cells, with day-3 embedding in 100% Matrigel for longer hair shafts.
- Characterization included histology and immunohistochemistry, SEM/TEM, RT-PCR arrays for ECM/adhesion and Wnt signaling, DNA microarray with GO/KEGG analysis, signaling inhibitors (MG132, LY294002, XAV-939), pigmentation agents (α-MSH, Nexinhib20), melanin quantification, and transplantation into nude mice.
- Statistical analyses used Student’s t tests or Tukey’s tests with P < 0.05; at least 12 hair follicloids per condition were cultured, with n = 20 for cell-ratio optimization and n = 12 for cell-number and Matrigel-concentration optimization, and experiments repeated with at least three donors.
Limitations
- Stated in the paper: hair follicloids lack hair cycles; sprouted follicles regressed within 1 month of culture; rodent embryonic skin cells were used, and human cell sources still require optimization, with ethical restrictions around human fetal/neonatal cells and heterogeneous cell populations complicating translation.
- Evident from the text: the in vitro model does not recapitulate full hair cycling or androgenic alopecia pathology, long-term culture shows follicle regression, and transplantation data come from nude mice rather than an immunocompetent or human system.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
23 of 47 cited sources checked · 10 supported · 0 flagged · accuracy not yet measured
- Findingown data
With two-step Matrigel supplement culture, hair shafts extended straightly because of hydrogel supports.
“However, with this two-step Matrigel supplement culture, the hair shafts extended straightly because of the hydrogel supports, which is consistent with a previous report on skin organoids (12).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [12] J. Lee, R. Böscke, P.-C. Tang, B. H. Hartman, S. Heller, K. R. Koehler, Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 22, 242– 254 (2018). 29298425 PMC5806130 source
The Results (Specialized epidermal and dermal cellular compartments arise in skin organoids; page unavailable) report that follicles grew outward in Matrigel droplets rather than wrapping around floating organoids. The Experimental Procedures (PSC differentiation and Image analysis; pages unavailable) describe Matrigel in the medium and later embedding. The paper does not say the hair shafts extended straight or establish that hydrogel support caused their direction of growth. (LLM, conf=0.88)
Experimental — accuracy not yet measured
- Findingown data
Expression levels of Apc, Axin1, Lef, Lrp5, and Wnt10b were significantly increased in hair follicloids.
“Specifically, the expression levels of genes involved in hair follicle development—Apc, Axin1, Lef, Lrp5, and Wnt10b (32, 35)—were significantly increased in hair follicloids (Fig. 5, A and B).”
Quote in the paperSubstantial claim2 of 2 sources checked- cites [32] B. T. MacDonald, K. Tamai, X. He, Wnt/beta-catenin signaling: Components, mechanisms, and diseases. Dev. Cell 17, 9– 26 (2009). 19619488 PMC2861485 source
The Perspectives section (page unavailable) says Wnt signaling regulates hair follicle development, and the pathway sections identify APC, Axin, TCF/LEF, and LRP5 as signaling components. The review does not establish that every named gene, particularly Wnt10b, is involved in hair follicle development. The reported increase in hair follicloids is the citing authors’ Figure 5 result, not a finding attributed to this review. (LLM, conf=0.82)
Experimental — accuracy not yet measured - cites [35] S. Reddy, T. Andl, A. Bagasra, M. M. Lu, D. J. Epstein, E. E. Morrisey, S. E. Millar, Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a as a target of Sonic hedgehog in hair follicle morphogenesis. Mech. Dev. 107, 69– 82 (2001). 11520664 source
The Introduction links Lef1 to hair follicle development, and section 2.2 reports elevated Wnt10b expression in developing hair follicles. The citation is attached to the description of the genes as involved in follicle development, not to the hair-follicloid increase shown in the citing paper’s Fig. 5. Our copy does not establish that role for Apc, Axin1, or Lrp5. (LLM, conf=0.86)
Experimental — accuracy not yet measured
- Premisecitation
ECM extracts from fetal skin promote hair regeneration after transplantation with epithelial and mesenchymal cells.
“ECM extracts from the fetal skin promote hair regeneration after transplantation with epithelial and mesenchymal cells (43).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [43] S. M.-Y. Fan, C.-F. Tsai, C.-M. Yen, M.-H. Lin, W.-H. Wang, C.-C. Chan, C.-L. Chen, K. K. L. Phua, S.-H. Pan, M. V. Plikus, S.-L. Yu, Y.-J. Chen, S.-J. Lin, Inducing hair follicle neogenesis with secreted proteins enriched in embryonic skin. Biomaterials 167, 121– 131 (2018). 29567388 PMC6050066 source
Results §3.4 reports that embryonic skin extract gave adult fibroblasts hair-forming competence before they were combined with keratinocytes in patch assays. The methods describe injection of these epithelial and mesenchymal cells into mice. The source calls the material a cell-free embryonic skin extract, however, not a specifically isolated ECM extract. (LLM, conf=0.83)
Experimental — accuracy not yet measured
- Premisecitation
The up-regulation of NF-κB and PI3K-Akt pathways is consistent with previous studies reporting that these genes are crucial for early hair follicle development and de novo regeneration of hair follicles.
“which is consistent with previous studies reporting that these genes are crucial for the early development of hair follicles and de novo regeneration of hair follicles (44, 45).”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [44] R. Schmidt-Ullrich, T. Aebischer, J. Hulsken, W. Birchmeier, U. Klemm, C. Scheidereit, Requirement of NF-κB/Rel for the development of hair follicles and other epidermal appendices. Development 128, 3843– 3853 (2001). 11585809 source
- cites [45] Y. Chen, Z. Fan, X. Wang, M. Mo, S. B. Zeng, R.-H. Xu, X. Wang, Y. Wu, PI3K/Akt signaling pathway is essential for de novo hair follicle regeneration. Stem Cell Res. Ther. 11, 144 (2020). 32245516 PMC7118821 source
The Results and Conclusions report that PI3K/Akt signaling is essential for de novo hair follicle regeneration. This paper does not establish that NF-κB or PI3K/Akt is crucial for early hair follicle development; its Introduction instead identifies Wnt signaling as predominant in early morphogenesis. No page numbers are available in our copy. (LLM, conf=0.86)
Experimental — accuracy not yet measured
- Premisecitation
Mouse embryonic skin mesenchymal and epithelial cells are composed of heterogeneous cell populations.
