Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids

To date, investigation into generating skin in vitro has focused on inducing pure populations of skin cell types from mouse and human PSCs.

“To date, investigation into generating skin in vitro have focused on inducing pure populations of skin cell types—e.g. dermal fibroblasts or keratinocytes—from mouse and human PSCs (Coraux et al., 2003; Ehama et al., 2007; Guenou et al., 2009; Itoh et al., 2013; 2011; Mavilio et al., 2006).”

• cites Coraux et al., 2003 sourceNot checked — no full text of the source
• cites Ehama et al., 2007 sourceDisputed

  The Human keratinocyte culture section says the keratinocytes were isolated from neonatal or adult foreskins, not induced from pluripotent stem cells. The Introduction and Discussion describe combining human epidermal cells with murine dermal papilla cells by grafting to generate hair follicle-like structures, rather than generating skin in vitro from PSC-derived cell populations. Our copy provides no page numbers for these sections. (LLM, conf=0.86)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites Guenou et al., 2009 sourceNot checked — no full text of the source

SB/BMP treated aggregates, without subsequent FGF and LDN treatment, produced epidermal keratinocyte cysts largely devoid of surrounding mesenchymal tissue.

“We knew from our previous work that SB/BMP treated aggregates, without subsequent FGF and LDN treatment, produced epidermal keratinocyte cysts largely devoid of surrounding mesenchymal tissue (Koehler et al., 2013).”

• cites Koehler et al., 2013 sourcePartly supported

  The passage on BMP/SB-treated aggregates (section labeled Abstract; no page provided) says their outer epithelium develops into Krt5/p63+ epidermal epithelium, supporting the epidermal-fate portion of the claim. It does not say that these aggregates form keratinocyte cysts or are largely devoid of surrounding mesenchymal tissue. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

The bulb-like structures are reminiscent of nascent guard, awl, or auchene hair follicles.

“We discovered that these structures were extensions of the KRT5+ epidermis and were associated with SOX2+ dermal papilla (DP)-like cells (Figure 1C, D), reminiscent of nascent guard, awl, or auchene HFs (Driskell et al., 2009; Lesko et al., 2013).”

• cites Driskell et al., 2009 sourceSupported
• cites Lesko et al., 2013 sourceSupported

A 3D mouse embryonic stem cell culture can produce cranial surface epithelia (non-neural ectoderm), a precursor tissue of the skin epidermis.

“Recently, we developed a three-dimensional (3D) mouse embryonic stem cell (mESC) culture capable of producing cranial surface epithelia (also known as non-neural ectoderm), a precursor tissue of the skin epidermis.”

• cites Koehler and Hashino, 2014 sourceSupported
• cites Koehler et al., 2013 sourceSupported

The dermal cell diversity is consistent with late embryonic and early postnatal dermal development.

“Interestingly, by day 16, CD34+ cells were present throughout the dermal layer, while DLK1+ cells were localized to discrete regions, suggesting that a diverse population of dermal cells may emerge in skin organoids (Figure 3F, G), consistent with late embryonic and early postnatal dermal development (Driskell et al., 2013).”

• cites Driskell et al., 2013 sourceSupported

In animal models, rodent and human skin with hair follicles can be reconstituted by co-culturing mesenchymal cells with epithelial cells or HF-initiating dermal papilla cells.

“In animal models, rodent and human skin with HFs can be reconstituted by co-culturing mesenchymal cells with epithelial cells or, specifically, HF initiating dermal papilla (DP) cells (Asakawa et al., 2012; Chuong et al., 2007; Ehama et al., 2007; Ikeda et al., 2009; Nakao et al., 2007; Takagi et al., 2016; Toyoshima et al., 2012; Zheng et al., 2005a).”

• cites Asakawa et al., 2012 sourcePartly supported

  The Methods (Preparation and cultivation of bioengineered hair germ; page not provided) describe combining mouse embryonic epithelial and dermal cells to reconstitute hair follicle germs. The Results (Regeneration of hair follicles by intracutaneous transplantation; page not provided) report regenerated follicles in recipient mouse skin. These passages do not establish reconstitution of human skin with follicles or describe co-culturing epithelial cells specifically with isolated dermal papilla cells. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites Chuong et al., 2007 sourceNot checked — no full text of the source
• cites Ikeda et al., 2009 sourcePartly supported

  The paper’s introductory section says epithelial and mesenchymal cells can be used to reconstitute organ germs and identifies the whisker follicle as a possible application. It does not report reconstituting rodent or human skin with hair follicles, or describe co-culture with hair-follicle-initiating dermal papilla cells; its reported replacement experiment concerns teeth. (LLM, conf=0.79)

  Experimental — accuracy not yet measured

In vitro skin derivation strategies have focused on generating keratinocytes and fibroblasts from pluripotent stem cells in separate cultures first, then combining the two cell types to form a skin-like bilayer.

