Single-cell transcriptomics of human-skin-equivalent organoids

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

THBS and ANGPTL signaling are known to promote angiogenesis.

“both are known to promote angiogenesis,54,55”

• cites [54] Lawler, 2012, Molecular basis for the regulation of angiogenesis by thrombospondin-1 and -2, Cold Spring Harb. Perspect. Med. sourceDisputed

  The Abstract describes TSP-1 and TSP-2 as potent endogenous inhibitors of angiogenesis, contrary to the claim that THBS signaling promotes it. The paper acknowledges proangiogenic effects of TSP-1 in some experimental models, but says those effects are eclipsed in the intact molecule. The supplied passages do not address ANGPTL signaling. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [55] Oike, 2004, Angiopoietin-related/angiopoietin-like proteins regulate angiogenesis, Int. J. Hematol. sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Human epidermis is more heterogeneous than previously thought.

“human epidermis is more heterogeneous than previously thought.”

• cites [26] Solé-Boldo, 2020, Single-cell transcriptomes of the human skin reveal age-related loss of fibroblast priming, Commun. Biol. sourcePartly supported

  The Results (scRNA-seq analysis of sun-protected human skin; page unavailable) identify three keratinocyte clusters, supporting heterogeneity among epidermal cells. But the paper says these clusters reflect mainly differentiation and that all identified clusters are known skin constituents; it does not establish that the epidermis is more heterogeneous than previously thought. The Discussion (page unavailable) places the extension of previous findings in dermal fibroblast heterogeneity. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [27] Wang, 2020, Single cell transcriptomics of human epidermis identifies basal stem cell transition states, Nat. Commun. sourceSupported
• cites [28] Reynolds, 2021, Developmental cell programs are co-opted in inflammatory skin disease, Science sourceSupported
• cites [29] Cheng, 2018, Transcriptional programming of normal and inflamed human epidermis at single-cell resolution, Cell Rep. sourceSupported

May combine two claims.

HSE composition and culture conditions vary greatly from lab to lab, which can affect interpretation of similar experiments.

“their composition and culture conditions vary greatly from lab to lab, which can affect interpretation of similar experiments.”

• cites [9] Atwood, 2021, Fostering a healthy culture: biological relevance of in vitro and ex vivo skin models, Exp. Dermatol. sourceSupported
• cites [10] Lee, 2020, Hair-bearing human skin generated entirely from pluripotent stem cells, Nature sourceCould not verify

  The Methods (page unavailable) describe this paper’s skin-organoid culture protocol and its validation by another laboratory. Neither those passages nor the passages found by searching for variation in skin-equivalent composition and culture conditions say that HSEs vary greatly between laboratories or that such variation affects interpretation of similar experiments. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites [11] Zhang, 2021, Using bioprinting and spheroid culture to create a skin model with sweat glands and hair follicles, Burns Trauma sourceCould not verify

  The Background (page unavailable) identifies reproducible skin constructs as an unresolved challenge, and the Discussion (page unavailable) describes limitations of this paper’s own culture conditions. Neither passage says that HSE composition and culture conditions vary greatly between laboratories or that such variation affects interpretation of similar experiments. Searches for interlaboratory variation and interpretation found no passage addressing that claim. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [16] Pourchet, 2017, Human skin 3D bioprinting using scaffold-free approach, Adv. Healthc. Mater. sourceNot checked — no full text of the source
• cites [18] El Ghalbzouri, 2009, Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products, Biomaterials sourceNot checked — no full text of the source
• cites [19] Li, 2015, Generation of genetically modified organotypic skin cultures using devitalized human dermis, J. Vis. Exp. sourceNot checked — no full text of the source

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

HSEs show marked molecular differences in basal and terminal gene expression, suggesting epidermal differentiation is not quite analogous to in vivo counterparts.

“Many studies defining HSEs have shown marked molecular differences in basal and terminal gene expression that suggest that epidermal differentiation is not quite analogous to the in vivo counterparts.”

• cites [20] Andreadis, 2001, Keratinocyte growth factor induces hyperproliferation and delays differentiation in a skin equivalent model system, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. sourcePartly supported

  The Discussion reports that KGF caused suprabasal proliferation in skin equivalents, whereas KGF-expressing mice showed no evidence of it. The ‘KGF delays differentiation’ section also reports altered differentiation-marker expression in KGF-treated equivalents versus control equivalents. These findings support a difference under KGF treatment, but not the broader claim that HSEs generally show marked basal and terminal gene-expression differences from in vivo epidermis. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [21] Jevtić, 2020, Impact of intercellular crosstalk between epidermal keratinocytes and dermal fibroblasts on skin homeostasis, Biochim. Biophys. Acta. Mol. Cell Res. sourceNot checked — no full text of the source

May combine two claims.

HSEs lack many components of the in vivo system, such as vasculature and immune cells, which limits the size of cultures and their response to experimental stimuli.