“Because both mesenchymal and epithelial cells from mouse embryonic skin are composed of heterogeneous cell populations (52, 53)”
Quote in the paperSubstantial claim2 of 2 sources checked- cites [52] P. K. Singhal, S. Sassi, L. Lan, P. Au, S. C. Halvorsen, D. Fukumura, R. K. Jain, B. Seed, Mouse embryonic fibroblasts exhibit extensive developmental and phenotypic diversity. Proc. Natl. Acad. Sci. U.S.A. 113, 122– 127 (2016). 26699463 PMC4711836 source
The Significance section reports that mouse embryonic fibroblasts are highly heterogeneous, supporting the mesenchymal-cell portion at a broader level. The cited passages do not establish heterogeneity specifically in embryonic skin or in its epithelial cells. (LLM, conf=0.87)
Experimental — accuracy not yet measured - cites [53] S. Liu, H. Zhang, E. Duan, Epidermal development in mammals: Key regulators, signals from beneath, and stem cells. Int. J. Mol. Sci. 14, 10869– 10895 (2013). 23708093 PMC3709707 source
- Findingown data
Global gene expression analysis showed that remodeling of ECMs seen in in vivo hair follicles, including laminins, fibronectins, and collagens, was activated in hair follicloids.
“The global gene expression analysis showed that the remodeling of the ECMs seen in in vivo hair follicles (49), including laminins, fibronectins, and collagens, was activated in the hair follicloids (Fig. 4B).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [49] S. C. de Groot, M. M. W. Ulrich, C. G. Gho, M. A. Huisman, Back to the future: From appendage development toward future human hair follicle neogenesis. Front. Cell Dev. Biol. 9, 661787 (2021). 33912569 PMC8075059 source
The sections on HF induction and organogenesis describe changes in laminin expression during hair follicle development, and the section on HF induction describes a change in collagen VII distribution. These support the cited point about ECM changes in in vivo follicles, but our copy does not establish the claimed involvement of fibronectins. The hair-follicloid gene-expression result is attributed to the citing paper’s Fig. 4B, not to this source. (LLM, conf=0.82)
Experimental — accuracy not yet measured
- Findingown data
Hair shaft length reached ~3 mm in culture at 23 days.
“The hair shaft length reached ~3 mm in culture.”
- Also stated in: § Control over the spatial distribution of cells for hair follicle neogenesis in vitro · plain
“The length of hair shafts reached ~3 mm at 23 days (Fig. 2B).”
Quote in the paperNot a substantial claim - Also stated in: § Control over the spatial distribution of cells for hair follicle neogenesis in vitro · plain
- Findingown data
De novo hair follicles with typical morphological features emerged in aggregates of epithelial and mesenchymal cells, termed hair follicloids.
“The de novo hair follicles with typical morphological features emerged in aggregates of the two cell types, termed hair follicloids”
Quote in the paperSubstantial claim - Findingnone given
Typical trichogenic signaling pathways were up-regulated in hair follicloids.
“Typical trichogenic signaling pathways were up-regulated in hair follicloids.”
Quote in the paperSubstantial claim - Findingown data
Hair shafts sprouted with near 100% efficiency in vitro.
“hair shafts sprouted with near 100% efficiency in vitro”
- Also stated in: § Control over the spatial distribution of cells for hair follicle neogenesis in vitro · plain
“A key finding of this study was that the supplement of Matrigel [or other extracellular matrices (ECMs), as described later] notably improved the sprouting efficiency to ~1.0 (~300 of 300 HFGs) (Fig. 1).”
Quote in the paperSubstantial claim - Also stated in: § Control over the spatial distribution of cells for hair follicle neogenesis in vitro · plain
- Findingown data
Matrigel had to be supplemented before dense and compacted aggregate formation.
“However, Matrigel had to be supplemented before dense and compacted aggregate formation.”
Quote in the paperSubstantial claim - Findingown data
In the presence of Matrigel, cells formed an aggregate composed of a core of epithelial cells surrounded by mesenchymal cells, not a dumbbell-shaped aggregate.
“The cells did not form a dumbbell-shaped aggregate but formed an aggregate composed of a core of epithelial cells surrounded by mesenchymal cells (Fig. 2A).”
Quote in the paperSubstantial claim - Findingown data
Supplement of Matrigel or other extracellular matrices notably improved sprouting efficiency to ~1.0 (~300 of 300 HFGs).
“A key finding of this study was that the supplement of Matrigel [or other extracellular matrices (ECMs), as described later] notably improved the sprouting efficiency to ~1.0 (~300 of 300 HFGs) (Fig. 1).”
Quote in the paperSubstantial claim - Findingown data
Hair cuticles and melanin granules were observed in cross sections of generated hair shafts similarly to those in native hair shafts.
“Hair cuticles and melanin granules were observed in the cross sections of hair shafts similarly to those in the native hair shafts (Fig. 3D).”
Quote in the paperSubstantial claim - Findingown data
Generated hair shafts had hair shaft-specific features, including the hair cuticle and medulla.
“The hair shafts generated had hair shaft–specific features, including the hair cuticle (Fig. 3B) and medulla (Fig. 3C).”
Quote in the paperSubstantial claim - Findingown data
Genes up-regulated >1.5-fold include Fbln1, Fn1, Lama3, Lamb3, Lamc1, Col1a1, Col4a1, Col4a2, Col5a1, Vcan, Tgfbi, Itgb1, Mmp2, Mmp9, Mmp11, and Mmp15.
“The genes up-regulated >1.5-fold include fibulin-1 (Fbln1), fibronectin (Fn1), laminin (Lama3, Lamb3, and Lamc1), collagen (Col1a1, Col4a1, Col4a2, and Col5a1), versican (Vcan), transforming growth factor–beta–induced protein ig-h3 (Tgfbi), integrin β1 (Itgb1), and matrix metalloproteinases (MMPs) (Mmp2, Mmp9, Mmp11, and Mmp15).”
Quote in the paperSubstantial claim - Findingown data
FGF2 slightly improved the frequency of appearance of hair follicle sprouting (6 of 300 HFGs).
“FGF2 slightly improved the frequency of appearance of hair follicle sprouting (6 of 300 HFGs).”
Quote in the paperNot a substantial claim - Findingown data
Sprouting occurred only in <1% of HFGs (1 of 300 HFGs) in the initial condition.
“Although the sprouting occurred only in <1% of HFGs (1 of 300 HFGs), this matured hair follicle formation encouraged us to seek an approach for in vitro hair follicle neogenesis.”
Quote in the paperSubstantial claim - Findingown data
FGF2 increased the expression of versican and Tgfβ2 in HFGs.
“Among them, fibroblast growth factor 2 (FGF2) was effective for HFGs to increase the expression of two trichogenic genes, versican and Tgfβ2 (fig. S1D), which is consistent with a previous study (15).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [15] T. Kageyama, A. Nanmo, L. Yan, T. Nittami, J. Fukuda, Effects of platelet-rich plasma on in vitro hair follicle germ preparation for hair regenerative medicine. J. Biosci. Bioeng. 130, 666– 671 (2020). 32859524 source
- Findingown data
A hair shaft-like protruding fiber appeared in vitro from dumbbell-shaped HFGs after 6 days of culture.