“To date, in vitro skin derivation strategies have focused on generating keratinocytes and fibroblasts from PSCs in separate cultures first, then combining the two types of cells to form a skin-like bilayer (Gledhill et al., 2015; Itoh et al., 2013; Oh et al., 2013; Sun et al., 2014).”

• cites Gledhill et al., 2015 sourceSupported
• cites Itoh et al., 2013 sourceSupported
• cites Sun et al., 2014 sourceNot checked — no full text of the source

The ITGα8+ αSMA+ muscle-like structures could indicate arrector pili muscle induction.

“Restricting analysis to HFs with normal morphology, we identified ITGα8+ αSMA+ muscle-like structures with elongated nuclei in the organoid dermis (Figure 5C, C′), which could indicate arrector pili muscle induction (Driskell et al., 2013).”

• cites Driskell et al., 2013 sourcePartly supported

  The Abstract (page unavailable) identifies the arrector pili muscle (APM) as a structure formed by the upper dermal fibroblast lineage, supporting the possibility of APM formation. The Methods > Antibodies section lists Itga8 and alpha-smooth muscle actin antibodies, but the passages do not establish that ITGα8+ αSMA+ elongated structures identify APM or demonstrate its induction in organoids. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

KRT15 and SOX9 were expressed broadly in the organoid HF outer root sheath as well as the bulge-like region.

“We found that the HF bulge and epithelial stem cells markers, KRT15 and SOX9, were expressed broadly in the organoid HF outer root sheath as well as the bulge-like region (Morris et al., 2004; Nowak et al., 2008; Figure 6B–B″″).”

• cites Morris et al., 2004 sourceNot checked — no full text of the source
• cites Nowak et al., 2008 sourceSupported

The majority of SG-like structures observed in the skin organoids were bifurcates, reminiscent of guard hairs.

“The majority of SG-like structures observed in the skin organoids were bifurcates, reminiscence of guard hairs (Driskell et al., 2009).”

• cites Driskell et al., 2009 sourceSupported

Skin organoids constituting epidermal and dermal layers can be derived from a homogeneous population of mouse pluripotent stem cells in a 3D culture.

“Here, we describe the derivation of skin organoids, constituting epidermal and dermal layers, from a homogeneous population of mouse pluripotent stem cells in a 3D culture.”

• AssumptionRests on an assumption: It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures.
• AssumptionRests on an assumption: To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in co-culture with stem cell-derived cells, combined with in vivo transplantation.
• AssumptionRests on an assumption: A chemically defined means of generating folliculogenic skin from pluripotent stem cells in vitro has been intangible.

By day 14 of differentiation, DLK1 was detected in cells around the epidermis.

“By day 14 of differentiation, a dermal fibroblast-like cell marker, DLK1, was detected in cells around the epidermis (Figure 3F).”

Skin organoids spontaneously produce de novo hair follicles.

“We show that skin organoids spontaneously produce de novo hair follicles in a process that mimics normal embryonic hair folliculogenesis.”

• Also stated in: § Introduction · plain

  “In this article, we report that the mouse PSC-derived skin organoids recapitulate key steps of integumentary development and have the capacity to generate de novo HFs.”

• AssumptionRests on an assumption: It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures.
• AssumptionRests on an assumption: To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in co-culture with stem cell-derived cells, combined with in vivo transplantation.
• AssumptionRests on an assumption: A chemically defined means of generating folliculogenic skin from pluripotent stem cells in vitro has been intangible.

When aggregates were treated with CHIR on day 8, presumptive HFs formed alongside inner ear organoids.

“Moreover, when we treated the aggregates with CHIR on day 8, as used previously to encourage inner ear organoid induction (DeJonge et al., 2016), we found that presumptive HFs formed alongside inner ear organoids (Figure S2C).”

KRT5 expression, indicative of basal keratinocytes, was first observed in patches of the ECAD+ TFAP2α+ epithelium on day 10 of differentiation.