“Many components of the in vivo system are lacking, such as vasculature and immune cells, which limits the size of cultures and their response to experimental stimuli.”

• cites [9] Atwood, 2021, Fostering a healthy culture: biological relevance of in vitro and ex vivo skin models, Exp. Dermatol. sourceSupported

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

Previous work spatially resolved four distinct basal stem cell populations within human IFE and delineated multiple spinous and granular cell populations contributing to a hierarchical differentiation lineage.

“we spatially resolved four distinct basal stem cell populations within human IFE and delineated multiple spinous and granular cell populations that contributed to a hierarchical differentiation lineage”

• cites [27] Wang, 2020, Single cell transcriptomics of human epidermis identifies basal stem cell transition states, Nat. Commun. sourcePartly supported

  The Discussion reports at least four spatially distinct basal stem-cell populations in human neonatal epidermis and describes a differentiation hierarchy leading through spinous cells to granular keratinocytes. However, the Results section on epidermal heterogeneity identifies spinous clusters but describes granular keratinocytes as one GRN community, not multiple delineated granular populations. The claim also omits the neonatal restriction. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

Human skin spheroids resemble fetal facial skin but have long incubation period and small size, not ideal for genetic manipulation or grafting.

“Although these skin spheroids resemble fetal facial skin, their long incubation period and small size are not ideal for genetic manipulation of individual cell types or for grafting in the clinic.”

• cites [10] Lee, 2020, Hair-bearing human skin generated entirely from pluripotent stem cells, Nature sourcePartly supported

  The Abstract reports that the organoids resemble second-trimester human facial skin and require 4–5 months of incubation. The grafting section describes organoids placed in 1–2 mm incisions in mice, but the paper does not say that their incubation time or small size makes them unsuitable for manipulating individual cell types or for clinical grafting. The Discussion instead envisages clinical use after further control of off-target cell lineages. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

BAS-III cells typically sit atop the rete ridges in vivo, whereas BAS-IV cells lie at the bottom of rete ridges.

“BAS-III cells typically sit atop the rete ridges in vivo, whereas BAS-IV cells lie at the bottom of rete ridges.27”

• cites [27] Wang, 2020, Single cell transcriptomics of human epidermis identifies basal stem cell transition states, Nat. Commun. sourceSupported

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

The outermost epithelial layer of the murine epidermis is the periderm, which forms during stratification at E11.5 and disaggregates between E16 and E17, when barrier formation occurs.

“the outermost epithelial layer of the murine epidermis is the periderm, which forms during stratification at E11.5 and disaggregates between E16 and E17, when barrier formation occurs.70”

• cites [70] Hammond, 2019, Periderm: life-cycle and function during orofacial and epidermal development, Semin. Cell Dev. Biol. sourceSupported

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

HSEs can be completely submerged in medium for multiple days prior to raising them to an air-liquid interface to promote stratification, generated with immortalized keratinocyte cell lines, or seeded onto collagen layers.

“HSEs can be completely submerged in medium for multiple days prior to raising them to an air-liquid interface to promote stratification,75 generated with immortalized keratinocyte cell lines,76 or seeded onto collagen layers.77”

• cites [75] Roger, 2019, Bioengineering the microanatomy of human skin, J. Anat. sourceSupported
• cites [76] Reijnders, 2015, Development of a full-thickness human skin equivalent in vitro model derived from TERT-immortalized keratinocytes and fibroblasts, Tissue Eng. Part A sourceSupported
• cites [77] Mieremet, 2019, Characterization of human skin equivalents developed at body’s core and surface temperatures, J. Tissue Eng. Regen. Med. sourceSupported

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

Melanoma-like, neuronal-like, and muscle-like cells were found using scRNA-seq of kidney organoids, which were consistent with previous observations using conventional methods in this system.

“melanoma-like, neuronal-like, and muscle-like cells were found using scRNA-seq of kidney organoids,72 which were consistent with previous observations using conventional methods in this system.”

• cites [72] Subramanian, 2019, Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation, Nat. Commun. sourcePartly supported

  The Results section on the 49 kidney organoid states reports melanoma-like, neuron-like, and muscle-like cells in single-cell profiles and says these populations had been reported previously. Our copy does not specify that the previous observations used conventional methods, so that detail is unsupported. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Matrigel is used in the majority of organoid systems.

“Matrigel is used in the majority of organoid systems4”

• cites [4] Corrò, 2020, A brief history of organoids, Am. J. Physiol. Cell Physiol. sourcePartly supported

  In “Other Organoid Types” (page not provided), the review says the vast majority of adult stem cell–derived organoids are cultured in Matrigel. The “3D Culture Models” section also calls Matrigel the most commonly used scaffold. Neither passage establishes that a majority of all organoid systems use it, so the claim extends beyond the stated scope. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

The periderm temporally expresses different marker genes as the epidermis differentiates, such as Krt17 during early stages and Krt6 during later stages.