“In the present study, we found that a hair shaft–like protruding fiber appeared in vitro from the dumbbell-shaped HFGs after 6 days of culture (fig. S1, A and B).”
Quote in the paperSubstantial claim - Findingreasoning
At the highest cell number, the sprouting number significantly dropped.
“However, at the highest cell number, the sprouting number significantly dropped, which could be attributed to the shortage of oxygen and nutrients.”
Quote in the paperSubstantial claim - Findingown data
The sprouting number increased by increasing the total number of cells at the constant 1:1 mixing ratio.
“The sprouting number was increased by increasing the total number of cells at the constant mixing ratio (epithelial and mesenchymal cells, 1:1) (Fig. 2E).”
Quote in the paperSubstantial claim - Findingown data
Supplementation with either MG132 or LY294002 completely suppressed in vitro hair follicle sprouting.
“Supplementation with either inhibitor completely suppressed in vitro hair follicle sprouting (Fig. 4D), indicating that both NF-κB and PI3K/Akt cascades are critical mechanisms in hair follicle morphogenesis.”
Quote in the paperSubstantial claim - Findingown data
The number of up-regulated Wnt signaling-related genes was higher in hair follicloids than in Matrigel (−).
“The number of up-regulated Wnt signaling–related genes was higher than those in the Matrigel (−) (Fig. 5A).”
Quote in the paperSubstantial claim - Findingown data
Gray hair shafts were generated in hair follicloids when exposed to an antagonist of Rab27a, Nexinhib20.
“Gray hair shafts were generated in hair follicloids when exposed to an antagonist of Rab27a, Nexinhib20 (fig. S4, A to C).”
Quote in the paperSubstantial claim - Findingown data
Hair follicle sprouting with Matrigel and other ECMs was closely associated with formation of a core-shell-shaped configuration rather than a dumbbell-shaped configuration.
“The hair follicle sprouting with Matrigel and the other ECMs was closely associated with the formation of a core-shell–shaped configuration rather than dumbbell-shaped (Fig. 7).”
Quote in the paperSubstantial claim - Findingown data
Collagen I resulted in hair follicle sprouting at 96% efficiency.
“It should be noted that collagen I, the most abundant ECM in the body, resulted in hair follicle sprouting at 96% efficiency, which would provide a robust, stable, and affordable system (fig. S5B).”
Quote in the paperSubstantial claim - Findingown data
Culture media with different viscosities prepared by adding methylcellulose did not produce hair follicle sprouting.
“Culture media with different viscosities were prepared by adding methylcellulose, a cell inert polymer, and tested for hair follicloid formation, but no hair follicle sprouting was observed (fig. S7).”
Quote in the paperSubstantial claim - Findingown data
Hair follicle sprouting was virtually independent of the ECM types used in this study.
“Because hair follicle sprouting was virtually independent of the ECM types used in this study”
Quote in the paperSubstantial claim - Findingown data
Hair follicloids cultured in the presence of α-MSH exhibited increased expression of TYRP1, TYRP2, TYR, and MITF at 6 days of culture.
“Hair follicloids cultured in the presence of α-MSH exhibited an increased expression of four pigmentation genes—TYRP1, TYRP2, TYR, and MITF—at 6 days of culture (Fig. 6D).”
Quote in the paperSubstantial claim - Findingcitation
When adhesivity between heterogeneous cell types is comparable to that between homogeneous cell types, the cells form an aggregate with more adhesive cells at the core and fewer adhesive cells in the outer layer.
“In contrast, when adhesivity between the heterogeneous cell types is comparable to that between homogeneous cell types, the cells form an aggregate composed of more adhesive cells at the core and fewer adhesive cells in the outer layer.”
Quote in the paperSubstantial claim - Findingreasoning
Monte Carlo simulations based on the differential adhesion hypothesis revealed that two cell types separated and formed a dumbbell-shaped aggregate when adhesivity between heterogeneous cell types was weak compared with that between homogeneous cell types.
“Monte Carlo simulations based on the hypothesis revealed that two types of cells separated from each other and eventually formed a dumbbell-shaped aggregate when the adhesivity between heterogeneous cell types is weak compared with that between homogeneous cell types.”
Quote in the paperSubstantial claim - Findingown data
In hair follicloids, NF-κB and PI3K-Akt signaling pathways were up-regulated in the presence of either Matrigel or collagen I.
“The transcriptome analysis of the hair follicloids revealed that signal pathways such as NF-κB and PI3K-Akt pathway were up-regulated in the presence of either Matrigel (Fig. 4C) or collagen I (fig. S6)”
Quote in the paperNot a substantial claim - Findingown data
Highly efficient hair shaft sprouting occurs using collagen IV, laminin-entactin complex, growth factor-reduced Matrigel, and collagen I.
“The results revealed that highly efficient hair shaft sprouting occurs using collagen IV, laminin-entactin complex, growth factor–reduced Matrigel, and collagen I, suggesting that growth factors and laminin, the major component of Matrigel, are not directly involved in the hair follicle sprouting in the hair follicloids (fig. S5A).”
Quote in the paperSubstantial claim - Findingown data
During long-term observation of hair follicloids with Matrigel or collagen I, the sprouted follicles appeared to regress within 1 month of culture.
“During long-term observation of hair follicloids with Matrigel or collagen I, the sprouted follicles appeared to regress within 1 month of culture (fig. S5C).”
Quote in the paperSubstantial claim - Findingown data
The most enriched pathways associated with hair follicle and epidermis development had significant P values (P < 0.05).
“The most enriched pathways associated with hair follicle and epidermis development were selected from “gene ontology” and “pathways” and had significant P values (P < 0.05).”
Quote in the paperNot a substantial claim - Findingnone given
The proposed organoid culture system generates hair follicles and hair shafts with almost 100% efficiency.
“In summary, we proposed an organoid culture system that generates hair follicles and hair shafts with almost 100% efficiency.”
Quote in the paperNot a substantial claim - Findingown data
Hair shaft generation was observed in the presence of MG132 and LY294002 after 6 days.
“Hair shaft generation was observed in the presence of MG132 and LY294002 after 6 days.”
Quote in the paperSubstantial claim - Findingown data
Successful hair follicloid formation was observed in the microdevice in 9 days of culture.
“Successful hair follicloid formation was observed in the microdevice in 9 days of culture (fig. S9).”
Quote in the paperSubstantial claim - Findingown data
Core-shell aggregates prepared through two-step seeding, with an epithelial core and a mesenchymal shell, did not produce hair follicle sprouting, with or without low concentrations of ECMs.
“No hair follicle sprouting occurred with this approach, neither with nor without low concentrations of ECMs (fig. S8).”
Quote in the paperSubstantial claim - Findingnone given
Hair follicloids lack hair cycles.
“The limitation of hair follicloids is the lack of hair cycles.”