“We first observed KRT5 expression, indicative of basal keratinocytes, in patches of the ECAD+ TFAP2α+ epithelium on day 10 of differentiation (Figure 3B and S3B).”

C57BL/BJ iPSCs generated HF-bearing skin organoids.

“The C57BL/BJ iPSCs also generated HF-bearing skin organoids (Figure S2B), suggesting that robust induction of HF-producing skin organoids is not limited to one cell line.”

The percentage of aggregates generating fifteen or more HFs was significantly greater under SB/BMP-FGF/LDN full-treatment compared to SB/BMP-LDN conditions (45 ± 7% vs 13 ± 7%, P < 0.01).

“However, we noted that the percentage of aggregates generating fifteen or more HFs was significantly greater under the SB/BMP-FGF/LDN full-treatment condition compared to SB/BMP-LDN conditions (Table S1b; 45 ± 7% (SB/BMP-FGF/LDN) vs. 13 ± 7% (SB/BMP-LDN) of organoids, respectively; P < 0.01).”

KRT10 expression was noticed in spinous-like epidermal cells by day 14 of differentiation.

“We then noticed KRT10 expression in spinous-like epidermal cells by day 14 of differentiation (Figure 3C).”

At day 28, HFs with SOX2-negative hair bulb region were present, reminiscent of zigzag HFs.

“Interestingly, however, at the later stage of differentiation by day 28, we also noticed the presence of HFs with SOX2-negative hair bulb region, reminiscent of zigzag HFs (Figure 4E–E″ and S4B, B′).”

By day 14, KRT5 was expressed in the epithelium in both R1 and Atoh1/nGFP organoids.

“By day 14, KRT5 was expressed in the epithelium in both R1 and Atoh1/nGFP organoids (n = 6 aggregates per cell line, 3 experiments; Figure 2A, B).”

COL3α1 and COL4α1 were present by day 12 of differentiation in the culture system.

“In our culture system, we noticed the presence of Collagen type III, alpha-1 chain (COL3α1) and Collagen type IV, alpha-1 chain (COL4α1) by day 12 of differentiation (Figure S3C), which are known extracellular matrix (ECM) proteins localized at the epidermal-dermal interface (Figure 3F′, G′).”

SB/BMP-LDN treated aggregates generated HF-bearing skin organoids, albeit inconsistently from batch-to-batch (46 ± 13% of organoids with HF-like bulbs per batch, n = 61 organoids, 8 experiments).

“Interestingly, SB/BMP-LDN treated aggregates generated HF-bearing skin organoids, albeit inconsistently from batch-to-batch (46 ± 13% of organoids with HF-like bulbs per batch, n = 61 organoids, 8 experiments).”

By day 18 of differentiation, all developing HFs contained SOX2+ dermal condensates/DPs, indicating development of nascent guard, awl, or auchene HFs.

“By day 18 of differentiation, all of the developing HFs contained SOX2+ dermal condensates/DPs, indicating the development of nascent guard, awl, or auchene HFs (Figure 4D–D″).”

Mature epidermal markers were typically absent from sections of the epithelium abutting non-epithelial cells, such as TUJ1+ neurons.

“Interestingly, we noted that these mature epidermal markers were typically absent from sections of the epithelium abutting non-epithelial cells, such as TUJ1+ neurons, suggesting that the composition of non-epithelial tissues may affect keratinocyte differentiation (Figure 3D–E′ and S3B′).”

Uncovered or partially covered C57BL/BJ iPSC aggregates produced higher number of HFs compared to fully covered aggregates bearing only 0-2 HFs.

“Moreover, as Atoh1/nGFP aggregates, uncovered or partially covered C57BL/BJ iPSC aggregates produced higher number of HFs compared to fully covered aggregates bearing only 0–2 HFs (data not shown).”

TEM revealed proper organization of the HF in the skin organoid, showing all HF lineages formed from the outermost dermal sheath to the center of the hair shaft medulla.

“The proper organization of the HF in the skin organoid was revealed by transmission electron microscopy (TEM), showing all of the HF lineages formed from the outermost dermal sheath to the center of the hair shaft medulla (Figure J–J″).”

CD34 was expressed in the dermal layer at later stages of differentiation as well (day 26).

“CD34 was expressed in the dermal layer at later stages of differentiation as well (day 26; Figure S3C′).”

R1 mESC aggregates occasionally form protruding bulb-like structures that differ in morphology from inner ear organoids.

