The empowering influence of air-liquid interface culture on skin organoid hair follicle development

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

ALI has previously been used for culturing other human organoids, such as cerebral organoids, where it improves organoid survival and functional development.

“ALI has previously been used for culturing other human organoids, such as cerebral organoids, where it is demonstrated to not only improve organoid survival but also organoid functional development [15].”

• cites [15] Giandomenico, 2019, Cerebral organoids at the air–liquid interface generate diverse nerve tracts with functional output, Nat Neurosci sourceSupported

May combine two claims.

SKO generation mimics embryogenesis, co-inducing epithelial and mesenchymal populations and facilitating their natural interaction to form HFs.

“The process of SKO generation, mimics embryogenesis, co-inducing the development of epithelial and mesenchymal populations, and thereby facilitating their natural interaction to form HFs [13].”

• cites [13] Lee, 2018, Hair follicle development in mouse pluripotent stem cell-derived skin organoids, Cell Rep sourceSupported

May combine two claims.

Mice and pigs have critical differences in skin morphology and disease processes from human skin, complicating research translation.

“Furthermore, human skin models are necessary to reduce the reliance on animal models such as mice and pigs, where the skin morphology and disease processes have critical differences from that of human skin, complicating research translation [1, 2].”

• cites [1] Zomer, 2018, Skin wound healing in humans and mice: challenges in translational research, J Dermatol Sci sourceNot checked — no full text of the source
• cites [2] Hosseini, 2022, Skin biomechanics: a potential therapeutic intervention target to reduce scarring, Burns & trauma sourceCould not verify

  The Review’s ‘Emerging therapeutic approaches’ section describes mouse and pig models, and notes a human-like scarring phenotype in a pig model. Our copy does not say that mice and pigs have critical differences in skin morphology or disease processes that complicate translation to humans (searched for species differences, animal-model limitations, and translation). (LLM, conf=0.72)

  Experimental — accuracy not yet measured

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

In early gestation, HF patterning is initiated by mesenchymal signals together with epithelial Wnt signaling to induce hair placodes.

“In early gestation, HF patterning is initiated by signals from the mesenchyme, in conjunction with epithelial Wnt signaling, to induce the formation of thickened regions of epithelium, known as hair placodes [11, 12].”

• cites [11] Wang, 2012, Dynamic signals for hair follicle development and regeneration, Stem Cells Dev sourceNot checked — no full text of the source
• cites [12] Fuchs, 2007, Scratching the surface of skin development, Nature sourceSupported

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

The pioneering protocol by Lee et al. involves forming hiPSC aggregates, maintaining them for 2 days in maintenance media, then stepwise modulation of TGFβ/FGF pathways to drive skin development.

“The pioneering protocol for human skin organoid generation, by Lee et al., involves forming hiPSC aggregates, maintaining these for 2 days in maintenance media, and then performing stepwise modulation of the TGFβ/FGF pathways to drive skin development [8].”

• cites [8] Lee, 2020, Hair-bearing human skin generated entirely from pluripotent stem cells, Nature sourceSupported

May combine two claims.

When human SKOs were planarized and transferred to ALI at Day 85 to 100, maturation of SKO epidermal layers was enhanced, with increased stratum corneum formation and expression of loricrin and filaggrin.

“Similarly, when human SKOs were planarized and transferred to an ALI at Day 85 to 100 of culture, the maturation of the SKO epidermal layers was enhanced, as indicated by significantly increased stratum corneum formation and expression of mature keratinocyte markers, loricrin, and filaggrin [16].”

• cites [16] Jung, 2022, Wnt-activating human skin organoid model of atopic dermatitis induced by Staphylococcus aureus and its protective effects by Cutibacterium acnes, Iscience sourcePartly supported

  The Results (Air-liquid interface (ALI)-skin organoids) say ALI culture began around days 85–100 and improved epidermal maturation. They attribute the significant increases in cornified stratum formation and loricrin and filaggrin expression specifically to a subsequent dry-culture phase, rather than to transfer to ALI alone. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

Hair follicles rely on epithelial-mesenchymal crosstalk to develop and are usually missing in other 2D or 3D skin culture systems.