Quote in the paperSubstantial claim - Premisecitation
We have reported an approach for engineering HFG-like aggregates in vitro through self-organization of embryonic dorsal epithelial and mesenchymal cells in a laboratory-made microwell array plate.
“We have reported an approach for engineering HFG-like aggregates in vitro through the self-organization of embryonic dorsal epithelial and mesenchymal cells in a laboratory-made microwell array plate (14).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [14] T. Kageyama, C. Yoshimura, D. Myasnikova, K. Kataoka, T. Nittami, S. Maruo, J. Fukuda, Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine. Biomaterials 154, 291– 300 (2018). 29156398 source
- Interpretationown data
Microenvironmental reprogramming via control over epithelial-mesenchymal interactions enabled hair follicle induction in vitro.
“This study revealed that microenvironmental reprogramming via control over these interactions enabled hair follicle induction in vitro.”
Quote in the paperSubstantial claim - Premisecitation
It remains challenging to induce hair neogenesis in in vitro culture, including the generation of matured hair follicles and the extension of long hair shafts.
“However, it remains challenging to induce hair neogenesis in in vitro culture, including the generation of matured hair follicles (e.g., dermal papilla and the bulge region) and the extension of long hair shafts (11).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [11] M. X. Lei, L. J. Schumacher, Y. C. Lai, W. T. Juan, C. Y. Yeh, P. Wu, T. X. Jiang, R. E. Baker, R. B. Widelitz, L. Yang, C. M. Chuong, Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells. Proc. Natl. Acad. Sci. U.S.A. 114, E7101– E7110 (2017). 28798065 PMC5576784 source
- Interpretationreasoning
Involvement of Matrigel components such as ECMs between the cells may be crucial to induce core-shell aggregates and subsequent hair follicle sprouting.
“These results imply that the involvement of Matrigel components such as ECMs between the cells may be crucial to induce core-shell aggregates and subsequent hair follicle sprouting.”
Quote in the paperSubstantial claim - Interpretationown data
PCR array results imply that hair follicloids reprogrammed their niches via activation of the secretion and reconstitution of ECMs.
“Collectively, the PCR array results imply that the hair follicloids reprogrammed their niches via the activation of the secretion and reconstitution of ECMs.”
Quote in the paperSubstantial claim - Interpretationown data
These results suggest that hair follicloids partially mimic normal embryonic hair folliculogenesis.
“These results suggest that the hair follicloids partially mimic normal embryonic hair folliculogenesis.”
Quote in the paperSubstantial claim - Interpretationcitation
The spatial distribution of the two cell types may be explained by the differential adhesion hypothesis governed by interfacial energies.
“The mechanism that determines the spatial distribution of the two cell types may be explained by the differential adhesion hypothesis governed by interfacial energies (47, 48).”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [47] R. A. Foty, M. S. Steinberg, The differential adhesion hypothesis: A direct evaluation. Dev. Biol. 278, 255– 263 (2005). 15649477 source
- cites [48] Y. Sun, Q. Wang, Modeling and simulations of multicellular aggregate self-assembly in biofabrication using kinetic Monte Carlo methods. Soft Matter 9, 2172– 2186 (2013). source
- Interpretationown data
Growth factors and laminin, the major component of Matrigel, are not directly involved in hair follicle sprouting in hair follicloids.
“suggesting that growth factors and laminin, the major component of Matrigel, are not directly involved in the hair follicle sprouting in the hair follicloids (fig. S5A).”
Quote in the paperSubstantial claim - Interpretationown data
Secretion of ECMs and their remodeling to form niches during spontaneous tissue formation may be critical for hair follicle sprouting.
“These results indicate that the secretion of ECMs and their remodeling to form niches during the spontaneous tissue formation may be critical for hair follicle sprouting.”
Quote in the paperSubstantial claim - Interpretationreasoning
Hair orientation in hair follicloids may be determined by the initial core-shell configuration composed of a core of epithelial cells surrounded by mesenchymal cells.
“in hair follicloids may be determined by this initial core-shell configuration composed of a core of epithelial cells surrounded by mesenchymal cells.”
Quote in the paperSubstantial claim - Premisecitation
A microdevice with an array of 1.0-mm-diameter round-bottom wells made of oxygen-permeable silicone rubber could produce hundreds to thousands of dumbbell-shaped HFGs under improved oxygen concentration conditions.
“In our previous study, we showed that a microdevice with an array of 1.0-mm-diameter round-bottom wells made of oxygen-permeable silicone rubber could produce hundreds to thousands of dumbbell-shaped HFGs under improved oxygen concentration conditions (14).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [14] T. Kageyama, C. Yoshimura, D. Myasnikova, K. Kataoka, T. Nittami, S. Maruo, J. Fukuda, Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine. Biomaterials 154, 291– 300 (2018). 29156398 source
- Classified as probably not a substantial claim. 27
- May combine two claims. 34
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationEMIs drive stomach specification, kidney duct patterning, lung development, lacrimal gland bud formation, and teeth bud formation.
“For example, EMIs drive stomach specification (1), duct patterning of the kidney (2) and lung (3), and bud formation of the lacrimal gland (4) and teeth (5).”
Quote in the paperNot a substantial claim1 of 3 sources checked- cites [1] T. H. Kim, R. A. Shivdasani, Stomach development, stem cells and disease. Development 143, 554– 565 (2016). 26884394 PMC4760317 source
- cites [2] O. Pleniceanu, O. Harari-Steinberg, B. Dekel, Concise review: Kidney stem/progenitor cells: Differentiate, sort out, or reprogram? Stem Cells 28, 1649– 1660 (2010). 20652959 PMC2996087 source
- cites [3] J. M. Shannon, B. A. Hyatt, Epithelial-mesenchymal interactions in the developing lung. Annu. Rev. Physiol. 66, 625– 645 (2004). 14977416 source
Classified as probably not a substantial claim.
PremisecitationEpithelial-mesenchymal interactions trigger the development of various tissues and organs through specific signaling pathways and transcription factors.
“During the early developmental stages, epithelial-mesenchymal interactions (EMIs) trigger the development of various tissues and organs through specific signaling pathways and transcription factors.”
Quote in the paperSubstantial claim1 of 3 sources checked- cites [1] T. H. Kim, R. A. Shivdasani, Stomach development, stem cells and disease. Development 143, 554– 565 (2016). 26884394 PMC4760317 source
- cites [2] O. Pleniceanu, O. Harari-Steinberg, B. Dekel, Concise review: Kidney stem/progenitor cells: Differentiate, sort out, or reprogram? Stem Cells 28, 1649– 1660 (2010). 20652959 PMC2996087 source
The Developmental Nephrology section and Figure 1 describe reciprocal mesenchymal–ureteric bud interactions in kidney development, including WNT signals and transcription factors. They support the kidney example, but our copy does not establish the broader claim that such interactions trigger development of various tissues and organs. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [3] J. M. Shannon, B. A. Hyatt, Epithelial-mesenchymal interactions in the developing lung. Annu. Rev. Physiol. 66, 625– 645 (2004). 14977416 source
Classified as probably not a substantial claim.