“While investigating the otic culture system using the R1 mESC line, we observed the occasional formation of protruding bulb-like structures that differed in morphology compared to inner ear organoids (Figure 1B).”

HFs reminiscent of early stage embryonic HFs (stages 1-4) could be identified in skin organoids at any time point between differentiation days 18-26.

“Subsequently, HFs reminiscent of early stage embryonic HFs (stages 1–4; Figure 4A) could be identified in skin organoids at any time point between differentiation days 18–26 (Figure 4C–D″), suggesting that new HF induction is continuous during this time period.”

From day 16, a DLK1+ fibrotic region emerged in the dermal layer.

“As the skin organoids differentiate (from day 16), a DLK1+ fibrotic region emerged in the dermal layer.”

Hair germs were visible as early as days 14-18 of differentiation by double-staining with EDAR and LHX2.

“In our in vitro system, hair germs were visible as early as days 14–18 of differentiation by double-staining with EDAR and LHX2 (Figure 4B and S4A, A′).”

HFs never developed when aggregates were grown in SB/BMP conditions (n = 12 aggregates, 3 experiments).

“Upon reexamination, we confirmed that HFs never developed when aggregates were grown in SB/BMP conditions (n = 12 aggregates, 3 experiments).”

The Atoh1/nGFP mESC line was highly folliculogenic.

“Surprisingly, while testing our otic induction method on other cell lines, we noted that an Atoh1/nGFP mESC line (Lumpkin et al., 2003; Oshima et al., 2010) was highly folliculogenic (Figure 2 and Table S1a).”

Mouse PSC-derived skin organoids have the capacity to generate de novo hair follicles.

“In this article, we report that the mouse PSC-derived skin organoids recapitulate key steps of integumentary development and have the capacity to generate de novo HFs.”

CD34 was detected from day 12 surrounding the epidermis.

“Likewise, CD34, another fibroblast-like cell marker, was detected from day 12 surrounding the epidermis (Figure 3G).”

ITGα8+ αSMA+ muscle-like structures with elongated nuclei were identified in the organoid dermis.

“Restricting analysis to HFs with normal morphology, we identified ITGα8+ αSMA+ muscle-like structures with elongated nuclei in the organoid dermis (Figure 5C, C′), which could indicate arrector pili muscle induction (Driskell et al., 2013).”

Aggregates in each category sorted by amount of exterior non-epithelial tissue produced HFs.

“Aggregates in each category, which were sorted by the amount of exterior non-epithelial tissue, produced HFs (Figure 2E; center pie graph).”

Skin organoids develop stratified epidermis.

“Remarkably, skin organoids develop stratified epidermis (Figure S3A, A′).”

Induced Brachyury+ mesendodermal cells and ECAD+ PAX8+ epithelial cells were observed during days 5-8 of differentiation of Atoh1/nGFP cells, similar to R1.

“Similar to the R1 cell line, we observed induced Brachyury+ mesendodermal cells and ECAD+ PAX8+ epithelial cells during days 5–8 of differentiation of Atoh1/nGFP cells (Figure S1).”

Around day 25, robust expression of Loricrin and FLG was observed in granular and cornified layers of the organoid epidermis, respectively.

“Later, around day 25, we observed robust expression of Loricrin and FLG in granular and cornified layers of the organoid epidermis, respectively (Figure 3D–E′ and S3B′).”

Without CHIR treatment, Atoh1/nGFP cells produced a significantly higher percentage of HF-bearing aggregates than R1 aggregates (83 ± 5% vs 18 ± 8%, P < 0.0001).

“Without CHIR treatment, however, we found that Atoh1/nGFP cells produced a significantly higher percentage of HF-bearing aggregates than R1 aggregates (Atoh1/nGFP: 83 ± 5% of aggregates with HF-like bulbs per batch, n = 193 organoids, 10 experiments, P < 0.0001, Table S1a).”

Pearl-shaped adipocytes started to appear on day 26, mimicking the maturation of fat in the hypodermis in vivo.

“Pearl-shaped adipocytes started to appear on day 26 (Figure 3H–I′), mimicking the maturation of fat in the hypodermis in vivo.”

The R1 cell line displayed relatively low frequency of HF production (18 ± 8% of aggregates with HF-like bulbs per batch, n = 84 organoids, 6 experiments).