“The periderm temporally expresses different marker genes as the epidermis differentiates, such as Krt17 during early stages and Krt6 during later stages.70”

• cites [70] Hammond, 2019, Periderm: life-cycle and function during orofacial and epidermal development, Semin. Cell Dev. Biol. sourceSupported

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

Transplantation of HSEs onto burn patients or recent transplantation of HSEs to cure junctional epidermolysis bullosa demonstrate their clinical importance and remains the gold standard.

“transplantation of HSEs onto burn patients or recent transplantation of HSEs to cure junctional epidermolysis bullosa demonstrate their clinical importance and remains the gold standard.69”

• cites [69] Hirsch, 2017, Regeneration of the entire human epidermis using transgenic stem cells, Nature sourcePartly supported

  The section ‘Regeneration of a functional epidermis by transgenic epidermal cultures’ (page not provided) says keratinocyte cultures have long been used to treat severe burns and reports regeneration of functional epidermis in one patient with junctional epidermolysis bullosa. The paper does not establish that HSE transplantation is the gold standard, and its JEB result is a single-patient treatment rather than a general cure. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

GLUT1 is a downstream target gene of hypoxia and HIF1A.

“a downstream target gene of hypoxia and HIF1A,65”

• cites [65] Firth, 1995, Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements, J. Biol. Chem. sourcePartly supported

  The introductory passage (page unavailable) lists glucose transporters among genes regulated by oxygen. The paper does not identify GLUT1 in that passage or establish that it is a downstream target of HIF1A; its HIF-1 analysis concerns LDH-A and other genes. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

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

NECTIN signaling has a role in cell adhesion and skin morphogenesis.

“given its role in cell adhesion and skin morphogenesis.56”

• cites [56] Okabe, 2004, Expression patterns of nectins and afadin during epithelial remodeling in the mouse embryo, Dev. Dyn. sourcePartly supported

  The Introduction describes the nectin–afadin system as a component of cell–cell adhesion and links nectin-1 mutations to abnormalities of skin, hair, and teeth. The Ectodermal Stratified Epithelia section reports nectin-1 expression in developing skin but says its role there remains elusive. The paper therefore supports a possible role in skin morphogenesis, not an established role for nectin signaling in that process. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

The presence of abnormal cell states and altered differentiation patterns in organoid cultures have been observed in a variety of tissues, including skin, using more conventional methods.

“The presence of abnormal cell states and altered differentiation patterns in organoid cultures have been observed in a variety of tissues,4 including skin,9 using more conventional methods.”

• cites [4] Corrò, 2020, A brief history of organoids, Am. J. Physiol. Cell Physiol. sourcePartly supported

  The section ‘Strengths and Limitations of 3D Organoid Cultures Over Conventional Models’ (page unavailable) reports abnormal cellular stress and impaired cell-type specification in cortical organoids. It does not establish that these changes were observed across a variety of tissues or specify the conventional methods claimed. The skin example is separately cited in the citing sentence, so it is not attributed to this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [9] Atwood, 2021, Fostering a healthy culture: biological relevance of in vitro and ex vivo skin models, Exp. Dermatol. sourcePartly supported

  In “What organotypic cultures can and can’t do” (page unavailable), the paper says 3D skin cultures can lack a normal stratum corneum, epidermal barrier, or lipid composition. It also reports impaired expression of differentiation markers, but that finding concerns monolayer keratinocyte cultures, not organoids. The paper therefore supports limitations in skin cultures, but not the specific claim that abnormal cell states and altered differentiation patterns were observed in skin organoids. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

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

scRNA-seq analysis of human intestinal and brain organoids used random forest classifiers to identify the cell types in their organoid cultures.

“scRNA-seq analysis of human intestinal and brain organoids used random forest classifiers to identify the cell types in their organoid cultures;73,74”

• cites [73] Fujii, 2018, Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition, Cell Stem Cell sourceNot checked — no full text of the source
• cites [74] Velasco, 2019, Individual brain organoids reproducibly form cell diversity of the human cerebral cortex, Nature sourceSupported

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

Recent studies using scRNA-seq to characterize organoid cultures of other tissue types have also identified abnormal cell populations present in their organoid cultures.

“Recent studies using scRNA-seq to characterize organoid cultures of other tissue types have also identified abnormal cell populations present in their organoid cultures.”

• cites [72] Subramanian, 2019, Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation, Nat. Commun. sourceSupported

May combine two claims.

GelHSEs had the highest EMT score, followed by FibHSEs and in vivo dataset.

“GelHSEs had the highest EMT score, followed by FibHSEs and the in vivo dataset”

May combine two claims.

BAS-1 and BAS-8 were primarily composed of HSE-specific BAS cells, whereas BAS-4 was primarily found in in vivo state.