PremisecitationThe hair follicle is a unique mini organ that repetitively and dynamically undergoes cyclic regeneration postnatally.
“However, the hair follicle is a unique mini organ that repetitively and dynamically undergoes cyclic regeneration postnatally (6, 7).”
Quote in the paperSubstantial claim1 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationSkin organoids including hair follicles and sebaceous glands were generated in vitro using mouse pluripotent stem cells through self-organization of epidermal and dermal layers.
“Skin organoids including hair follicles and sebaceous glands were generated in vitro using mouse pluripotent stem cells through self-organization of epidermal and dermal layers (12).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [12] J. Lee, R. Böscke, P.-C. Tang, B. H. Hartman, S. Heller, K. R. Koehler, Hair follicle development in mouse pluripotent stem cell-derived skin organoids. Cell Rep. 22, 242– 254 (2018). 29298425 PMC5806130 source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationHuman induced pluripotent stem cells were used to generate skin organoids with appendages including hair follicles in 4 to 5 months of culture.
“More recently, human induced pluripotent stem cells were used to generate skin organoids with appendages including hair follicles in 4 to 5 months of culture (13).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [13] J. Y. Lee, C. C. Rabbani, H. Y. Gao, M. R. Steinhart, B. M. Woodruff, Z. E. Pflum, A. Kim, S. Heller, Y. L. Liu, T. Z. Shipchandler, K. R. Koehler, Hair-bearing human skin generated entirely from pluripotent stem cells. Nature 582, 399– 404 (2020). 32494013 PMC7593871 source
Classified as probably not a substantial claim.
PremisecitationHFG-like aggregates were reconstructed in culture using dissociated embryonic epithelial and mesenchymal cells.
“Hair follicle germ (HFG)–like aggregates were reconstructed in culture using dissociated embryonic epithelial and mesenchymal cells (10).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [10] K. E. Toyoshima, K. Asakawa, N. Ishibashi, H. Toki, M. Ogawa, T. Hasegawa, T. Irie, T. Tachikawa, A. Sato, A. Takeda, T. Tsuji, Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches. Nat. Commun. 3, 784 (2012). 22510689 PMC3337983 source
Classified as probably not a substantial claim. May combine two claims.
PremisecitationWhen transplanted into the skin of nude mice, HFGs generated de novo hair follicles, implicating that HFGs have hair neogenesis capability.
“When transplanted into the skin of nude mice, HFGs generated de novo hair follicles, implicating that HFGs have hair neogenesis capability.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [10] K. E. Toyoshima, K. Asakawa, N. Ishibashi, H. Toki, M. Ogawa, T. Hasegawa, T. Irie, T. Tachikawa, A. Sato, A. Takeda, T. Tsuji, Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches. Nat. Commun. 3, 784 (2012). 22510689 PMC3337983 source
May combine two claims.
PremisecitationSkin 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.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [13] J. Y. Lee, C. C. Rabbani, H. Y. Gao, M. R. Steinhart, B. M. Woodruff, Z. E. Pflum, A. Kim, S. Heller, Y. L. Liu, T. Z. Shipchandler, K. R. Koehler, Hair-bearing human skin generated entirely from pluripotent stem cells. Nature 582, 399– 404 (2020). 32494013 PMC7593871 source
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
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationBoth MMP11 and MMP15 degrade fibronectin and laminin.
“both MMP11 and MMP15 degrade fibronectin and laminin (30).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [30] P. Lu, K. Takai, V. M. Weaver, Z. Werb, Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb. Perspect. Biol. 3, a005058 (2011). 21917992 PMC3225943 source
In “Protein Components of the ECM Are Degraded by Proteinases” (page unavailable), the paper says MMP-3 and MMP-10 target fibronectin and laminin. Searches for MMP-11 and MMP-15 found no passage saying either degrades those proteins. Naming different MMPs does not establish that MMP-11 or MMP-15 cannot do so. (LLM, conf=0.78)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationMMP2 and MMP9 are gelatinases whose main substrates are gelatin and collagen type IV.
“MMP2 and MMP9 are gelatinase, of which the main substrates are gelatin and collagen type IV (29).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [29] A. Nikolov, N. Popovski, Role of gelatinases MMP-2 and MMP-9 in healthy and complicated pregnancy and their future potential as preeclampsia biomarkers. Diagnostics 11, 480 (2021). 33803206 PMC8001076 source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationWnt signaling is one of the fundamental mechanisms regulating embryonic development and is closely associated with hair follicle morphogenesis and differentiation.
“Wnt signaling is one of the fundamental mechanisms regulating embryonic development and is closely associated with hair follicle morphogenesis and differentiation (32–34).”
Quote in the paperNot a substantial claim2 of 3 sources checked- cites [32] B. T. MacDonald, K. Tamai, X. He, Wnt/beta-catenin signaling: Components, mechanisms, and diseases. Dev. Cell 17, 9– 26 (2009). 19619488 PMC2861485 source
The Introduction describes Wnt signaling as fundamental to embryonic development, and Perspectives identifies hair follicle development and homeostasis as examples of Wnt-regulated biology. The section on Wnt target genes says Wnt regulates differentiation generally, but our copy does not specifically connect it to hair follicle differentiation. No page numbers are available in our copy. (LLM, conf=0.82)
Experimental — accuracy not yet measured - cites [33] T. Andl, S. T. Reddy, T. Gaddapara, S. E. Millar, WNT signals are required for the initiation of hair follicle development. Dev. Cell 2, 643– 653 (2002). 12015971 source
- cites [34] X. Lim, R. Nusse, Wnt signaling in skin development, homeostasis, and disease. Cold Spring Harb. Perspect. Biol. 5, a008029 (2013). 23209129 PMC3552514 source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationTGFBI is a secreted RGD motif-containing protein that binds to ECMs such as collagen types I, II, and IV as well as proteoglycans.
“TGFBI is a secreted Arg-Gly-Asp (RGD) motif-containing protein that also binds to ECMs such as collagen types I, II, and IV as well as proteoglycans (28).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [28] B. Reinboth, J. Thomas, E. Hanssen, M. A. Gibson, Beta ig-h3 interacts directly with biglycan and decorin, promotes collagen VI aggregation, and participates in ternary complexing with these macromolecules. J. Biol. Chem. 281, 7816– 7824 (2006). 16434404 source
The introductory passage (page unavailable) identifies βig-h3/TGFBI as an extracellular-matrix protein and says it binds several collagen types; the discussion specifically mentions collagen I binding. The paper also reports direct binding to the proteoglycans biglycan and decorin. Our copy does not identify collagen II or IV as binding partners or explicitly establish that the protein is secreted and contains an RGD motif; it lists RGD-CAP as an alternative name. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe cells used contained melanoblasts, which differentiate into melanocyte stem cells following melanocytes in the hair matrix.