“Hair follicles (HFs) are a key example of an appendage that relies on epithelial-mesenchymal crosstalk to develop and are therefore usually missing in other 2D or 3D skin culture systems [7].”

• cites [7] Hosseini, 2022, Biofabrication of human skin with its appendages, Advanced healthcare materials sourceSupported

May combine two claims.

Bilayer skin equivalent models have limited functional and anatomical relevance due to their simplicity, rarely forming mature structures such as skin appendages.

“However, these bilayer ‘skin equivalent’ models have limited functional and anatomical relevance due to their simplicity, rarely resulting in the formation of mature structures such as skin appendages, which are crucial for human skin function [7].”

• cites [7] Hosseini, 2022, Biofabrication of human skin with its appendages, Advanced healthcare materials sourcePartly supported

  The Introduction says current skin models lack important biological and structural features, particularly appendages; the Conclusions and Outlook says appendages are essential to skin function. These passages support the general limitation, but our copy does not specifically identify bilayer skin equivalents as the models affected or establish that their simplicity is the reason they rarely form mature appendages. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

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

Further crosstalk between epithelium and early dermal papilla drives additional invagination and maturation, leading to a mature HF with sebaceous glands, inner and outer root sheath regions, and a hair shaft.

“Further crosstalk between the epithelium and the early dermal papilla drives additional invagination and maturation, eventually leading to the development of a mature HF with sebaceous glands, inner and outer root sheath regions, and a hair shaft [7, 10].”

• cites [7] Hosseini, 2022, Biofabrication of human skin with its appendages, Advanced healthcare materials sourceSupported
• cites [10] Lee, 2021, Skin organoids: a new human model for developmental and translational research, Exp Dermatol sourcePartly supported

  In “The Challenge of Growing Skin Appendages in a Dish” (page unavailable), the paper says the epidermis and dermis exchange cues during development and that skin organoids produce hair follicles. It does not describe crosstalk specifically with an early dermal papilla driving further invagination, or identify sebaceous glands, root sheaths, and a hair shaft as the resulting structures. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

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

Hair placode epithelium invaginates into the dermis and signals surrounding mesenchymal cells to cluster and form the early dermal papilla.

“The hair placode epithelium invaginates into the dermis while signaling to surrounding mesenchymal cells to cluster and form the early dermal papilla.”

• cites [7] Hosseini, 2022, Biofabrication of human skin with its appendages, Advanced healthcare materials sourceSupported
• cites [10] Lee, 2021, Skin organoids: a new human model for developmental and translational research, Exp Dermatol sourcePartly supported

  In “The Challenge of Growing Skin Appendages in a Dish” (page unavailable), the paper describes chemical cues exchanged between the epidermis and dermis during development and discusses the establishment of hair placodes. It does not say that placode epithelium invaginates into the dermis or signals mesenchymal cells to cluster into an early dermal papilla. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

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

De novo generation of skin substitutes with specialized cell types poses a significant challenge in the field.

“De novo generation of skin substitutes with specialized cell types poses a significant challenge in the field [7].”

• cites [7] Hosseini, 2022, Biofabrication of human skin with its appendages, Advanced healthcare materials sourceSupported

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

ALI culture has been widely employed for developing hiPSC-derived organoids such as cerebral organoids, kidney organoids, and intestinal organoids.

“ALI culture has been widely employed for developing hiPSC-derived organoids, such as cerebral organoids, kidney organoids, and intestinal organoids [15, 26, 27].”

