Mosaic partial epidermal reprogramming remodels neighbors and niches to refine skin homeostasis and repair

Domain: neuroscience
Published: 2026-01-30
Editorial outcome: minor_revision

Abstract
Adult stem cells and their niches communicate intricately for tissue maintenance and regeneration. However, effectively coordinating these complex interactions is challenging. Here, we demonstrate that transient dedifferentiation of a fraction of epithelial stem cell progenies orchestrates beneficial changes within the entire skin’s cellular networks to favor repair. We achieved this by inducing a mosaic and reversible expression of reprogramming factors (Oct-4, Sox2, Klf4, and c-Myc) in the mouse epidermis. This in vivo partial epidermal reprogramming not only affected the partially reprogrammed cells, but also their microenvironment, including neighboring epithelial cells and T cells, conferring widespread healing characteristics even in the absence of injury. When a wound was introduced, these collective changes accelerated re-epithelialization in both wild-type and a hyperglycemic mouse disease model. Furthermore, the effects extended to dermal healing, leading to reduced scarring and altered angiogenesis. In conclusion, our work reveals that mosaic partial reprogramming of the epidermis influences various cell types within the skin during homeostasis and repair, leading to enhanced cutaneous wound healing.

Introduction
Adult stem cells (ASCs) maintain homeostasis by regulating tissue turnover. They are activated upon injury, the physiological damage that disrupts homeostasis, to heal the tissue1. The injury is either completely regenerated; in others, it is repaired with excessive fibrosis, forming scars—collagen-rich nonfunctional tissue2–4; or in some cases, the injury remains unrepaired. Previous studies have explored the regulation and development of injury repair processes and identified several factors that determine regenerative capacity. First, the proliferative ability of ASCs and their progeny is crucial5–7. In addition, sophisticated spatial and temporal control of communication between ASCs and their niches is critical, because the healing process involves re-establishment of complex organ structures and functions beyond restoration of cell numbers8–10. These niches include mesenchymal cells that modulate the extracellular matrix (ECM) for precise cell proliferation and migration, and immune cells that produce inflammatory responses against damage2,11–13.

Despite numerous studies highlighting the significance of stem cell-niche interactions in regeneration, understanding the entire network is challenging due to its spatial and temporal complexity. Additionally, manipulation of the network for optimal outcomes has been difficult, as manipulation of individual factors can have unforeseen consequences. For instance, accelerating repair processes, which should be beneficial, can sometimes lead to detrimental effects such as increased fibrosis or scarring14–16. Therefore, effective regulation of stem cell-niche communication requires further investigation.

Cellular reprogramming by Oct-4, Sox2, Klf4, and c-Myc (OSKM), known as the Yamanaka factors, has emerged as a valuable tool for the manipulation of cell fate17–19. Originally, OSKM were used only in vitro because their forced expression in vivo lead to teratoma formation due to the production of induced pluripotent stem cells20,21. However, it was found that transient and reversible OSKM expression could induce partial reprogramming, which confers cellular plasticity while retaining the original cell identity and functionality. This process did not induce pluripotency, indicating that in vivo partial reprogramming could prevent teratoma formation22–24. Partial reprogramming in mice rejuvenated progeria or physiological aging22,23,25–27. Furthermore, recent studies have revealed that in vivo partial reprogramming promotes tissue repair in various organs, such as the muscle, liver, and intestine28–30. Another study observed a reduction in fibrosis and scarring after injury15. Even the adult heart and nervous system, which are composed of post-mitotic cells and therefore do not heal well, have shown regeneration via partial reprogramming31,32.

To enhance tissue regeneration, short-term OSKM expression partially dedifferentiates resident somatic cells in a cell-autonomous manner at the cellular level29,30,32. However, the influence of partially reprogrammed cells on their in vivo environment is poorly understood. It is hence unclear whether previously reported improvements in tissue regeneration by partial reprogramming are solely mediated by proliferation and dedifferentiation of somatic cells, or if an interplay between the dedifferentiated cells and the surrounding environment plays a crucial role in the process. Understanding such interplay between partially reprogrammed cells and their neighbors/niches in complex in vivo systems will be essential for possible future practical applications of in vivo partial reprogramming to promote tissue homeostasis and repair.

In this study, we addressed this gap in knowledge by establishing a genetically engineered mouse model that allows partial epidermal reprogramming in a mosaic manner. We transiently induced mosaic OSKM expression, coupled with the fluorescent reporter mCherry, specifically in the epithelial cells of the skin—the body’s primary protective barrier against external threats. Using this model, we demonstrate that various types of skin cells enter an injury-responsive-like state upon partial reprogramming of only a small fraction of epidermal cells. Enhanced cutaneous healing was also observed. We conclude that short-term mosaic OSKM expression in the epidermis is sufficient to coordinate the cellular networks in the skin, directing them to be beneficial for healing, and may provide an avenue for the control of skin homeostasis and repair processes.

Results
In vivo mosaic partial epidermal reprogramming through transient OSKM induction
To explore how in vivo partial reprogramming affects the function of skin epithelial stem cells and their progeny during homeostasis and repair, we transiently expressed OSKM in epidermal keratinocytes of mice. We crossed mice carrying the LoxP-STOP-LoxP-rtTA-EGFP and TetO-OSKM-mCherry cassettes33 with a Krt14-CreER transgene that is expressed in the interfollicular epidermal (IFE) cells34–36 (Supplementary Fig. 1a–c). In the absence of tamoxifen and doxycycline (Dox), neither EGFP nor mCherry expression was detectable (Supplementary Fig. 1d). After Dox treatment for 3 days, reprogrammable mice (Epi-iOSKM mice) exhibited global GFP and mCherry mosaic expression in the epidermis (Fig. 1a, b; Supplementary Fig. 1b–d) with no histological abnormalities (Supplementary Fig. 1e, f). Mice without the TetO-OSKM-mCherry cassette (control mice) expressed only rtTA-EGFP and not mCherry (Fig. 1b). We also observed the induction of Oct4, Sox2, Klf4, and c-Myc expression in the mCherry+ cells of Epi-iOSKM mice, and quantified that the proportion of Sox2+ cells was 21.91 ± 3.97% in basal epidermal cells and 25.55 ± 3.43% in suprabasal epidermal cells, confirming effective induction of the reprogramming factors (Fig. 1c; Supplementary Fig. 1g–j). Furthermore, the pluripotency marker Nanog was not detected, indicating that these cells were not fully reprogrammed (Supplementary Fig. 2a). After Dox withdrawal, mCherry was absent (Supplementary Fig. 2b), and no cell death was detected (Supplementary Fig. 2c, d), confirming OSKM was transiently induced only during periods of Dox administration.