“BAS-1 and BAS-8 were primarily composed of HSE-specific BAS cells, whereas BAS-4 was primarily found in the in vivo state”

May combine two claims.

Sender EGF signaling is expanded to BAS and early SPN populations in HSEs, coinciding with appearance of VIM+ BAS cells.

“sender EGF signaling is expanded to the BAS and early SPN populations in the HSEs, coinciding with the appearance of VIM+ BAS cells”

May combine two claims.

A subset of pathways (LAMININ, CD99, CDH1, EPHB, MPZ) show similar signaling profiles across in vivo and HSE tissues, whereas other pathways show marked differences.

“A subset of these pathways, such as LAMININ, CD99, CDH1, EPHB, and MPZ, show similar signaling profiles across the in vivo and HSE tissues, whereas the other pathways show marked differences”

May combine two claims.

Keratinocyte stratification occurs under both GelHSE and FibHSE conditions by day 7, with tightly packed columnar basal cell layer, multiple irregular polyhedral squamous cell layers, several flattened granular cell layers, and a thin stratum corneum.

“Keratinocyte stratification occurs under both conditions by day 7, where the HSEs show a tightly packed columnar basal cell layer, multiple irregular polyhedral squamous cell layers, several flattened granular cell layers, and a thin stratum corneum”

May combine two claims.

Splicing kinetics support two distinct differentiation trajectories, a BAS-SPN-GRN and a BAS-XENO-GRN trajectory, with uniform velocity streams flowing from one state to the next.

“The splicing kinetics further support two distinct differentiation trajectories, a BAS-SPN-GRN and a BAS-XENO-GRN trajectory, possessing uniform velocity streams flowing from one state to the next (Figure 5E).”

May combine two claims.

Both HSE cultures have high levels of LAMC2 and LGALS1 expression in all BAS populations and lower expression levels in more differentiated keratinocytes.

“Both HSE cultures have high levels of LAMC2 and LGALS1 expression in all BAS populations and lower expression levels in more differentiated keratinocytes”

May combine two claims.

CellChat detected 18 significant signaling pathways in in vivo dataset and HSEs recapitulated 16 of 18 pathways.

“CellChat detected 18 significant signaling pathways in the in vivo dataset and the HSEs recapitulated 16 of the 18 pathways”

May combine two claims.

BAS-III keratinocyte proportions are much higher and BAS-IV proportions are lower in the xenograft than in the in vivo setting.

“the proportions of BAS-III and BAS-IV keratinocytes are not similar to each other, with BAS-III proportions being much higher and BAS-IV being lower in the xenograft than in the in vivo setting (Figures 4D and 4G)”

May combine two claims.

BAS keratinocytes expressing KRT15 were placed at the beginning of the trajectory, and cells expressing FLG were placed toward one of the trajectory termini.

“BAS keratinocytes expressing KRT15 were placed at the beginning of the trajectory, and cells expressing the terminal differentiation gene FLG were placed toward one of the trajectory termini”

May combine two claims.

Both HSEs had a higher proportion of BAS-III and HSE-I cells and a lower proportion of SPN-2, SPN-4, SPN-5, and GRN-2 compared with in vivo state.

“Both HSEs had a higher proportion of BAS-III and HSE-I cells and a lower proportion of the spinous (SPN) cell clusters SPN-2, SPN-4, and SPN-5, and the granular (GRN) cluster GRN-2 compared with the in vivo state.”

May combine two claims.

AREG-EGFR signaling is overrepresented in both HSEs, and AREG ligand is expressed in most HSE-cultured keratinocytes.

“AREG-EGFR signaling is overrepresented in both HSEs, and the AREG ligand is expressed in most HSE-cultured keratinocytes”

May combine two claims.

CDH1 is negatively correlated with VIM and shows higher expression in VIM- HSE keratinocytes compared with in vivo state.

“The epithelial cell marker CDH1 is negatively correlated with VIM and shows higher expression in VIM- HSE keratinocytes compared with the in vivo state”

May combine two claims.

Many major in vivo cellular states were found in in vitro HSEs, including the full complement of in vivo basal cell states.

“Remarkably, many of the major in vivo cellular states were found in the in vitro HSEs, including the full complement of in vivo basal cell states.”

May combine two claims.

An HSE cell state clustered separately from in vivo cells and was annotated HSE-1.

“Intriguingly, an HSE cell state clustered separately from the in vivo cells and was annotated HSE-1”

May combine two claims.

HSE-1 was placed at a distinct trajectory terminus away from GRN cell states, generating a BAS-SPN-HSE differentiation trajectory.

“Intriguingly, HSE-1 was placed at a distinct trajectory terminus away from the GRN cell states, generating a BAS-SPN-HSE differentiation trajectory”

May combine two claims.