“The R1 cell line displayed relatively low frequency of HF production (18 ± 8% of aggregates with HF-like bulbs per batch, n = 84 organoids, 6 experiments, Table S1a).”

A dermal layer was induced on the surface of skin organoids concurrently with epidermal layer development.

“Concurrently with epidermal layer development, a dermal layer was induced on the surface of skin organoids.”

Partial dissociation and re-plating organoids in Matrigel droplets did not improve skin organoid longevity.

“Partial dissociation and re-plating organoids in Matrigel droplets did not improve skin organoid longevity (data not shown).”

The frequency of accompanying induction of dermis and HFs appears to be cell-line dependent.

“In our culture system, the induction of epidermal cysts is highly reproducible across three PSC lines, yet the frequency of accompanying induction of dermis and HFs appears to be cell-line dependent.”

Between 35-50 days, HFs appeared to have arrested growth.

“Between 35–50 days, HFs appeared to have arrested growth and a dense layer of adipocytes forms on the surface of the organoids.”

Minor modifications to the treatment concentrations and timing did not consistently improve or diminish the efficiency of HF induction.

“We found that minor modifications to the treatment concentrations and timing did not consistently improve or diminish the efficiency of HF induction.”

After 24 days of differentiation, SG-like structures were detected on organoid HFs by Oil Red O and LipidTOX staining.

“After 24 days of differentiation, we could detect SG-like structures on organoid HFs by Oil Red O and LipidTOX staining (Figure 5D–D″).”

EdU-incorporation was consistently observed in a subset of cells located in a bulge-like region near the SG in the upper outer root sheath.

“Despite the sparse labeling, we consistently observed EdU-incorporation in a subset of cells located in a bulge-like region near the SG in the upper outer root sheath (Figure 6A′, A″ and Video S2).”

EdU incorporation was less prevalent in regions of the HF above the matrix.

“EdU incorporation was less prevalent in regions of the HF above the matrix.”

Melanocyte induction was infrequent during production of Atoh1/nGFP skin organoid production (12/252 organoids).

“However, melanocyte induction was infrequent during production of Atoh1/nGFP skin organoid production (12/252 organoids; other cell lines were not quantified).”

Skin organoid HF shafts cannot shed as they do in vivo during a typical HF growth cycle.

“We noted that skin organoid HF shafts cannot shed as they do in vivo during a typical HF growth cycle, which may lead to abnormalities in skin organoid HFs transitioning from development stages into the growth cycle.”

These skin organoid structures can be generated de novo, without the use of embryonic tissue or undefined media.

“Our study demonstrates how these skin organoid structures can be generated de novo, without the use of embryonic tissue or undefined media.”

These Atoh1/nGFP+ ISL1+ cells were rarely seen after approximately day 20 of differentiation.

“However, these cells were rarely seen after approximately day 20 of differentiation (data not shown).”

HF growth rates gradually decreased day by day.

“We found that the HFs continued to grow until the experiment was terminated on day 32 while the growth rates gradually decreased day by day, and eventually, the HF matrix deteriorated around day 32, implying the possibility that the HFs undergo catagen (Figure 6D, E).”

HF-bearing skin can be generated in vitro from a homogenous source of mouse PSCs under serum-free conditions.

“Our findings demonstrate that HF-bearing skin can be generated in vitro from a homogenous source of mouse PSCs, under serum-free conditions.”

Follicular KRT15+ cells that co-expressed CD34 were rarely observed, indicating that organoid HFs likely do not contain adult HFSC-like cells.

“We rarely observed follicular KRT15+ cells that co-expressed CD34, indicating that organoid HFs likely do not contain adult HFSC-like cells (Figure 6B–B″″ and S4G–H′).”

p75+ SOX10+ neural crest-like cells were observed in the mesenchymal layer.

“Remarkably, we also observed p75+ SOX10+ neural crest-like cells in the mesenchymal layer (Figure 5G, S4D–E′, and Video S1).”

In our culture system, the induction of epidermal cysts is highly reproducible across three PSC lines.

“In our culture system, the induction of epidermal cysts is highly reproducible across three PSC lines, yet the frequency of accompanying induction of dermis and HFs appears to be cell-line dependent.”

The HF matrix deteriorated around day 32.

“We found that the HFs continued to grow until the experiment was terminated on day 32 while the growth rates gradually decreased day by day, and eventually, the HF matrix deteriorated around day 32, implying the possibility that the HFs undergo catagen (Figure 6D, E).”