Mosaic partial reprogramming of epidermal cells induces themselves and their neighbors to undergo lineage-preserved dedifferentiation
Despite recent remarkable findings on partial in vivo reprogramming, the influence of partially reprogrammed cells on their surroundings is unclear. We therefore utilized our mosaic partial reprogramming model to investigate both the effects of partial epidermal reprogramming on neighboring cells, as well as cell-autonomous effects. Firstly, we performed single-cell RNA sequencing (scRNA-seq) to explore the changes in the molecular and cellular characteristics of skin epithelial cells upon mosaic partial reprogramming. To characterize the respective cellular properties of the OSKM-mCherry-expressing IFE cells (mChpos cells) and neighboring IFE cells without OSKM-mCherry expression (mChneg cells), we sorted mChpos and mChneg epidermal cells from Epi-iOSKM mice (Supplementary Fig. 3a, b). We also collected epidermal cells from control mice and included these in our analysis. To identify the IFE cells, we used uniform manifold approximation and projection (UMAP) plots; this divided the IFE cells into 15 clusters (Supplementary Fig. 3c, d). Based on marker gene expression (Supplementary Fig. 3e), we annotated clusters 0, 1, 3, 4, 5, 6, and 14 as non-proliferative basal cells (Basal), clusters 2, 10, and 13 as proliferating cells (Prolif), and clusters 7, 8, 9, 11, and 12 as non-proliferative suprabasal cells (Supra) (Supplementary Fig. 3f). Interestingly, IFE cells from control mice were rarely found in clusters 2, 3, and 12. However, cells in these clusters were enriched for mChpos populations (Supplementary Fig. 3d). We hence annotated clusters 2, 3, and 12 as Prolif II, Basal II, and Supra II, respectively, potentially describing converted IFE cells upon cell-autonomous OSKM induction, whereas we defined other Prolif, Basal, and Supra cells as Prolif I, Basal I, and Supra I, respectively, representing canonical IFE cells (Fig. 1d).

Surprisingly, we found that the mChneg populations in the Epi-iOSKM epidermis exhibited a higher proportion of Basal II and Supra II cells than control epidermis (Fig. 1d, e). This implies the potential that cell-non-autonomous conversion occurs in cells lacking OSKM expression, causing the mChneg cells to change in a manner similar to mChpos cells. To compare the control-enriched IFE cell types and mChpos and mChneg-enriched IFE cell types, we validated the expression of epidermal differentiation markers. The epidermis comprises a Krt14+ basal layer with epidermal stem/progenitor cells, and a suprabasal layer with Krt10+ differentiated cells (Supplementary Fig. 4a)37. Krt10 expression was lower in Supra II than in Supra I, while the pattern of Krt14 expression showed the opposite trend (Fig. 1f and Supplementary Fig. 4b). Krt10 expression in the suprabasal IFE cells was lower in not only mChpos but also mChneg populations than in the control, demonstrating cell-non-autonomous effects (Fig. 1g). Consistently, immunohistochemical analysis confirmed that short-term OSKM expression generated cells that express less Krt10 in the suprabasal layer in both cell-autonomous and non-autonomous manners (Fig. 1h and Supplementary Fig. 4c). Expression of another suprabasal marker, Dsg1a38, was also downregulated in Epi-iOSKM suprabasal IFE cells in a similar manner (Supplementary Fig. 4d). Together with the reduction of the differentiation markers, partial reprogramming increased the Krt14+ basal layer thickness (Supplementary Fig. 4e). We also comprehensively examined the extent of differentiation in Supra IFE cells using pseudotime computed with Monocle 3. The pseudotime values, which are proportional to the expression levels of epidermal differentiation markers (Supplementary Fig. 4f–h), were lower for mChpos/mChneg than controls (Fig. 1i), demonstrating Supra cells in the Epi-iOSKM epidermis exhibit reduced epidermal differentiation characteristics.

We next found that the proportion of Prolif cells is higher in mChpos cells than mChneg cells and control (Fig. 1j). The differentially expressed genes (DEGs) that were only upregulated in mChpos cells—and not in mChneg cells—from the Epi-iOSKM epidermis showed that genes associated with cell division were dramatically upregulated only in autonomously OSKM-expressing cells (Supplementary Fig. 4i). Together, from immunohistochemistry, we observed increased density of Ki67+ cells in the Epi-iOSKM epidermis compared to control (Supplementary Fig. 4j), which was an effect of mChpos cells, not mChneg cells; because the ratio of Ki67+ cells was higher in mCherry-expressing cells than their wildtype neighbors and control epidermal cells (Fig. 1k).

To infer the cell state dynamics of epithelial stem cells and their progeny in the control and Epi-iOSKM epidermis, we performed RNA velocity analysis on our single-cell RNA-seq dataset. In addition to the previously reported dynamics within Basal/Prolif cells and within Supra cells39, control IFE cells exhibited the cell fate transition from Basal to Supra cells, implying epidermal differentiation. In contrast to this, in mChpos and mChneg cells of the Epi-iOSKM epidermis, cell fate transitions from Supra to Basal cells were observed (Fig. 1l and Supplementary Fig. 4k). These transitions, indicative of IFE lineage dedifferentiation, were further supported by gene expression patterns. Cell fate transition trends of mChpos and mChneg cells in Epi-iOSKM epidermis were from high expression of differentiation markers such as Krt10 and Dsg1a to elevated expression of epidermal stem cell markers such as Krt14 and Itga6, reversing the differentiation trends observed in control IFE cells (Fig. 1m and Supplementary Fig. 4l–n). Moreover, only in mChpos cells, the trajectory from Supra to Prolif cells was identified (Fig. 1l). While control and Epi-iOSKM mChneg cells exhibited a trajectory from Mki67-positive to Mki67-negative states, mChpos cells exhibited the reversed pattern (Fig. 1n), indicating induced proliferation in a cell-autonomous manner. Collectively, these observations suggest that mosaic and transient OSKM expression drives IFE cell lineage-preserved partial dedifferentiation in both cell-autonomous and non-autonomous manners, but induces proliferation of IFE cells solely in a cell-autonomous manner (Fig. 1o).