BAS cell marker KRT15 remains restricted to the BAS-most layer of HSEs, whereas KRT19 shows selective expansion in GelHSEs.

“The BAS cell marker KRT15 does remain restricted to the BAS -most layer of the HSEs, whereas KRT19 shows selective expansion in GelHSEs”

May combine two claims.

The partial VIM+ EMT-like state and remnant PSCA+ embryonic program are no longer detected in xenograft tissue.

“The two abnormal features of the HSE cultures, the partial VIM+ EMT- like state and remnant PSCA+ embryonic program, are no longer detected in the xenograft tissue (Figure 4I)”

May combine two claims.

HSE-specific keratinocytes constituted 0.6% of FibHSEs and 9.3% of GelHSEs.

“HSE-specific keratinocytes constituted 0.6% of FibHSEs and 9.3% of GelHSEs”

May combine two claims.

There is an expansion of KRT14+ cell layers and disrupted epidermal differentiation in GelHSE and FibHSE cultures.

“there is an expansion of KRT14+ cell layers and disrupted epidermal differentiation in the GelHSE and FibHSE cultures”

May combine two claims.

VIM shows high RNA expression in GelHSE BAS keratinocytes and VIM+ protein expression in GelHSE and FibHSE BAS keratinocytes.

“The mesenchymal marker VIM, which is normally restricted to fibroblasts, melanocytes, and Langerhans cells of in vivo skin, shows high RNA expression in GelHSE BAS keratinocytes and VIM+ protein expression in GelHSE and FibHSE BAS keratinocytes”

May combine two claims.

Many BAS and SPN velocity vectors for GelHSEs point toward HSE-1 cluster, with undefined flow between SPN and GRN clusters.

“many BAS and SPN velocity vectors for GelHSEs point toward the HSE-1 cluster, with an undefined flow of vectors between the SPN and GRN clusters”

May combine two claims.

Histologically, HSEs largely remain the same up through day 28, except for thickening of stratum corneum and general spreading out of keratinocytes at all epidermal layers.

“Histologically, the HSEs largely remain the same up through day 28, except for a thickening of the stratum corneum and a general spreading out of keratinocytes at all epidermal layers.”

May combine two claims.

BAS-I-BAS-IV represented approximately 27.3% of in vivo cells, 55.6% of FibHSEs, and 22.0% of GelHSEs.

“BAS-I–BAS-IV represented approximately 27.3% of the in vivo cells, 55.6% of FibHSEs, and 22.0% of GelHSEs”

May combine two claims.

In vivo epidermal dataset shows low entropy for BAS and GRN clusters, indicating stable states, whereas SPN clusters have high probabilities of transitioning to a new state.

“The in vivo epidermal dataset shows low entropy for the BAS and GRN clusters, indicating that these are stable states, whereas the SPN clusters have high probabilities of transitioning to a new state”

May combine two claims.

BAS-1 and BAS-8 clusters have higher expression of VIM than other clusters and have higher EMT score.

“The BAS-1 and BAS-8 clusters have a higher expression of VIM than the other clusters and have a higher EMT score”

May combine two claims.

The xenograft-unique clusters were designated XENO-1-XENO-3 and collectively comprise ~49.2% of the total xenograft cells.

“The xenograft-unique clusters were designated XENO-1–XENO-3 and collectively comprise ~49.2% of the total xenograft cells (Figure 4D).”

May combine two claims.

Histologically, the xenografts appear relatively normal, with some BAS keratinocytes adopting a cuboidal morphology.

“Histologically, the xenografts appear relatively normal, with some BAS keratinocytes adopting a cuboidal morphology (Figure 4B).”

May combine two claims.

XENO states are highly stable, along with the BAS-III state.

“The XENO states are highly stable, along with the BAS-III state”

May combine two claims.

The ratios of BAS-I and BAS-II cycling cells remained largely similar between in vivo tissue and FibHSEs, while GelHSEs had a reduction in cycling cells.

“The ratios of BAS-I and BAS-II cycling cells remained largely similar between the in vivo tissue and FibHSEs, while GelHSEs had a reduction in cycling cells.”

May combine two claims.

BAS-III cells are enriched in both HSEs, with FibHSEs possessing over 3.5 times as many cells in this cluster than in vivo tissue.

“BAS-III cells are enriched in both HSEs, with FibHSEs possessing over 3.5 times as many cells in this cluster than the in vivo tissue”

May combine two claims.

10 of 12 cell type proportions were significantly changed in GelHSEs compared with in vivo datasets, while only 6 of 12 were significantly different in FibHSEs.

“10 of 12 cell type proportions were significantly changed in GelHSEs compared with the in vivo datasets, while only 6 of 12 were significantly different in FibHSEs”

May combine two claims.

EREG and TGFA ligands specifically contribute to EGF signaling in HSEs, whereas HBEFG-EGFR signaling is reduced compared with in vivo state.