NFATc1 was specifically expressed by cells in the presumptive bulge region of organoid HFs between days 26-30.

“By contrast, NFATc1 was specifically expressed by cells in the presumptive bulge region of organoid HFs between days 26–30 (Figure 6C, C′).”

Folliculogenic Atoh1/nGFP mESC and C57BL/BJ miPSC aggregates contained less non-dermal tissues.

“By contrast, folliculogenic Atoh1/nGFP mESC and C57BL/BJ miPSC aggregates contained less non-dermal tissues.”

HFs continued to grow until the experiment was terminated on day 32.

“We found that the HFs continued to grow until the experiment was terminated on day 32 while the growth rates gradually decreased day by day, and eventually, the HF matrix deteriorated around day 32, implying the possibility that the HFs undergo catagen (Figure 6D, E).”

Elongated ITGα8+ αSMA+ cells with attachment points on the HFs and interfollicular epidermis, as expected for fully formed arrector pili muscles, were not observed.

“However, we did not observe elongated ITGα8+ αSMA+ cells with attachment points on the HFs and interfolliclar epidermis as would be expected for fully formed arrector pili muscles.”

Skin organoids assume a cyst conformation with radially oriented HFs.

“Lastly, it is notable that the skin organoids assume a cyst conformation with radially oriented HFs.”

No newly forming HFs were observed beyond days 32-35.

“We did not observe newly forming HFs beyond days 32–35.”

A dense layer of adipocytes forms on the surface of the organoids between 35-50 days.

“Between 35–50 days, HFs appeared to have arrested growth and a dense layer of adipocytes forms on the surface of the organoids.”

A diverse population of dermal cells may emerge in skin organoids.

“Interestingly, by day 16, CD34+ cells were present throughout the dermal layer, while DLK1+ cells were localized to discrete regions, suggesting that a diverse population of dermal cells may emerge in skin organoids (Figure 3F, G), consistent with late embryonic and early postnatal dermal development (Driskell et al., 2013).”

HFs generated within skin organoids after day 24 shared key features with HFs in embryonic stages 7-8.

“By dissecting the structural architecture of more mature-looking HFs after day 24 of differentiation, we discovered that the HFs generated within our skin organoids shared key features with HFs in embryonic stages 7–8.”

The composition of non-epithelial tissues may affect keratinocyte differentiation.

“Interestingly, we noted that these mature epidermal markers were typically absent from sections of the epithelium abutting non-epithelial cells, such as TUJ1+ neurons, suggesting that the composition of non-epithelial tissues may affect keratinocyte differentiation (Figure 3D–E′ and S3B′).”

The full-treatment of SB/BMP-FGF/LDN appears to be optimal for HF-bearing skin organoid formation.

“Thus, the full-treatment of SB/BMP-FGF/LDN appears to be optimal for HF-bearing skin organoid formation.”

The process of de novo hair follicle production in skin organoids mimics normal embryonic hair folliculogenesis.

“We show that skin organoids spontaneously produce de novo hair follicles in a process that mimics normal embryonic hair folliculogenesis.”

The pulse of GSK3β inhibition does not disrupt HF induction.

“Consequently, the pulse of GSK3β inhibition does not disrupt HF induction.”

Mouse PSC-derived skin organoids recapitulate key steps of integumentary development.

“In this article, we report that the mouse PSC-derived skin organoids recapitulate key steps of integumentary development and have the capacity to generate de novo HFs.”

• AssumptionRests on an assumption: It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures.
• AssumptionRests on an assumption: To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in co-culture with stem cell-derived cells, combined with in vivo transplantation.
• AssumptionRests on an assumption: A chemically defined means of generating folliculogenic skin from pluripotent stem cells in vitro has been intangible.

Skin organoids produced in our culture system can generate HFs similar to those seen in late embryonic and early postnatal development, consisting of all major units including HF matrix, inner root, outer root, and dermal sheath layers.

“Together, these findings reveal that skin organoids produced in our culture system can generate HFs similar to those seen in late embryonic and early postnatal development, consisting of all major units including HF matrix, inner root, outer root, and dermal sheath layers.”

New HF induction is continuous during days 18-26.

“Subsequently, HFs reminiscent of early stage embryonic HFs (stages 1–4; Figure 4A) could be identified in skin organoids at any time point between differentiation days 18–26 (Figure 4C–D″), suggesting that new HF induction is continuous during this time period.”