Mosaic partial reprogramming triggers an epidermis-wide repair response in the absence of injury
To investigate how partial reprogramming and particularly its cell-non-autonomous effects influence the properties of Epi-iOSKM epidermis, we performed additional scRNA-seq by collecting all epidermal cells from control and Epi-iOSKM mice without fluorescence sorting. After identifying IFE cells (Supplementary Fig. 5a), we found altered cell subtype composition between control and Epi-iOSKM epidermis (Supplementary Fig. 5b). Notably, genes related to the cellular response to external stimuli were highly upregulated in the Epi-iOSKM epidermis (Supplementary Fig. 5c). Other DEGs—those associated with protein synthesis, programmed cell death, and differentiation—were also related to the response of the tissue against the external stimulant (Supplementary Fig. 5c)40,41. This suggests that short-term OSKM expression recapitulates the response of the epidermis to injury, which disrupts the epidermal barrier and exposes keratinocytes to external stimuli. We then confirmed these main changes of the Epi-iOSKM epidermis in our initial scRNA-seq data, in which mChpos and mChneg cells were separated. Most alterations in the response of cells to external stimuli were confirmed in the pure mChneg and mChpos populations, albeit to different degrees (Fig. 2a).

To compare mChneg and mChpos cells with epithelial cells under actual wounding conditions, we integrated our data with a publicly available reference scRNA-seq dataset containing IFE cells from wounded and unwounded skin39 using the anchor-based reciprocal principal components analysis (RPCA) (Fig. 2b). In addition to the cell types annotated in the original study, we identified a cluster of wound-responsive (W.R) cells unique to wounded conditions (Supplementary Fig. 6a). In the integrated map, nine distinct clusters were identified in an unbiased manner (clusters 0–8, Fig. 2b). The trend of proportional changes in each cluster was similar across the two experimental designs: transition from control to mChneg or mChpos populations, and from unwounded to wounded conditions. Cells in clusters 0, 1, 3, and 7 exhibited higher proportions in the control and unwounded epidermis than those in the other conditions, whereas the opposite trend was observed for clusters 2, 4, and 6 (Fig. 2c). Among them, cluster 6 was highly enriched with W.R cells from the reference scRNA-seq data, and Basal II and Supra II from our scRNA-seq data, indicating that the three cell types share common features (Fig. 2d, e). Notably, Krt17, Krt16, and Krt6a, which are markers of active keratinocytes induced in injured skin42, were highly expressed in cluster 6 (Fig. 2f and Supplementary Fig. 6b, c). Their expression was upregulated in mChneg and mChpos cells from the Epi-iOSKM epidermis compared to control epidermis (Fig. 2g and Supplementary Fig. 6d). Krt17 and Krt6a protein expression was also increased in mChpos and mChneg cells of the Epi-iOSKM epidermis (Fig. 2h, i).

Wound repair progresses in several stages: the early response (including hemostasis and inflammation), proliferation (including re-epithelialization), and remodeling (Supplementary Fig. 6e)2,43. In the early response phase, the skin epidermis recognizes external stressors via hypoxia and inflammatory pathways (Supplementary Fig. 6f)44–46. Interestingly, even without external stressors, partial epidermal reprogramming enhanced hypoxia-associated pathways and cytokine-mediated inflammatory pathways in both cell-autonomous and non-autonomous manners (Fig. 2j, k). Consistently, the levels of HIF-1α and phospho-STAT3, hallmarks for hypoxia and inflammatory pathways respectively44,45,47, were increased in partially reprogrammed epidermis (Fig. 2l, m). Our interpretation of these findings is that the conversion of epidermal cells by partial reprogramming recapitulates the early response phase of wound repair, in addition to the following proliferation stage, in which Krt17+ and Krt6+ active keratinocyte function. Collectively, molecular alterations upon partial reprogramming mimic the skin repair processes despite the absence of injury in both cell-autonomous and non-autonomous manners.

Partial reprogramming confers plasticity within epidermal lineages
During repair, injury-responsive IFE cells acquire plasticity—the ability of cells to change their properties in response to the environmental cues (Supplementary Fig. 7a). After regeneration, the plastic cells lose their characteristics, such as Krt17/6a expression, and return to the original stable state (Supplementary Fig. 7b–d). Similarly, we confirmed that Epi-iOSKM epidermis acquired increased differentiation potency in comparison to control epidermis (Supplementary Fig. 7e). In addition, after Dox withdrawal for 10 days, Krt17 and Krt6a upregulation by transient OSKM expression was reversed in the Epi-iOSKM epidermis (Supplementary Fig. 7f, g), with the epidermis returning to a homeostatic state (Supplementary Fig. 7h). The fact that conversion under partial reprogramming is reversible and the tissue reacquires specific epidermal properties, as in the repair process, indicates that partial reprogramming imposes the potential only to become epidermal cells, and no other cell types in the in vivo microenvironment, unlike complete reprogramming-conferred plasticity to become multiple kinds of cells (Fig. 2n). The expression of IFE cell markers, and lack of expression of the pluripotent cell marker Nanog in the Epi-iOSKM epidermis after 3 days Dox treatment (Supplementary Figs. 2a and 7i–k) support our partial reprogramming model proposing induced cellular plasticity only within IFE cell lineages.