“EREG and TGFA ligands also specifically contribute to EGF signaling in the HSEs, whereas HBEFG-EGFR signaling is reduced compared with the in vivo state”

May combine two claims.

BAS-III and GRN-1 are still signaling hubs in GelHSE and FibHSE datasets; HSE-1-specific signaling exerts wide influence over GelHSEs, whereas all four BAS clusters actively signal in FibHSEs.

“While BAS-III and GRN-1 are still signaling hubs in the GelHSE and FibHSE datasets, HSE-1-specific signaling exerts a wide influence over GelHSEs, whereas all four BAS clusters actively signal in FibHSEs”

May combine two claims.

Several BAS cell markers are appropriately expressed in their corresponding cell states in xenografts compared with HSE cultures: PTTG1 with BAS-I, RRM2 with BAS-II, and ASS1 and KRT19 with BAS-III.

“Several BAS cell markers are now appropriately expressed in their corresponding cell states compared with the HSE cultures (PTTG1 with BAS-I, RRM2 with BAS-II, and ASS1 and KRT19 with BAS-III)”

May combine two claims.

In vivo epidermal dataset showed expected BAS-III and BAS-IV velocity vectors pointing toward SPN clusters and SPN velocity vectors pointing toward GRN clusters, reconstructing BAS-SPN-GRN differentiation trajectory.

“The in vivo epidermal dataset showed the expected BAS-III and BAS-IV velocity vectors pointing toward the SPN clusters and SPN velocity vectors pointing toward the GRN clusters, reconstructing the BAS-SPN-GRN differentiation trajectory”

May combine two claims.

Removal of EGF resulted in a significant decrease in VIM expression in FibHSE keratinocytes, whereas further EGF supplementation increased VIM expression.

“Removal of EGF resulted in a significant decrease in VIM expression in FibHSE keratinocytes (Figure 3H), whereas further EGF supplementation increased VIM expression”

May combine two claims.

SLUG (SNAI2) was present in nuclei of keratinocytes throughout all living layers of HSEs, while human abdominal skin had little to no observable staining.

“SLUG (SNAI2), an EMT-inducing transcription factor,49 was present in the nuclei of keratinocytes throughout all living layers of the HSEs, while human abdominal skin had little to no observable staining”

May combine two claims.

The proportions of BAS-III cells were enriched and BAS-IV cells were depleted compared with the in vivo state.

“the proportions of BAS-III cells were enriched and BAS-IV cells were depleted compared with the in vivo state”

May combine two claims.

The differentiation program appeared partially rescued with KRT10 expression in the SPN and GRN compartments compared with sporadic staining under normoxic conditions and LOR showing more restriction to the GRN layer.

“The differentiation program also appeared to be partially rescued with KRT10 expression in the SPN and GRN compartments compared with the sporadic staining under normoxic conditions and LOR showing more restriction to the GRN layer.”

May combine two claims.

BAS cell heterogeneity in our organoids fully mimics in vivo BAS cell heterogeneity during homeostasis, with most of the differentiated states also present.

“We have shown that BAS cell heterogeneity in our organoids fully mimics in vivo BAS cell heterogeneity during homeostasis, with most of the differentiated states also present.”

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

In the integrated In Vivo and Xenograft dataset, cluster XENO-4 had an average of 807 unique genes expressed and 1881 UMIs.

“Similarly, one cluster in the In Vivo and Xenograft integrated dataset, XENO-4, had an average of 807 unique genes expressed and 1881 UMIs.”

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

In the integrated In Vivo, GelHSE, and FibHSE dataset, cluster HSE-2 had an average of 469 unique genes expressed and 805 UMIs.

“one cluster in the In Vivo, GelHSE, and FibHSE integrated dataset, HSE-2, had an average of 469 unique genes expressed and 805 UMIs”

May combine two claims.

Expression of these genes was higher in the GelHSEs but still present in FibHSEs.

“Expression of these genes was higher in the GelHSEs but still present in FibHSEs”

May combine two claims.

Hypoxic HSEs showed a partially repaired BAS program with KRT15 showing more uniform BAS enrichment compared with normoxic conditions.

“The hypoxic HSEs showed a partially repaired BAS program with KRT15 showing more uniform BAS enrichment compared with normoxic conditions (Figure 5M).”

May combine two claims.

The most significantly enriched term for the xenograft-unique metacluster was hypoxia, and the most significantly depleted pathway was oxidative phosphorylation.

“The most significantly enriched term for the xenograft-unique metacluster identified hypoxia, whereas the most significantly depleted pathway was oxidative phosphorylation (Figure 5J)”

May combine two claims.

Mice have a distinct density of hair follicles and eccrine glands, a layer of striated muscle found beneath the hypodermis, a lack of melanocytes in the interfollicular epidermis, and absence of rete ridges.