The mouse skin organoids produced in our culture system recapitulate key features of skin differentiation on a time scale that roughly correlates with normal embryonic development.

“Thus, the mouse skin organoids produced in our culture system recapitulate key features of skin differentiation on a time scale that roughly correlates with normal embryonic development.”

The results support an approach to generate skin and skin appendages from PSCs wherein epidermal and dermal cells are co-induced in a single organoid unit.

“Our results support an approach to generate skin and skin appendages from PSCs wherein the epidermal and the dermal cells are co-induced in a single organoid unit.”

• AssumptionRests on an assumption: It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures.
• AssumptionRests on an assumption: To date, generation of hair follicles in vitro has only been possible using primary cells isolated from embryonic skin, cultured alone or in co-culture with stem cell-derived cells, combined with in vivo transplantation.
• AssumptionRests on an assumption: A chemically defined means of generating folliculogenic skin from pluripotent stem cells in vitro has been intangible.

Robust induction of HF-producing skin organoids is not limited to one cell line.

“The C57BL/BJ iPSCs also generated HF-bearing skin organoids (Figure S2B), suggesting that robust induction of HF-producing skin organoids is not limited to one cell line.”

Skin organoids may be capable of producing at least two types of HFs-SOX2+ guard, awl, or auchene HFs and SOX2− zigzag HFs-in a single skin organoid unit.

“These findings suggest that skin organoids may be capable of producing at least two types of HFs—SOX2+ guard, awl, or auchene HFs and SOX2− zigzag HFs—in a single skin organoid unit.”

HF induction seems to be dependent on the co-development of both epidermal and dermal cells.

“Not surprisingly, HF induction seems to be dependent on the co-development of both epidermal and dermal cells.”

Non-dermal tissues may be physically and/or chemically inhibitory to HF induction in floating aggregates.

“In particular, our data suggests that non-dermal tissues may be physically and/or chemically inhibitory to HF induction in floating aggregates.”

Organoid derived skin may be similar to skin in the aural region-ear canal, auricular, or surrounding skin tissue.

“Based on our analysis, it is difficult to pinpoint the precise anatomical location represented by skin organoids; however, our data suggest that organoid derived skin may be similar to skin in the aural region—ear canal, auricular, or surrounding skin tissue.”

Derivation of multiple germ layers within a single stem cell aggregate may be a general strategy to produce hair-bearing skin.

“These results suggest that the derivation of multiple germ layers within a single stem cell aggregate may be a general strategy to produce hair-bearing skin.”

Skin organoid HFs are capable of producing nascent HFSCs, reminiscent of HFSCs at a late embryonic (~E17.5) stage of development.

“Based on previous studies, these data suggest that skin organoid HFs are capable of producing nascent HFSCs, reminiscent of HFSCs at a late embryonic (~E17.5) stage of development (Horsley et al., 2008).”

• cites Horsley et al., 2008 sourceNot checked — no full text of the source

The rarity of cells expressing nuclear NFATc1 may indicate that the current culture conditions are not optimal for maturation of the bulge region stem cell niche.

“The rarity of cells expressing nuclear NFATc1 may indicate that the current culture conditions are not optimal for maturation of the bulge region stem cell niche.”

The inability of skin organoid HF shafts to shed may lead to abnormalities in skin organoid HFs transitioning from development stages into the growth cycle.

“We noted that skin organoid HF shafts cannot shed as they do in vivo during a typical HF growth cycle, which may lead to abnormalities in skin organoid HFs transitioning from development stages into the growth cycle.”

For our skin organoid system, the heterogeneity of the exterior tissue mass that forms during days 8-12 of differentiation likely underlies variability in HF induction.

“For our skin organoid system, the heterogeneity of the exterior tissue mass that forms during days 8–12 of differentiation, likely underlies variability in HF induction.”

Organoid HFs likely do not contain adult HFSC-like cells.

“We rarely observed follicular KRT15+ cells that co-expressed CD34, indicating that organoid HFs likely do not contain adult HFSC-like cells (Figure 6B–B″″ and S4G–H′).”

Melanocytes can develop from the neural crest-like cell population.

“In addition, melanocytes were visible around the epidermis as early as day 21 (Figure 5H) and migrated to and pigmented HFs in a subset of Atoh1/nGFP mESC-derived skin organoids by day 26 (Figure 5H′, H″ and S4F), suggesting that melanocytes can develop from the neural crest-like cell population.”