Cell-autonomous PI3K activation is necessary for the partial epidermal reprogramming
We then aimed to elucidate how partial reprogramming results in injury-responsive-like conversion of IFE cells. Firstly, we focused on the transition of OSKM-expressing mChpos cells. Because increased proliferation was only observed in these, and not in mChneg cells (Fig. 1j, k), we investigated Prolif cells from the scRNA-seq data by analyzing enriched pathways from DEGs that changed only in mChpos cells of the Epi-iOSKM epidermis. We found that the expression of genes involved in signaling pathways regulating pluripotency of stem cells was altered (Fig. 3a). We focused on these genes as candidates to elucidate cell-autonomous effects because induction of pluripotency is a major direct result of prolonged OSKM expression17–19, and it can explain the observed increase in proliferation in a cell-autonomous manner. Besides Pou5f1, Sox2, and c-Myc upregulation, which were directly induced as part of OSKM, we observed the downregulation of six genes associated with signaling pathways that induce cell proliferation (Fig. 3b). Of these, only Pik3r1, encoding the phosphoinositide 3-kinase (PI3K) subunit P85α, could account for the activation of the relevant signaling, because P85α inhibits PI3K activity (Fig. 3c)48,49. Pik3r1 gene expression in mChpos cells from the Epi-iOSKM epidermis was downregulated not only in Prolif, but also in Basal and Supra IFE cells (Fig. 3d). Besides Pik3r1, we investigated the expression of other genes that regulate PI3K activity and found that the expression of Shb, which activates PI3K (Fig. 3c)50,51, was upregulated in mChpos IFE cells. The expression of Pik3r1 and Shb was not altered in mChneg cells of the Epi-iOSKM epidermis (Fig. 3d). Next, we examined the changes induced in well-known cellular pathways upon partial reprogramming52 and observed a higher activation of the PI3K pathway in mChpos cells (Fig. 3e). Specifically, unlike other pathways activated in the Epi-iOSKM epidermis, such as the JAK-STAT, epidermal growth factor receptor (EGFR), and MAPK pathways, the PI3K pathway was not affected in mChneg cells (Fig. 3e; Supplementary Fig. 8a). Further, using immunohistochemistry, we observed increased AKT phosphorylation, a hallmark of PI3K pathway activation53, only in mChpos cells of Epi-iOSKM epidermis (Fig. 3f), confirming that PI3K activation was a cell-autonomous effect of OSKM expression. Well-known target genes of FOXO transcription factors, inhibited by the PI3K-AKT pathway, such as a cell cycle-related gene, Ccng2 (ref.54), and a modulator of the negative feedback loop of the pathway, Irs1 (ref.55), were downregulated in mChpos IFE cells, supporting PI3K downstream activation (Supplementary Fig. 8b).

Subsequently, we assessed whether the PI3K pathway functions upstream of injury-responsive-like molecular changes induced by transient OSKM activation. The dorsal skin of Epi-iOSKM mice was topically treated with the PI3K inhibitor LY29400256,57. This reversed some effects of the partial epidermal reprogramming: HIF-1α activation as part of the wound-responsive-like response in the epidermis of Epi-iOSKM mice was attenuated upon LY294002 treatment (Fig. 3g and Supplementary Fig. 8c). The upregulation of Krt17 and Krt6a, epithelial cell markers in wound repair, was also reversed (Fig. 3h and Supplementary Fig. 8d). Moreover, an increase in cell proliferation, which is a cell-autonomous effect of partial reprogramming, was attenuated by PI3K inhibition (Fig. 3i).

A previous study reported that the PI3K pathway is activated during wound healing58. We performed immunohistochemistry at several time points during wound healing, and observed increased AKT phosphorylation at 3- and 7-days post-wounding (dpw) (Supplementary Fig. 8e). However, phospho-AKT was not detected until 1 dpw (Supplementary Fig. 8f), corresponding to the beginning of the early response phase of wound repair. In contrast, HIF-1α expression was induced immediately upon wounding within 3 hours (Supplementary Fig. 8g) even in the absence of detectable phospho-AKT (Supplementary Fig. 8f). This suggests that the early response to the wounding via the HIF-1α pathway is independent of PI3K activation (Supplementary Fig. 8h). Thus, we conclude that partial reprogramming via cell-autonomous PI3K activation promotes wound-responsive-like pathways (Fig. 3j) but is distinct from the early response of keratinocytes to wounding.

EGFR signaling propagates the effects of partially reprogrammed cells to the neighbors
We next moved our focus to the conversion of neighboring cells. To test whether the changes in mChneg populations in the Epi-iOSKM epidermis were caused by direct effects of partially reprogrammed keratinocytes, we performed cell-cell interaction (CCI) analysis on our scRNA-seq data from IFE cells. Ligand-receptor interactions between IFE cells were modified by partial reprogramming (Supplementary Fig. 9a). Notably, signaling pathways associated with the ECM, such as FN1 and KLK, those activating EGFR, such as EPGN and EGF, and those promoting EGFR/ErbB3 complex signaling, such as NRG59, were significantly activated in the Epi-iOSKM epidermis (Fig. 4a and Supplementary Fig. 9b).

Next, we examined how OSKM-expressing cells modify cellular communication within the epidermis. We identified upregulated DEGs in mChpos IFE cells compared to controls, focusing on genes encoding proteins in the extracellular space that could potentially mediate CCIs (Fig. 4b). Analysis using both the REACTOME pathway database and the SMART protein domain database revealed a significant upregulation of EGFR-related genes (Fig. 4c). The expression of Epgn, Tgfa, and Areg, which encode ligands that activate EGFR, was upregulated in the Epi-iOSKM epidermis (Fig. 4d). We confirmed both cell-autonomous and non-autonomous activation of EGFR signaling in the epidermis of Epi-iOSKM mice by pathway analysis of scRNA-seq data (Fig. 4e) and immunohistochemistry of phospho-EGFR (Fig. 4f). The MAPK and JAK-STAT pathways, which are downstream signaling pathways of EGFR60, were similarly activated (Supplementary Fig. 9c).