“mice have a distinct density of hair follicles and eccrine glands, a layer of striated muscle found beneath the hypodermis, a lack of melanocytes in the interfollicular epidermis (IFE), and absence of rete ridges.”

• cites [1] Kolarsick, 2011, Anatomy and physiology of the skin, J. Dermatol. Nurses Assoc. sourceNot checked — no full text of the source
• cites [6] Schmook, 2001, Comparison of human skin or epidermis models with human and animal skin in in-vitro percutaneous absorption, Int. J. Pharm. sourceNot checked — no full text of the source

May combine two claims.

These differences impact epidermal homeostasis, wound repair, and the severity of certain skin disorders.

“These differences impact epidermal homeostasis, wound repair, and the severity of certain skin disorders”

• cites [5] Zomer, 2018, Skin wound healing in humans and mice: challenges in translational research, J. Dermatol. Sci. sourceNot checked — no full text of the source

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

One cluster in xenograft appeared to be low-quality cells likely apoptotic.

“We excluded one cluster from our downstream analysis that appeared to be low-quality cells that passed our initial quality control thresholds because the number of genes detected and UMIs appeared to be far lower than those of the other clusters, and the percentage of mitochondrial gene expression appeared to be higher, suggesting that these were likely apoptotic cells”

May combine two claims.

These states are robust to perturbations and remain a core lineage trajectory in HSEs.

“suggesting that these states are robust to perturbations and remain a core lineage trajectory in the HSEs”

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

Xenograft-unique signaling pathways indicate significant tissue remodeling, likely from the wounding process for engraftment, including enrichment for pathways that promote angiogenesis.

“Because xenograft-unique signaling pathways indicate significant tissue remodeling, likely from the wounding process for engraftment, including enrichment for pathways that promote angiogenesis”

Classified as probably not a substantial claim.

The extent to which HSEs can recapitulate cell type heterogeneity, cell-cell signaling, and differential gene expression of in vivo human skin remains unclear.

“The extent to which HSEs can recapitulate the cell type heterogeneity, cell-cell signaling, and differential gene expression of in vivo human skin remains unclear.”

May combine two claims.

Suprabasal KRT14+ cells are not fully functioning BAS cells and are likely differentiating without fully turning off the BAS cell state.

“suggesting that suprabasal KRT14+ cells are not fully functioning BAS cells and are likely to be differentiating without fully turning off the BAS cell state.”

May combine two claims.

Keratinocytes expressing an EMT signature separate out from other BAS populations and are primarily composed of HSE-specific BAS cells.

“indicating that the keratinocytes expressing an EMT signature separate out from the other BAS populations and are primarily composed of HSE-specific BAS cells”

May combine two claims.

The xenograft tissue more closely reflects the in vivo state compared with HSE cultures, with restored terminal differentiation, cell-cell adhesion, and partially restricted BAS programs.

“The xenograft-unique clusters notwithstanding, the xenograft tissue more closely reflects the in vivo state compared with the HSE cultures with restored terminal differentiation, cell-cell adhesion, and partially restricted BAS programs.”

May combine two claims.

NSG mice were chosen because of their ability to engraft skin at very high levels and perivascular infiltration of immune cells.

“NSG mice were chosen because of their ability to engraft skin at very high levels and perivascular infiltration of immune cells.”

• cites [53] Brehm, 2012, Human allograft rejection in humanized mice: a historical perspective, Cell. Mol. Immunol. sourceNot checked — no full text of the source

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

PSCA expression may indicate a remnant embryonic program reactivated by growth factors in culture medium.

“may indicate a remnant embryonic program that is reactivated as a result of growth factors in the culture medium.”

May combine two claims.

Terminal differentiation may be disrupted and HSE-1 may represent an alternative differentiation trajectory terminus in GelHSEs.

“suggesting that terminal differentiation may be disrupted and that HSE-1 may represent an alternative differentiation trajectory terminus in the GelHSEs.”

May combine two claims.

Human cells have more nuclear and mitochondrial RNA reads aligning to human reference genome, and same for mouse reads and mouse reference genome.

“Human cells have more nuclear and mitochondrial RNA reads aligning to a human reference genome, and the same is true for mouse reads and a mouse reference genome”

Classified as probably not a substantial claim.

For downstream analyses, cells were kept if they met the following filtering criteria per biological replicate per condition: >200 and <5000 genes/cell, and <10% mitochondrial gene expression.

“For downstream analyses, we kept cells which met the following filtering criteria per biological replicate per condition: >200 and <5000 genes/cell, and <10% mitochondrial gene expression.”

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

Hypoxia and angiogenesis are causally linked to wound repair, which collectively induces substantial molecular and morphological changes to tissues during repair.