“The cells used contained melanoblasts (37), which differentiate into melanocyte stem cells following melanocytes in the hair matrix.”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [37] W. Qiu, C. M. Chuong, M. Lei, Regulation of melanocyte stem cells in the pigmentation of skin and its appendages: Biological patterning and therapeutic potentials. Exp. Dermatol. 28, 395– 405 (2019). 30537004 PMC6488374 source
The Introduction says melanoblasts differentiate into melanocyte stem cells in the hair-follicle bulge, and that these stem cells give rise to mature melanocytes in the hair matrix. The paper does not establish that the citing study’s cells contained melanoblasts; nor does it describe stem cells arising after hair-matrix melanocytes. (LLM, conf=0.80)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationA mutation of melanosome transport-related genes including Rab27a causes hair graying through Griscelli syndrome, a rare autosomal recessive disorder.
“A mutation of melanosome transport–related genes including Rab27a causes hair graying through the so-called Griscelli syndrome, a rare autosomal recessive disorder (39).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [39] P. Bahadoran, E. Aberdam, F. Mantoux, R. Busca, K. Bille, N. Yalman, G. de Saint-Basile, R. Casaroli-Marano, J. P. Ortonne, R. Ballotti, Rab27a: A key to melanosome transport in human melanocytes. J. Cell Biol. 152, 843– 850 (2001). 11266474 PMC2195788 source
May combine two claims.
FindingreasoningThe mixing ratios of the two cell types significantly altered sprouting, and the sprouting number peaked when the mixing ratio of epithelial and mesenchymal cells was equal.
“The mixing ratios of the two cell types significantly altered the sprouting (Fig. 2D). The sprouting number peaked when the mixing ratio of epithelial and mesenchymal cells was equal, probably because EMIs might have been maximized.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataHair follicle sprouting was sensitive to the timing of Matrigel supplement; vigorous sprouting occurred when Matrigel was added 0 to 6 hours after seeding, but almost no sprouting occurred when it was added over 15 hours.
“Hair follicle sprouting was sensitive to the timing of Matrigel supplement (Fig. 2C). Vigorous sprouting was observed when the timing was 0 to 6 hours after seeding, but when it was over 15 hours, almost no sprouting was observed where the cells formed dumbbell-shaped HFGs.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataMultiple pigmented portions emerged in the single aggregates at 2 days of culture, from which hair follicles sprouted.
“Multiple pigmented portions emerged in the single aggregates at 2 days of culture, from which hair follicles sprouted (Fig. 2A, arrows, and movie S1).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataSupplement of Matrigel at a low concentration altered the spatial distribution of the two cell types in the aggregate at 2 days of culture and improved subsequent sprouting of hair follicles in vitro.
“The supplement of Matrigel at a low concentration notably altered the spatial distribution of the two types of cells in the aggregate at 2 days of culture, as shown in Fig. 4A, and improved subsequent sprouting of hair follicles in vitro.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataWhen hair organoids were transplanted into the dorsal skin of nude mice, they showed strong hair regeneration activity and hair cycles repeating for at least 10 months at approximately monthly intervals.
“It should be noted that when the hair organoids were transplanted into the dorsal skin of nude mice, they showed strong hair regeneration activity and hair cycles repeating for at least 10 months (at approximately monthly intervals; fig. S2).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThere was no significant difference in sprouting number between 1 and 5% Matrigel, but 10% Matrigel formed a hydrogel in which most cells remained encapsulated as single cells and did not form an aggregate, resulting in quite low sprouting.
“There was no significant difference in the sprouting number between 1 and 5% (v/v) of Matrigel, but Matrigel at 10% (v/v) formed a hydrogel in which most cells remained encapsulated as a single cell and did not form an aggregate, resulting in quite low sprouting (Fig. 2F).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataRT2 Profiler PCR array revealed that almost half the genes (39 of 80) were substantially up-regulated, while 1 was down-regulated in the presence of Matrigel compared with those without Matrigel.
“We used the RT2 Profiler polymerase chain reaction (PCR) array for ECM and adhesion molecules, which revealed that almost half the genes (39 of 80 genes) were substantially up-regulated, while 1 was down-regulated in the presence of Matrigel compared with those without Matrigel (Fig. 4B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe fibers became ~200 μm in length at 23 days of culture, with typical morphological features such as the hair cortex and microfibrils.
“The fibers became ~200 μm in length at 23 days of culture, which had typical morphological features such as the hair cortex and microfibrils (fig. S1C).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe Wnt signaling pathway was significantly up-regulated on Matrigel (+) compared to Matrigel (−), but there was no significant difference in Wnt signaling between day 0 and Matrigel (+).
“The Wnt signaling pathway was significantly up-regulated on Matrigel (+) compared to that on Matrigel (−), but there was no significant difference in Wnt signaling between day 0 [fresh cells from embryonic day 18 (E18) mice skin] and Matrigel (+).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataKEGG pathway analysis showed that the functioning of the NF-κB and PI3K-Akt signaling pathways was significantly different between D0 and Matrigel (+) and between Matrigel (−) and Matrigel (+).
“Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the functioning of the nuclear factor κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathways was significantly different between both D0 and Matrigel (+) as well as Matrigel (−) and Matrigel (+) (Fig. 4Ciii).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataGene ontology analysis showed that genes associated with hair follicle and epidermis development were differentially expressed between D0 and Matrigel (+) and between Matrigel (−) and Matrigel (+).
“Gene ontology analysis showed that genes associated with hair follicle and epidermis development were differentially expressed between both D0 and Matrigel (+) as well as Matrigel (−) and Matrigel (+) (Fig. 4Cii).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataHFG-like structures were generated in the outer layers of hair follicloids and contained Wnt10b-positive cells, as observed in embryonic skin.
“Hematoxylin and eosin (H&E) and immunofluorescence staining revealed that HFG-like structures were generated in the outer layers of hair follicloids and contained Wnt10b-positive cells, as observed in the embryonic skin (Fig. 5, C to E).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataAddition of XAV-939 to hair follicloids decreased the number of Wnt10b-positive cells and inhibited the formation of HFG-like structures and subsequent hair follicle sprouting.
“The addition of XAV-939 to hair follicloids decreased the number of Wnt10b-positive cells and inhibited the formation of HFG-like structures and subsequent hair follicle sprouting (Fig. 5F).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe volume of total melanin significantly decreased and the expression of pigmentation genes including TYRP1, TYRP2, TYR, and MITF decreased in the presence of Nexinhib20.
“The volume of total melanin significantly decreased (fig. S4D), and the expression of pigmentation genes including TYRP1, TYRP2, TYR, and MITF decreased in the presence of Nexinhib20 (fig. S4E).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataHair follicloids treated with α-MSH generated dark-black hair shafts with an increased total melanin volume.