EGFR signaling is well known as a key mechanism activated during skin repair to induce epidermal proliferation and migration61–63. Additionally, EGFR activation is known to upregulate HIF-1α and Krt1764–67. To assess whether EGFR signaling activation in mChpos and mChneg cells contributed to healing features induced by mosaic partial epidermal reprogramming, we topically treated the dorsal skin of Epi-iOSKM mice with AG1478, an EGFR inhibitor (Supplementary Fig. 9d). Upregulation of HIF-1α and Krt17, representative markers for wound-responsive-like activation, was significantly reversed by EGFR inhibition in the Epi-iOSKM epidermis (Fig. 4g, h and Supplementary Fig. 9e), demonstrating that EGFR signaling is essential to acquire healing features. For the next question, we investigated how EGFR signaling was activated in the Epi-iOSKM epidermis. We found that PI3K activation in mChpos cells is necessary for EGFR activation in the Epi-iOSKM epidermis (Fig. 4i). Furthermore, treatment of cultured keratinocytes with a PI3K activator induced EGFR activation (Supplementary Fig. 9f). These demonstrate that PI3K activation in OSKM-expressing IFE cells conferred healing characteristics to themselves, and activated EGFR signaling to induce healing features not only to themselves but also to their neighbors (Fig. 4j).

EGFR signaling in concert with WNT inhibition accelerates repair without altering proliferation
The cellular pathways identified in the Epi-iOSKM epidermis were associated with increased proliferation; EGF signaling, a key modulator for keratinocyte proliferation, as well as IL1 and VEGF signaling that can indirectly contribute, were significantly enriched60,62,63,68,69 (Fig. 4b, d). However, we did not observe any changes in the proliferation of mChneg cells (Fig. 1j, k). We hypothesized that an additional factor might regulate cell proliferation of mChneg cells. PROGENy pathway analysis revealed that WNT signaling was the only pathway showing significantly different activity levels in mChneg cells, but not in mChpos cells, of Epi-iOSKM mice compared to control (Fig. 3e). WNT signaling was downregulated in mChneg cells of Epi-iOSKM mice compared to both control epidermal cells and mChpos cells of Epi-iOSKM epidermis (Supplementary Fig. 10a).

WNT signaling, known to activate cell proliferation70, emerged as a promising candidate regulating the proliferation of mChneg cells. To investigate how WNT signaling was inhibited in wild type cells of Epi-iOSKM epidermis, we examined the RNA levels of secreted WNT regulatory molecules, which can broadly modulate the WNT pathway activity in the epidermis. First, we found that the expression of Wnt4, Wnt6, Wnt16, and Rspo3, a Wnt signaling enhancer70,71, was lower in IFE cells of the Epi-iOSKM epidermis than in control epidermis (Supplementary Fig. 10b). Next, we examined the expression of well-known WNT inhibitory molecules. Among these, the number of cells expressing Sfrp1 was much higher than those expressing other inhibitory genes in the control epidermis, suggesting that Sfrp1 is the major inhibitory modulator of WNT ligand activity in the epidermis (Supplementary Fig. 10c)72,73. The expression of Sfrp1, encoding the key repressor protein, significantly increased in mChpos cells (Supplementary Fig. 10c). Collectively, the gene expression of secreted proteins that modulate WNT signaling activity was altered toward a tendency to suppress the signal (Supplementary Fig. 10d).

To examine the effects of EGF activation in combination with WNT inhibition, we treated HaCaT human keratinocytes with EGF and the WNT secretion inhibitor LGK97474. This condition recapitulates the in vivo microenvironment of mChneg cells in the Epi-iOSKM epidermis, consisting of high EGF and low WNT ligand concentrations. Keratinocyte proliferation was increased by treatment with EGF alone but was not affected by treatment with both EGF and LGK974 (Supplementary Fig. 10e). Conversely, the wound healing rates of in vitro scratch wounds were promoted by the treatment of EGF with or without LGK974 (Supplementary Fig. 10f). These findings indicate that simultaneous EGF treatment and WNT inhibition switch keratinocytes to a state beneficial for healing while avoiding an increase in cell proliferation.

In conclusion, unlike mChpos cells, which acquired healing features and increased proliferative ability via activation of PI3K and EGFR signaling pathway, mChneg cells exhibited: (1) healing features via upregulation of the EGFR pathway, (2) downregulation of the WNT pathway, and (3) no changes in proliferation (Supplementary Fig. 10g). These findings suggest that EGFR pathway activation, combined with WNT pathway inhibition in mChneg epidermal cells of Epi-iOSKM mice converted them into a wound-responsive-like, but non-proliferative state.

Partial epidermal reprogramming activates immune niches
Having shown that partial reprogramming of IFE cells can influence the surrounding environment by inducing cellular plasticity in neighboring keratinocytes, similar to injury responses, we next determined whether and how partial reprogramming affects other cell types. We specifically focused on immune cells, as our REACTOME analysis showed that chemokines known to modulate skin immunity were significantly upregulated in mChpos cells of the Epi-iOSKM epidermis (Fig. 4c and Supplementary Fig. 11a). Also, keratinocytes activated by skin injury are known to promote T cell immunity75–77. T-cell responses, in turn, reciprocally activate epidermal keratinocytes, contributing to wound re-epithelialization (Fig. 5a)75–79.

We found that partial epidermal reprogramming, which we propose to resemble the wound repair process, promotes T-cell immunity in the skin. Specifically, the proportion of T cells in the epidermis and the numbers of both epidermal and dermal T cells increased in the skin of Epi-iOSKM mice (Fig. 5b, c and Supplementary Fig. 11b) without a corresponding increase in T cell proliferation (Supplementary Fig. 11c, d), suggesting that partial epidermal reprogramming primarily promotes T cell recruitment. Furthermore, the ratio of rounded T cells in the epidermis—activated epidermal T cells known to be crucial for wound re-epithelialization—47,75,76,80, also increased (Fig. 5c). To identify how the partially reprogrammed epithelial cells acted on immune cells, we analyzed increased cytokines after wounding using the reference scRNA-seq data containing IFE cells from wounded and unwounded skin (Fig. 5d)39. We then assessed how their expression was altered by partial reprogramming of IFE cells. Notably, genes encoding ligands reported to activate dendritic epidermal T cells (DETCs) and dermal γδ T cells during injury repair, including Ccl8, Ccl20, Cxcl10, Cxcl16, Il1b, and Tnf81–89, were upregulated upon partial epidermal reprogramming (Fig. 5e and Supplementary Fig. 11e–g).