“hypoxia and angiogenesis are causally linked to wound repair, which collectively induces substantial molecular and morphological changes to tissues during repair.78,79”

• cites [78] Botusan, 2008, Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice, Proc. Natl. Acad. Sci. USA sourceNot checked — no full text of the source
• cites [79] Gurtner, 2008, Wound repair and regeneration, Nature sourceNot checked — no full text of the source

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

Allowing more time for the graft to heal would presumably return it to a fully homeostatic state.

“allowing more time for the graft to heal would presumably return it to a fully homeostatic state”

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

MIF signaling has been shown to be upregulated during wound healing in mice.

“has been shown to be upregulated during wound healing in mice.57”

• cites [57] Gilliver, 2011, MIF: a key player in cutaneous biology and wound healing, Exp. Dermatol. sourceNot checked — no full text of the source

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

Human clinical studies were approved by the Ethics Committee of the University of California, Irvine.

“Human clinical studies were approved by the Ethics Committee of the University of California, Irvine.”

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

We cannot rule out wound repair as the major cause of transcriptional changes in our xenograft HSEs.

“While we cannot rule out wound repair as the major cause of transcriptional changes in our xenograft HSEs”

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

Each cohort of organoids initiated on separate days used cells from a distinct subject.

“Each cohort of organoids initiated on separate days used cells from a distinct subject.”

Classified as probably not a substantial claim.

Informed consent was obtained from all participants.

“We have obtained informed consent from all participants.”

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

No personal information was collected for this study.

“No personal information was collected for this study.”

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

Inappropriate signals from the dermis may also be the cause of the BAS defects.

“Inappropriate signals from the dermis may also be the cause of the BAS defects.”

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

HSEs have long served as models of human IFE in place of murine skin.

“HSEs have long served as models of human IFE in place of murine skin.7,18,66,67”

• cites [7] Bell, 1983, The reconstitution of living skin, J. Invest. Dermatol. sourceNot checked — no full text of the source
• cites [18] El Ghalbzouri, 2009, Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products, Biomaterials sourceNot checked — no full text of the source
• cites [66] Augustin, 1997, Use of dermal equivalent and skin equivalent models for identifying phototoxic compounds in vitro, Photodermatol. Photoimmunol. Photomed. sourceNot checked — no full text of the source
• cites [67] Gu, 2020, Development of a new eczema-like reconstructed skin equivalent for testing child atopic dermatitis-relieving cosmetics, J. Cosmet. Dermatol. sourceNot checked — no full text of the source

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

All maintenance, care, and experiments have been approved and abide by regulatory guidelines of the Institutional Animal Care and Use Committee of the University of California, San Diego.

“All maintenance, care, and experiments have been approved and abide by regulatory guidelines of the Institutional Animal Care and Use Committee of the University of California, San Diego.”

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

All cells and organoids are of male origin.

“As such, all cells and organoids are of male origin.”

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

This is true of any supervised machine learning algorithm and can be misleading when examining cellular heterogeneity.

“This is true of any supervised machine learning algorithm and can be misleading when examining cellular heterogeneity.”

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

Genes that were expressed in less than 3 cells were excluded.

“Genes that were expressed in less than 3 cells were excluded.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

Matrigel more than likely induces effects similar to those observed here.

“and more than likely induces effects similar those observed here”

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

Cluster XENO-4 was excluded from downstream analysis.

“This cluster was also excluded from downstream analysis.”

May combine two claims.

Because the epidermis is not directly supplied with blood, in vivo oxygen levels range between 0.5% and 8%.

“Because the epidermis is not directly supplied with blood, in vivo oxygen levels range between 0.5% and 8%.63,64”

• cites [63] Evans, 2006, Oxygen levels in normal and previously irradiated human skin as assessed by EF5 binding, J. Invest. Dermatol. sourceNot checked — no full text of the source
• cites [64] Rezvani, 2011, Loss of epidermal hypoxia-inducible factor-1α accelerates epidermal aging and affects re-epithelialization in human and mouse, J. Cell Sci. sourceNot checked — no full text of the source

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

This difference may be due to the variations in culturing methods.

“This difference may be due to the variations in culturing methods.”

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

Oxidative phosphorylation has been shown to be down-regulated in response to hypoxia.

“which has been shown to be down-regulated in response to hypoxia.58”

• cites [58] Rodríguez-Enríquez, 2010, Oxidative phosphorylation is impaired by prolonged hypoxia in breast and possibly in cervix carcinoma, Int. J. Biochem. Cell Biol. sourceNot checked — no full text of the source

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

Following exclusion of debris and singlet/doublet discrimination, cells were gated on viability for downstream scRNA-seq.

“Following exclusion of debris and singlet/doublet discrimination, cells were gated on viability for downstream scRNA-seq.”

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

All human studies were performed in strict adherence to the Institutional Review Board (IRB) guidelines of the University of California, Irvine (2009-7083).

“All human studies were performed in strict adherence to the Institutional Review Board (IRB) guidelines of the University of California, Irvine (2009–7083).”