“Hair follicloids treated with α-MSH generated dark-black hair shafts (Fig. 6B and movie S4) with an increased total melanin volume (Fig. 6C).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn the presence of Matrigel, embryonic epithelial and mesenchymal cells separated and self-organized into a core-shell aggregate within 2 days of culture, then generated epithelial cysts in the core and HFG-like structures containing Wnt10b-positive epithelial cells in the outer layer.
“In the presence of Matrigel (non–gel forming), the two types of cells separated from each other and self-organized into a core-shell aggregate within 2 days of culture; following which the aggregates generated epithelial cysts in the core and HFG-like structures containing Wnt10b-positive epithelial cells in the outer layer.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe generated hair follicles had specific features including bulges, sub-bulge, dermal papillae, melanosomes, adipose tissues, and cuticles.
“These hair follicles had specific features, including bulges, sub-bulge, dermal papillae, melanosomes, adipose tissues, and cuticles.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataAfter an additional 6 days of culture, hair follicle sprouting occurred, and hair shafts growing from HFGs became elongated.
“After an additional 6 days of culture, hair follicle sprouting occurred, and hair shafts growing from HFGs became elongated.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationSome crucial mechanisms of EMIs, including the effect of Wnt signaling on hair follicle morphogenesis, have been elucidated using animal models.
“Over the past several decades, some crucial mechanisms of EMIs, including the effect of Wnt signaling on hair follicle morphogenesis (9), have been elucidated using animal models.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [9] D. M. Chen, A. Jarrell, C. T. Guo, R. Lang, R. Atit, Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation. Development 139, 1522– 1533 (2012). 22434869 PMC3308184 source
May combine two claims.
PremisecitationIn this approach, two types of rodent cells initially formed a single aggregate where the two cell types were distributed randomly and subsequently spatially separated and formed HFG-like structures during 3 days of culture.
“In this approach, the two types of rodent cells initially formed a single aggregate where the two cell types were distributed randomly and subsequently spatially separated and formed HFG-like structures during 3 days of culture.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [14] T. Kageyama, C. Yoshimura, D. Myasnikova, K. Kataoka, T. Nittami, S. Maruo, J. Fukuda, Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine. Biomaterials 154, 291– 300 (2018). 29156398 source
May combine two claims.
PremisecitationWhen transplanted into the skin of nude mice, HFGs efficiently generated de novo hair follicles in a few weeks and represented normal hair cycles.
“When transplanted into the skin of nude mice, the HFGs efficiently generated de novo hair follicles in a few weeks and represented normal hair cycles.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [14] T. Kageyama, C. Yoshimura, D. Myasnikova, K. Kataoka, T. Nittami, S. Maruo, J. Fukuda, Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine. Biomaterials 154, 291– 300 (2018). 29156398 source
May combine two claims.
PremisecitationHFGs generated hair follicles and shafts in vitro in long-term culture, but the appearance of hair follicles was quite rare.
“Furthermore, HFGs generated hair follicles and shafts in vitro in a long-term culture, but the appearance of hair follicles was quite rare.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [14] T. Kageyama, C. Yoshimura, D. Myasnikova, K. Kataoka, T. Nittami, S. Maruo, J. Fukuda, Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine. Biomaterials 154, 291– 300 (2018). 29156398 source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationFibulin-1 is a secreted glycoprotein that binds to a number of ECMs including fibronectin, versican, and basal membrane proteins.
“Fibulin-1 is a secreted glycoprotein that binds to a number of ECMs including fibronectin, versican, and basal membrane proteins (27).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [27] R. Timpl, T. Sasaki, G. Kostka, M. L. Chu, Fibulins: A versatile family of extracellular matrix proteins. Nat. Rev. Mol. Cell Biol. 4, 479– 489 (2003). 12778127 source
May combine two claims.
PremisecitationMG132 is a proteosome inhibitor that inhibits NF-κB signaling, while LY294002 is a PI3K/Akt signaling antagonist.
“MG132 is a proteosome inhibitor, which inhibits NF-κB signaling (31), while LY294002 is a PI3K/Akt signaling antagonist.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [31] S. Nakajima, H. Kato, S. Takahashi, H. Johno, M. Kitamura, Inhibition of NF-κB by MG132 through ER stress-mediated induction of LAP and LIP. FEBS Lett. 585, 2249– 2254 (2011). 21627972 source
May combine two claims.
Interpretationown dataBoth NF-κB and PI3K/Akt cascades are critical mechanisms in hair follicle morphogenesis.
“indicating that both NF-κB and PI3K/Akt cascades are critical mechanisms in hair follicle morphogenesis.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationXAV-939, a Wnt signaling antagonist, reportedly suppresses the expression of glycogen synthase kinase-3β and Wnt10b.
“XAV-939, a Wnt signaling antagonist, reportedly suppresses the expression of glycogen synthase kinase–3β and Wnt10b (36).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [36] D. W. Liu, K. S. Mai, Y. J. Zhang, W. Xu, Q. H. Ai, Wnt/β-catenin signaling participates in the regulation of lipogenesis in the liver of juvenile turbot (Scophthalmus maximus L.). Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 191, 155– 162 (2016). 26545985 source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationMelanocytes are activated by α-MSH and produce melanosomes in the skin and hair follicles for pigmentation.
“Melanocytes are activated by α–melanocyte-stimulating hormone (α-MSH) and produce melanosomes in the skin and hair follicles for pigmentation (38).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [38] M. De Luca, W. Siegrist, S. Bondanza, M. Mathor, R. Cancedda, A. N. Eberle, Alpha melanocyte stimulating hormone (alpha MSH) stimulates normal human melanocyte growth by binding to high-affinity receptors. J. Cell Sci. 105, 1079– 1084 (1993). 8227196 source
May combine two claims.
InterpretationreasoningSupplementation of ECMs increases adhesivity between heterogeneous cell types, probably through integrin, to form a core-shell aggregate, which increases the contact area between the two cell regions to enhance EMIs.
“Our results imply that the supplement of ECMs increases the adhesivity between heterogeneous cell types (probably through integrin) to form a core-shell aggregate, which increases the contact area between two cell regions to enhance EMIs.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim. May combine two claims.
PremisecitationAccording to the differential adhesion hypothesis, an aggregate configuration composed of two cell types depends on the balance of cell adhesivity between homogeneous and heterogeneous cell types and eventually reaches a state that minimizes system energy.