Moreover, the skin of Epi-iOSKM mice mimicked aspects of the immune niche in the repair state. Among the immune cytokines, Ccl20 exhibited the greatest increase in expression (Fig. 5e and Supplementary Fig. 11e–h). CCL20 is a chemokine that attracts T cells expressing its sole receptor, CCR6. During repair, the CCL20-CCR6 axis is activated, recruiting RORγt+ T cells near the wound edge82,84,90,91, to induce re-epithelialization79. Similarly, in the skin of Epi-iOSKM mice, the number of RORγt+ cells increased (Fig. 5f). Furthermore, intradermal injection of a CCR6 antagonist reversed the increase of RORγt+ cells in Epi-iOSKM skin (Fig. 5f). This indicates that partial epidermal reprogramming increased RORγt+ T cells in the skin via the CCL20-CCR6 axis (Fig. 5g). Collectively, partially reprogrammed epithelial cells not only had similar molecular and cellular identities to the injured epithelium (Figs. 2–4) but also functioned similarly in epithelial-immune crosstalk (Fig. 5).

Prolonged OSKM expression leads to epidermal hyperplasia accompanied by skin inflammation
Previous studies have reported that in vivo OSKM induction leads to detrimental effects including premature death and teratoma formation20,21, but we did not observe any deleterious effects, such as mortality and tumorigenesis, in our Epi-iOSKM mice after Dox treatment for 3 days (Fig. 5h and Supplementary Fig. 12a). To investigate the effects of prolonged OSKM expression in the epidermis, we administered Dox for 9 days in Epi-iOSKM mice. These mice exhibited pathogenic skin morphologies, including swelling of the facial skin, bumps and rashes in the dorsal skin, and exfoliated or swollen paws (Fig. 5h). No significant changes were observed in the other organs, and none of the mice died (Supplementary Fig. 12a). Furthermore, we examined the potential for teratoma development but did not observe tumor formation in the skin or the expression of Nanog, a pluripotent stem cell marker92, or Krt18, an epidermal cancer marker (Supplementary Fig. 12b, c)93.

We have shown that the activation of epithelial-immune crosstalk in the Epi-iOSKM skin resembles processes during wound healing. Notably, these interactions are also excessively activated in certain skin inflammatory diseases (Fig. 5a)4,79,94–96. Immunostaining for Krt17, Krt6a, and Ki67, observed in hyperimmune epidermis95,97, revealed a significant increase in the number of IFE cells expressing these proteins in the skin of Epi-iOSKM mice after 9 days of Dox treatment (Supplementary Fig. 12d–f). Although tumors did not develop, prolonged OSKM expression resulted in the thickening of the dorsal epidermis and the stratum corneum (Fig. 5i). Additionally, the number of T cells in the dermis dramatically increased (Fig. 5j). Moreover, following 10 days of Dox withdrawal after a 9-day administration period, the mice exhibited other pathologies, such as dry skin, scaly ears, and hair loss, along with abnormal skin histology characterized by excessive thickening of the stratum corneum and an increased number of CD3+ T cells in both the epidermis and dermis (Supplementary Fig. 12g–i). These persistent inflammatory phenotypes were recovered after 30 days of Dox withdrawal (Supplementary Fig. 12j, k), and pluripotency was still not induced (Supplementary Fig. 12l), suggesting that 9 days of Dox administration caused transient disease phenotypes without fatal consequences. Collectively, prolonged OSKM expression in the epidermis resulted in inflammatory skin pathologies, whereas transient mosaic partial reprogramming induced only regenerative characteristics without adverse effects.

Partial reprogramming promotes re-epithelialization
Since we had shown that short-term partial reprogramming of epithelial cells induces the skin to exhibit healing characteristics in several ways, we next examined whether this approach improves skin repair upon actual injury. To assess this, we created 6-mm splinted full-thickness excisional wounds, which induce healing kinetics similar to those of humans by preventing the rapid wound contraction commonly observed in mice with loose skin98, during short Dox administration for 3 days (Fig. 6a). This revealed that partial epidermal reprogramming accelerated re-epithelialization, as evidenced by significantly increased lengths of the migrating epithelial tongues at 10 dpw (Fig. 6b). Additionally, wound closure rates were increased in Epi-iOSKM mice, as confirmed by a decrease in the number of days reaching 50% of the wound area (wound half-life) from control = 10.58 to Epi-iOSKM = 9.15 (Supplementary Fig. 13a). Functionally, trans-epidermal water loss, an indicator of epidermal barrier function, was reduced, further supporting enhanced healing outcomes (Fig. 6c).

Partial epidermal reprogramming reduces scar formation
During cutaneous wound repair, epidermal stem cells and their niches in the dermis actively interact with each other2,10,12,44,45,75. Interestingly, granulation tissue formation was promoted in Epi-iOSKM mice compared to controls at 3 and 10 dpw (Supplementary Fig. 13b, c), suggesting partial epidermal reprogramming enhances dermal healing. Thus, we next examined how dermal scarring was influenced in Epi-iOSKM skin. After wound closure, at 20 dpw, the increase in dermal thickness associated with scarring was diminished in the skin of the Epi-iOSKM mice (Fig. 6d). Epidermal thickness also decreased (Supplementary Fig. 13d). In addition, the scars formed in Epi-iOSKM mice had intermediate characteristics between unwounded healthy dermal tissues and scars from control mice (Fig. 6e), suggesting enhanced dermal healing. Consistently, the scarred areas of Epi-iOSKM mice contained more mature collagen fibers that demonstrated a complex structure similar to the normal dermis before wounding (Supplementary Fig. 13e)99. Together, the amount of collagen deposition, as detected by aniline blue signals in trichrome staining100,101, decreased in Epi-iOSKM mice, indicating a reduction in scar formation (Supplementary Fig. 13f). Immunohistochemical analysis further revealed that collagen III, the immature and thin collagen102 that only appeared in scars and was not found in unwounded skins, was significantly less present in scarred areas of Epi-iOSKM mice than those of control, supporting less scarring in Epi-iOSKM mice (Fig. 6f and Supplementary Fig. 13g). The percentage of collagen I+ area, the mature and thick collagen103, in the wounded dermal area did not change significantly, but the relative amount of collagen I deposition, normalized to epidermal lengths, was reduced in Epi-iOSKM mice (Supplementary Fig. 13h). Taken together, the ratio of collagen III to collagen I was significantly decreased in Epi-iOSKM mice, suggesting that immature collagen III, typically observed in skin undergoing repair, was converted more rapidly to mature collagen I, which is found in the repaired skin (Supplementary Fig. 13i)104. Collectively, mosaic partial reprogramming of epithelial cells in the skin accelerated re-epithelialization and reduced dermal scarring, thereby broadly enhancing skin repair.