• cites [15] Giandomenico, 2019, Cerebral organoids at the air–liquid interface generate diverse nerve tracts with functional output, Nat Neurosci sourcePartly supported

  The Methods sections on ALI-CO culture and generation of cerebral organoids describe using an air-liquid interface to culture cerebral organoids, supporting that example. However, the human pluripotent stem cell Methods section says the organoids shown were generally generated from H9 human embryonic stem cells, not hiPSCs. This source does not establish widespread use; the other organoid examples have separate citations in the citing sentence. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [26] Takasato, 2015, Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis, Nature sourceNot checked — no full text of the source
• cites [27] Yoshida, 2020, Generation of intestinal organoids derived from human pluripotent stem cells for drug testing, Sci Rep sourcePartly supported

  The Results section (no page number provided) confirms that the paper generated hiPSC-derived intestinal organoids. But the Methods describe embedding cells in Matrigel and overlaying them with culture medium; the paper does not identify this as air–liquid interface (ALI) culture or address cerebral and kidney organoids. It therefore supports the intestinal-organoid example, not the claimed use of ALI culture. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

SOX2 is a key protein in the dermal papilla for the development of HFs in mammals.

“SOX2 is a key protein in the dermal papilla for the development of HFs in mammals [21].”

• cites [21] Driskell, 2009, Sox2-positive dermal papilla cells specify hair follicle type in mammalian epidermis, Development sourcePartly supported

  The section on Sox2 expression in the dermal papilla reports Sox2 in developing mouse hair follicles, and the paper’s opening summary reports that Sox2-positive dermal papilla cells specify particular follicle types. But the reconstitution experiment removed Sox2-positive cells, not Sox2 protein itself; zigzag follicles still formed. The paper therefore does not establish that SOX2 protein is broadly required for hair-follicle development in mammals. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

The hypodermis layer contributes to the mechanical and thermoregulatory properties of normal skin.

“The Hypodermis layer not only contributes to the mechanical and thermoregulatory properties of the normal skin”

• cites [22] Zimoch, 2021, Bio-engineering a prevascularized human tri-layered skin substitute containing a hypodermis, Acta Biomater sourceSupported
• cites [25] Schmidt, 2013, Intradermal adipocytes mediate fibroblast recruitment during skin wound healing, Development sourceNot checked — no full text of the source

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

The hypodermis layer supports fibroblast proliferation during skin wound healing.

“but also supports fibroblast proliferation during skin wound healing [22, 25].”

• cites [22] Zimoch, 2021, Bio-engineering a prevascularized human tri-layered skin substitute containing a hypodermis, Acta Biomater sourceSupported
• cites [25] Schmidt, 2013, Intradermal adipocytes mediate fibroblast recruitment during skin wound healing, Development sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Previous studies reported enhanced maturation, development, and function of human organoids in the laboratory.

“These previous studies have reported enhanced maturation, development, and function of human organoids in the laboratory [16, 28].”

• cites [16] Jung, 2022, Wnt-activating human skin organoid model of atopic dermatitis induced by Staphylococcus aureus and its protective effects by Cutibacterium acnes, Iscience sourceSupported
• cites [28] Abo, 2022, Air-liquid interface culture promotes maturation and allows environmental exposure of pluripotent stem cell–derived alveolar epithelium, JCI Insight sourcePartly supported

  The Results section reports greater maturation-marker expression in human iAT2 cells cultured at an air–liquid interface than in 3D spheres, and greater barrier function than in submerged 2D culture. The Discussion describes functional AT2 characteristics. However, the enhanced maturation and function were observed in air–liquid interface cultures, not in the 3D organoids themselves; the paper does not establish enhanced organoid development. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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

Hypoxic culture can rescue some aberrant signaling programs in human-skin-equivalent organoids and mimic physiological levels, thus improving the maturation of skin equivalents.

“Stabell et al. reported that hypoxic culture can rescue some aberrant signaling programs in human-skin-equivalent organoids and mimic physiological levels, thus improving the maturation of skin equivalents [34].”