“The hypothesis states that an aggregate configuration composed of two cell types depends on the balance of cell adhesivity between homogeneous and heterogeneous cell types and eventually reaches to minimize the energy in the system.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [47] R. A. Foty, M. S. Steinberg, The differential adhesion hypothesis: A direct evaluation. Dev. Biol. 278, 255– 263 (2005). 15649477 source
- cites [48] Y. Sun, Q. Wang, Modeling and simulations of multicellular aggregate self-assembly in biofabrication using kinetic Monte Carlo methods. Soft Matter 9, 2172– 2186 (2013). source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationMatrigel consists of basement membrane extracts from mouse sarcoma, including laminin, collagen IV, heparin sulfate proteoglycans, entactin/nidogen, and a number of growth factors.
“Matrigel has been widely used in organoid research and consists of basement membrane extracts from mouse sarcoma, including laminin, collagen IV, heparin sulfate proteoglycans, entactin/nidogen, and a number of growth factors (42).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [42] C. I. L. Sciences. Corning Matrigel matrix. Frequently asked questions (2019).
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenStatistical analyses for optimization of cell ratio, cell number, and Matrigel concentration used Tukey's tests, with P < 0.05 considered significant.
“Statistical analyses for the optimization of the cell ratio, cell number, and Matrigel concentration were conducted using Tukey’s tests, and results with P < 0.05 were considered statistically significant.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Study design and conductreasoningExperiments were repeated using at least three different donors because of anticipated variability between donors.
“Because we anticipated variabilities between different donors, we repeated the experiments using at least three different donors.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
Study design and conductnone givenAt least 12 hair follicloids per condition were cultured and used for calculations of the number of hair follicles sprouting.
“We cultured at least 12 hair follicloids per condition, which were used for the calculations of the number of hair follicles sprouting.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductreasoningThe care and handling of mice conformed with the requirements of the committee on animal care and use at Yokohama National University, Central Institute for Experimental Animals, and National Institute of Health Sciences.
“The care and handling of mice conformed with the requirements of the committee on animal care and use at Yokohama National University, Central Institute for Experimental Animals, and National Institute of Health Sciences.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Study design and conductnone givenStatistical analyses of PCR array, RT-PCR, and total melanin volume data used two-tailed Student's t test, with P < 0.05 considered significant.
“Statistical analyses of the PCR array, RT-PCR, and total melanin volume data were conducted using two-tailed Student’s t test, and results with P < 0.05 were considered significant.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe average absorbance was determined using at least three samples.
“We determined the average absorbance using at least three samples.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim. May combine two claims.
Study design and conductnone givenRelative gene expression levels were determined using the 2−ΔΔCt method and presented as means ± SE of at least three independent experiments.
“Relative gene expression levels were determined using the 2−ΔΔCt method and presented as the means ± SE values of at least three independent experiments.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningThe organoid culture approach is scalable because hair follicloids form through self-organization of cells when the two cell types are aggregated in culture medium supplemented with a low concentration of ECM.
“This approach is scalable, because hair follicloids are formed through the self-organization of cells when the two types of cells are aggregated in culture medium supplemented with a low concentration of ECM.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe animal study was approved by the committee on animal care and use at Yokohama National University (permit numbers 2019-04 and 2019-06), Central Institute for Experimental Animals (permit number 20077), and National Institute of Health Sciences (permit number 782).
“The committee on animal care and use at Yokohama National University (permit number: 2019-04 and 2019-06), Central Institute for Experimental Animals (permit number: 20077), and National Institute of Health Sciences (permit number: 782) approved the animal study.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
The development of the hair follicle is coordinated by epithelial-mesenchymal interactions.
“In addition, the development of the hair follicle is coordinated by EMIs.”
The paper relies on this without support.
Cell-cell contact between epithelial and mesenchymal cells is critical to induce epithelial-mesenchymal interactions.
“because cell-cell contact between epithelial and mesenchymal cells is critical to induce EMIs.”
The paper relies on this without support.
Cell aggregation is critical to induce epithelial-mesenchymal interactions.
“Because cell aggregation is critical to induce EMIs, we expected that it would be better to add Matrigel after the cells were sufficiently aggregated as previously reported (18).”
The paper relies on this without support.
Fully elucidating molecular mechanisms for epithelial-mesenchymal interactions remains challenging because of the crowded in vivo environment.
“fully elucidating molecular mechanisms for EMIs remains challenging because of the crowded in vivo environment.”
The paper relies on this without support.
Skin organoid culture requires multiple differentiation steps, takes a relatively long period of time, contains unexpectedly differentiated cells and their secreted proteins, and is limited to relatively short lengths of hair shafts.
“However, skin organoid culture requires multiple differentiation steps starting with ectoderm induction on the surface of cell aggregates, takes a relatively long period of time, contains unexpectedly differentiated cells and their secreted proteins, and is limited to relatively short lengths of hair shafts.”
The paper relies on this without support.
Rodent embryonic skin cells have a shorter period of hair generation, higher hair inducing activity, and greater availability than human cells.
“In this study, rodent embryonic skin cells were used because of their shorter period of hair generation, higher hair inducing activity, and availability compared to cells of human origin.”
Cites
Wnt/β-Catenin Signaling: Components, Mechanisms, and Diseases
MacDonald, B. T., Tamai, K., & He, X. (2009) · Developmental Cell · doi:10.1016/j.devcel.2009.06.016 · Cited by 5,583 · see citing works
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- Authors
- Kageyama, T., Shimizu, A., Anakama, R., Nakajima, R., Suzuki, K., Okubo, Y., & Fukuda, J.
- Affiliations
- Yokohama National University (JP)
- National Institute of Health Sciences (JP)
- Corresponding author
- Fukuda, Junji
- Venue
- Science Advances
- Published
- 2022-10-21
- Volume
- 8
- Issue
- 42
- Pages
- eadd4603-eadd4603
- ISSN
- 2375-2548 (ISSN-L)
- PMC
- PMC9586475
- OpenAlex
- W4307038220
- Cited by
- 73
- References
- 53
- Citations by year
- 2026: 18 · 2025: 24 · 2024: 17 · 2023: 13 · 2022: 1
- FWCI
- 13.66
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Science Advances (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- Institutional Repositories DataBase (IRDB) (publishedVersion, cc-by)
- PubMed Central (submittedVersion, cc-by)
- Yokohama National University Repository (Yokohama National University) (submittedVersion)
- Funders
- Sumitomo Foundation
- Japan Agency for Medical Research and Development (19213841)
- Kanagawa Institute of Industrial Science and Technology (19K21107, 18K18971)
- Hoyu Science Foundation
- Japan Society for the Promotion of Science (20K20208, 20H05744, 18K18971, 19K21107, 20H02535)
- Japan Science and Technology Agency (JPMJPR19H2)
- Precursory Research for Embryonic Science and Technology (JPMJPR19H2)
- Topics
- Health Sciences › Medicine › Urology › Hair Growth and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Skin and Cellular Biology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › dental development and anomalies
- Keywords
- hair follicle induction, hair follicle morphogenesis, three-dimensional microenvironment, melanosome transport, gray hair
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.
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