Extensive alterations in wound repair processes induced by partial epidermal reprogramming
We next investigated the mechanistic link between partial epidermal reprogramming and the enhancement of epidermal and dermal repair. Our first question was how transient partial reprogramming until 1 dpw could promote healing at later stages (10–20 dpw). The second question was how partial reprogramming of the epidermis influences dermal scarring. To elucidate how the effects of partial epidermal reprogramming may expand spatiotemporally, we examined changes across multiple cell types in Epi-iOSKM skin during intermediate stages of wound repair (Fig. 7a and Supplementary Fig. 14a).

First, we examined alterations in the epidermis during healing. While partial epidermal reprogramming induced cell-autonomous proliferation under homeostatic conditions (Fig. 1j, k), the number of proliferating IFE cells was not altered in Epi-iOSKM skin during wound repair (Supplementary Fig. 14b). In contrast, the levels of HIF-1α, which we had found to be increased by partial reprogramming in cell-autonomous and non-autonomous manners (Figs. 2l and 7b), were still elevated near the wound edges at 3 and 7 dpw in Epi-iOSKM mice (Fig. 7c and Supplementary Fig. 14c), even though Dox administration had been stopped. Notably, HIF-1α levels were more increased near old epidermal cells that had experienced partial reprogramming than in regenerated migrating epithelial tongues (Fig. 7c, d). This was surprising because in the absence of wounding, HIF-1α activation in Epi-iOSKM epidermis disappeared after Dox withdrawal for 6 days (Fig. 7e). The different reversibility of HIF-1α activation between no wounding and wounding suggests that partially reprogrammed cells synergize with actual wounds to maintain and enhance the reversible effect observed without wounding.

We next examined dermal alterations in healing Epi-iOSKM skin. During wound healing, three main types of dermal niches interplay with epidermal cells: mesenchymal, immune, and vascular cells. We first assessed the differentiation of mesenchymal cells into myofibroblasts, but detected no notable changes in immunohistochemical analysis for α-SMA, a marker of myofibroblasts (Supplementary Fig. 14d). We then investigated alterations in immune niches. Macrophage infiltrations were unaffected by partial epidermal reprogramming (Supplementary Fig. 14e). However, T cell numbers in wounded area were increased. At 3 dpw, the number of epidermal T cells located proximal to the wound edge was higher in the Epi-iOSKM skin than in the control skin (Supplementary Fig. 14f). At 10 dpw, there were more infiltrated T cells in the wounded area of Epi-iOSKM mice (Supplementary Fig. 14g).

Lastly, we examined the vascular niche by performing immunohistochemical analysis for CD31, a marker for endothelial cells. Interestingly, the pattern of angiogenesis was significantly altered following partial epidermal reprogramming (Fig. 7f–h), correlating with earlier changes in HIF-1α signaling (Fig. 7b–d), which is known to promote neovascularization. In the dermis near the initial wound site, blood vessels in Epi-iOSKM skin were more closely localized to the epidermis at 10 dpw (Fig. 7f, g), consistent with the increase of HIF-1α in the same area at earlier time points (Fig. 7b–d). In the wound bed near the newly generated epithelium, control mice exhibited higher HIF-1α levels compared to the initial wound site during earlier stages of healing (Fig. 7b–d). In contrast, in Epi-iOSKM mice, HIF-1α signals were comparable near the initial wound site and the newly generated epithelium, with elevated levels at both locations (Fig. 7b–d). By 10 dpw, Epi-iOSKM skin, which did not exhibit different HIF-1α levels according to the location, showed a dramatic reduction in neovascularization in the dermis near the leading edge of migrating tongues compared with controls (Fig. 7f, h). Despite decreased neovascularization at 10 dpw in the wounded area near leading edges, after wound closure, CD31+ cell areas in the dermis between control and Epi-iOSKM mice became similar (Supplementary Fig. 14h), suggesting that angiogenesis in Epi-iOSKM skin was not completely blocked but instead occurred in an altered pattern.

HIF-1α activation is essential for enhancing wound healing and attenuating scar formation in Epi-iOSKM skin
Previous studies have reported that forced activation of HIF-1α accelerates re-epithelialization105–107. In our model, HIF-1α activation induced by partial reprogramming was sustained after wounding (Fig. 7c), suggesting it may continue to promote re-epithelialization even after transient reprogramming has ceased. Moreover, the altered patterns of HIF-1α activation correlated with reorganized angiogenesis (Fig. 7b–h), potentially influencing dermal repair. Notably, appropriate regulation of neovascularization during wound repair is known to reduce scar formation108–111.

When a wound occurs in Epi-iOSKM mice, HIF-1α is activated twice in the epidermis—first by partial reprogramming and again by the wound itself. To disrupt the synergistic effect of these two stimuli, we transiently inhibited HIF-1α activity by treating the HIF-1α inhibitor PX-478 once at the time of wounding (Fig. 7i). HIF-1α activity decreased at 1 dpw but recovered by 10 dpw as expected (Supplementary Fig. 15a, b). Interestingly, transient HIF-1α inhibition during the early repair phase reversed the accelerated re-epithelialization in Epi-iOSKM mice, bringing it to levels comparable to controls (Fig. 7j, k). Moreover, temporal inhibition of HIF-1α markedly reduced angiogenesis. Without inhibition, control mice displayed widespread angiogenesis across the wound bed, whereas Epi-iOSKM mice showed angiogenesis concentrated near the original wound site (Fig. 7f–h). Following PX-478 treatment, however, both groups exhibited a pronounced reduction in angiogenesis, with low CD31 expression at newly formed dermis near both the initial wound sites and leading edges (Fig. 7j, l and Supplementary Fig. 15c, d). These findings indicate that HIF-1α is a key regulator of neovascularization patterns during wound repair in both control and Epi-iOSKM mice.