• cites [34] Stabell, 2023, Single-cell transcriptomics of human-skin-equivalent organoids, Cell Rep sourcePartly supported

  In Results, “Hypoxia partially drives transcriptome-wide changes in xenograft-unique cells” (page unavailable), 3% oxygen partially repaired basal and differentiation programs, and the authors suggest that physiological oxygen levels likely improve those programs. The paper does not show that hypoxic culture rescues the aberrant signaling pathways described separately in Results, “HSEs exhibit abnormal signaling associated with EMT”; it also frames the improvement as likely rather than established maturation. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

Transplantation of HF-containing transplants contributes to faster healing and less scarring compared to non-HFs-containing transplants.

“Plotczyk M et al. showed that transplantation of HF-containing transplants contributes to faster healing and less scarring as compared to non-HFs-containing transplants [32].”

• cites [32] Plotczyk, 2023, Anagen hair follicles transplanted into mature human scars remodel fibrotic tissue, NPJ Regenerative Medicine sourcePartly supported

  The Results and Discussion report remodeling of mature scars after hair-follicle transplantation, including reduced collagen I fibers. They do not establish faster healing or less scarring compared with transplants without hair follicles: the Discussion says a sham transplantation with non-hairy skin was not possible. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

May combine two claims.

Matured SKOs formed a multilayered epithelium with high K10 expression in the outer suprabasal layer, whereas K14 expression was detected in the basal layer of the epidermis and HFs regions.

“Matured SKOs formed a multilayered epithelium with a high level of K10 expression in the outer (suprabasal) layer, whereas K14 expression was detected in the basal layer of the epidermis and the HFs regions (Figure 3e).”

May combine two claims.

T-SKOs were consistently larger than F-SKOs and could grow up to around 4 mm in size by day 115.

“The transwell culture resulted in the development of SKOs with similar sizes, and T-SKOs were consistently larger than F-SKOs and could grow up to around 4 mm in size by day 115 (Figure 2).”

May combine two claims.

Hair follicles are smaller and shorter in F-SKO compared with T-SKOs, with HFs in transwell culture exhibiting greater elongation over time and reaching up to 1 mm by Day 115.

“It was found that the hair follicles are smaller and shorter in the F-SKO compared with T-SKOs.”

• Also stated in: § ALI culture results in accelerated development of skin organoid · plain

  “Compared with the floating system, HFs exhibited greater elongation in transwell culture over time, reaching a length of up to 1 mm by Day 115 (Figure 2).”

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

HF placodes were observed within the dermal layer potentially due to their invagination into the dermis.

“The HF placodes were observed within the dermal layer potentially due to their invagination into the dermis (Figure 3b).”

May combine two claims.

HFs in T-SKOs exhibit features suggestive of hair shafts, resembling characteristics of HF development.

“Additionally, the HFs in T-SKOs exhibit features suggestive of hair shafts (dark and elongated structure), resembling the characteristics of HF development (Figure 2, images from Day 95 and Day 115).”

May combine two claims.

In the following timepoints, the number of hair placodes increased, leading to their transition into HFs.

“In the following timepoints, the number of hair placodes increased, leading to their transition into HFs.”

May combine two claims.

F-SKOs were spherical, while T-SKOs displayed a domed structure and became large and fused within the transwell.

“While the F-SKOs were spherical, the T-SKOs displayed a domed structure and became large and fused within the transwell.”

May combine two claims.

On Days 72, 82, and 92, C32 T-SKOs showed a higher quantity of HFs, whereas C32 F-SKOs exhibited greater numbers on Days 102 and 112.

“On Days 72, 82, and 92, the C32 T-SKOs showed a higher quantity of HFs, whereas the C32 F-SKOs exhibited greater numbers on Days 102, and 112 (Figure 4b).”

• AssumptionRests on an assumption: The number of hair placodes and HFs within individual SKOs is an indicator of SKO development and function.

May combine two claims.

In the transwell group, the number of HFs increased from Day 72 onward and peaked on Day 102, Day 112, and Day 82 for P111, P112, and C32 hiPSC lines, respectively.

“In the transwell group, the number of HFs increased from Day 72 onward and peaked on Day 102, Day 112, and Day 82 for P111, P112, and C32 hiPSC lines, respectively (Figure 4b).”

• AssumptionRests on an assumption: The number of hair placodes and HFs within individual SKOs is an indicator of SKO development and function.

May combine two claims.

Transwell cultures allow longitudinal observation of individual HF growth over time, while floating SKOs undergo rotational movement making continuous tracking impractical.

“The transwell cultures allow for longitudinal observation of individual HF growth over time, while the floating SKOs undergo rotational movement making the continuous tracking impractical.”

May combine two claims.

The hair placodes became discernible around Day 65 and quantifiable from Day 72.

“The hair placodes became discernible on SKOs under the microscope around Day 65 of differentiation and were quantifiable from Day 72, allowing quantification from this timepoint.”

May combine two claims.

HF length increased over time, with HFs protruding from the organoids by Day 120.

“The HF length was increased over time, with HFs protruding from the organoids by Day 120 (Figure 5d, red arrows).”

May combine two claims.

For both culture conditions, hair placode induction occurred on Day 65, and HFs reached in vitro maturation by Day 120.

“For both culture conditions, hair placode induction occurred on Day 65, and HFs reached in vitro maturation by Day 120.”

May combine two claims.

Sebaceous glands are positive for PPAR-gamma but negative for K15 marker.

“sebaceous glands are also positive for peroxisome proliferator-activated receptor (PPAR)-gamma (red color), but negative for cytokeratin-15 (K15, green color) marker.”

May combine two claims.

Pigmented HFs exhibit a dark coloration and protrude from the T-SKOs.

“The pigmented HFs exhibit a dark coloration and protrude from the T-SKOs (Figure 6a).”

The quote only partly states this claim.

In hiPSC-derived skin organoids, ALI culture at an early timepoint resulted in enhanced hair follicle length compared to classical floating culture.

“We found that using ALI culture at this early timepoint resulted in enhanced HF production, length, and morphology”

May combine two claims.

SKOs lack blood vessels and immune cells.

“the SKOs still lack other cell populations, including blood vessels and immune cells”

Classified as probably not a substantial claim. May combine two claims.

SKOs were randomly selected and divided into two groups, F-SKO and T-SKO.

“SKOs were randomly selected and divided into two groups, namely Floating SKO (F-SKO) and Transwell SKO (T-SKO).”

Classified as probably not a substantial claim.

On Day 12 of differentiation, SKOs were randomly divided between the ALI and floating culture conditions.

“On Day 12 of differentiation, the SKOs were randomly divided between the two culture conditions (ALI or floating) (Figure 1).”

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

For immunofluorescence analysis and histology, at least three biological replicates were prepared and analyzed.

“For immunofluorescence analysis and histology, at least three biological replicates were prepared and analyzed.”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

The ALI transwell setup recapitulates the physiological and anatomical conditions of skin, with the outer layer exposed to air and the dermis connected and nourished by underlying tissues.

“This setup recapitulates the physiological and anatomical conditions of the skin, while the outer layer is exposed to air, the dermis is connected and nourished by underlying tissues.”

Classified as probably not a substantial claim.

The small size of current organoids remains a limiting factor that restricts their ability to fully recapitulate the late stages of human organ development.

“Nevertheless, the small size of current organoids remains a limiting factor that restricts their ability to fully recapitulate the late stages of human organ development [19].”

• cites [19] Qian, 2019, Brain organoids: advances, applications and challenges, Development sourceNot checked — no full text of the source

May combine two claims.

Human primary tissue organoids range in size from a few hundred micrometers to a few millimeters and do not grow beyond a few millimeters in culture.

“Human primary tissue organoids range in size from a few hundred micrometers to a few millimeters (mm) and do not grow beyond a few millimeters in culture.”

• cites [19] Qian, 2019, Brain organoids: advances, applications and challenges, Development sourceNot checked — no full text of the source

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

An optimized SKO differentiation protocol using direct embryoid body formation from hiPSCs results in cystic SKOs that develop sensory neural networks, HFs, and sweat gland-like structures.

“Recently, an optimized SKO differentiation protocol was established by using direct embryoid body formation from hiPSCs as an immediate differentiation step, resulting in the formation of cystic SKOs that develop sensory neural networks, HFs, and sweat gland-like structures [9].”

• cites [9] Shafiee, 2024, Development of physiologically relevant skin organoids from human induced pluripotent stem cells, Small sourceNot checked — no full text of the source

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

The presence of cartilaginous regions indicates the need for further optimization of differentiation protocols to prevent off-target chondral tissue development.

“Dense cartilaginous regions were identified within the SKOs (Figure 5a, black asterisks) indicating the need for further optimization of differentiation protocols to prevent off-target (chondral) tissue development.”

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

The results on arrector pili muscles and Merkel cells within the SKOs are indicative and preliminary.

“However, the production of arrector pili muscles and Merkel cells within the SKOs requires further investigation, as these results are indicative and preliminary.”

May combine two claims.

Transwell culture promoted differentiation of epidermal layers and maturation of HFs.

“The results indicate that SKOs could be successfully generated from both culture systems, and that transwell culture promoted differentiation of epidermal layers and maturation of HFs.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

The KRT20-positive cells are Merkel cells.

“KRT20-positive cells were suggestive of Merkel cells adjacent to HFs (Figure 5j, and5k, white asterisks indicating the HFs).”

Probably not a substantial claim: may not pass S4 (based).

ALI culture provides a biomimetic microenvironment mimicking the skin's physiological condition.

“ALI culture provides a biomimetic microenvironment mimicking the skin's physiological condition.”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

The ALI system offers a supporting scaffold that promotes growth, differentiation, and stratification of keratinocytes while facilitating epithelial-mesenchymal interactions critical for HF morphogenesis.

“The ALI system offers a supporting scaffold that promotes growth, differentiation, and stratification of keratinocytes while facilitating epithelial-mesenchymal interactions critical for HF morphogenesis.”

Classified as probably not a substantial claim.

In vitro pigmented skin models provide a research platform to advance investigations into pigment-related skin disorders and devise new strategies for restoring hair pigmentation.

“Successfully achieving in vitro pigmented skin models would also provide a research platform to advance investigations into pigment-related skin disorders and devise new strategies for restoring hair pigmentation.”

Classified as probably not a substantial claim.

Generation of advanced-stage HFs with extended length enables detailed investigation of composition, development, and structural integrity providing insights into new skin therapeutics or cosmetics.

“the generation of advanced-stage HFs with extended length enables detailed investigation of composition, development, and structural integrity providing insights into new skin therapeutics or cosmetics.”

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

Human HFs with arrector pili muscle contraction play an important role in body temperature regulation.

“Human HFs with arrector pili muscle contraction, play an important role in body temperature regulation [30].”

• cites [30] Madaan, 2018, Review of hair follicle dermal papilla cells as in vitro screening model for hair growth, Int J Cosmet Sci sourceNot checked — no full text of the source

May combine two claims.

Epithelial stem cells within the HFs migrate and support wound re-epithelialization.

“the epithelial stem cells within the HFs migrate, and support wound re-epithelialization [33].”

• cites [33] Plotczyk, 2022 sourceNot checked — no full text of the source

May combine two claims.

The presence of HFs within engineered grafts provides better cosmetic outcomes and improves skin graft take rate.

“The presence of HFs within engineered grafts could not only provides better cosmetic outcomes but also improves skin graft take rate [31].”

• cites [31] Yang, 2015, Comparison between hair follicles and split-thickness skin grafts in cutaneous wound repair, Int J Clin Exp MedNot checked — no full text of the source