We next examined whether HIF-1α, which is essential for the alterations in re-epithelization and angiogenesis in Epi-iOSKM mice, also contributes to scar reduction. At 20 dpw, dermal thickness in the scarred area of PX-478 treated Epi-iOSKM mice was significantly thicker than that that of untreated Epi-iOSKM mice and was comparable to that of PX-478 treated control mice (Fig. 7m). Consistently, the alterations in collagen I deposition observed in Epi-iOSKM skin were restored by PX-478 treatment (Supplementary Fig. 15e). In addition, the reduced proportion of collagen III+ area observed in Epi-iOSKM mice was reversed by HIF-1α inhibition during partial epidermal reprogramming (Fig. 7n), indicating that elevated HIF-1α levels are essential for the reduction in scarring. Collectively, increased HIF-1α activation upon partial epidermal reprogramming—particularly the atypical pattern observed in the old epidermis near the initial wound site rather than in the newly generated epithelium (Fig. 7b–d)—substantially contributes to the widespread changes that promote wound repair.

Partial epidermal reprogramming enhances impaired wound healing under hyperglycemic conditions
We next investigated potential clinical implications by generating a mouse model of hyperglycemia, the most widely used model for impaired wound healing, through streptozotocin injection into male mice112 (Fig. 8a and Supplementary Fig. 16a). This disease model exhibited decelerated wound closure (Fig. 8b). Consistent with our findings in wild-type mice, short-term mosaic OSKM induction in diabetic mice accelerated wound closure compared with diabetic controls. At 11 dpw, wound areas were significantly smaller in diabetic Epi-iOSKM mice than in diabetic controls, bringing them to levels comparable to those of vehicle-treated controls (Fig. 8b).

It is known that hyperglycemia impairs cell proliferative capacity113, and consistently, our diabetic model exhibited reduced epidermal cell proliferation. Interestingly, this defect was restored in Epi-iOSKM mice (Fig. 8c). These suggests that the effects of partial epidermal reprogramming on diabetic wound healing differ from those observed in the wild type healing model: in the normal healing, partial reprogramming did not alter epidermal proliferation during repair (Supplementary Fig. 14b). We next evaluated HIF-1α activity, another key factor for epithelial activation. HIF-1α levels were elevated in diabetic Epi-iOSKM mice compared with diabetic controls, similar to the effects observed in the non-diabetic condition (Supplementary Fig. 16b). Collectively, these results indicate that partial epidermal reprogramming can counteract diabetes-induced impairments in epidermal repair.

We additionally found that hyperglycemia impaired re-epithelialization, neovascularization, and granulation tissue formation after wounding, consistent with previous reports113–115. These defects were also restored by partial epidermal reprogramming (Fig. 8d–f and Supplementary Fig. 16c, d). Diabetic Epi-iOSKM mice exhibited faster re-epithelialization than diabetic controls (Fig. 8d, e), demonstrating that the beneficial effects observed in normal mice (Fig. 6b) can also be applied in a disease context. In contrast, in the case of angiogenesis, the effects of partial epidermal reprogramming differed under hyperglycemic conditions, which are characterized by a marked reduction in vascularization. The total CD31+ area in the dermis increased in diabetic Epi-iOSKM mice compared to diabetic controls, partially rescuing the vascularization defect (Fig. 8d, f), in contrast to the non-diabetic condition, where Epi-iOSKM mice exhibited an altered pattern of neovascularization (Fig. 7f–h) but no changes in the total amount (Fig. 7l). We next examined collagen deposition after wound closure under hyperglycemic conditions but did not detect significant differences between Epi-iOSKM and control skin (Supplementary Fig. 16e–g). However, decreased granulation tissue formation observed in diabetes was recovered by partial epidermal reprogramming (Supplementary Fig. 16d). Collectively, under impaired healing conditions by diabetes, partial epidermal reprogramming exhibited distinct effects compared to normal conditions, ultimately improving the defective healing processes (Fig. 8g).

Methods
Animals
K14-CreERT (JAX Strain #005107)123, ROSA26-rtTA(neo);OKMSCh250 (JAX Strain #031012)33 mice were obtained from Jackson Laboratory. Epi-iOSKM mice and their littermates as controls, used for each experiment, were obtained by crossing K14-CreERT and ROSA26-rtTA(neo);OKMSCh250 mice. All experiments were performed on 7–9-week-old mice in the 2nd telogen phase of the hair growth cycle. All control mice received the same doses of tamoxifen and doxycycline as the Epi-iOSKM mice. Both male and female mice were used for all experiments except the scRNA-seq experiments and hyperglycemic disease modeling, which were performed on male mice. All mice were bred and maintained in the animal care facility of the Pohang University of Science and Technology (POSTECH) Biotech Center per the institutional ethical guidelines. Mice were group-housed with bedding and a red hut under specific pathogen-free conditions, maintained on a 12-h light-dark cycle at an ambient temperature of 22 °C and 45–65% relative humidity. All animal protocols and experiments were approved by the POSTECH Institutional Animal Care and Use Committee (POSTECH-2021-0115, POSTECH-2022-0080, POSTECH-2023-0067, POSTECH-2023-0156 and POSTECH-2024-0140).

Cell lines
HaCaT human keratinocytes124 were donated by Dr. Kyong-Tai Kim (POSTECH). HaCaT cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with high glucose (4.5 g/L glucose, 4 mM L-glutamine, sodium pyruvate; Cytiva, SH30243.01) supplemented with 10% fetal bovine serum (FBS, Corning, 35-015-CV) and 100 U/ml penicillin-streptomycin (Gibco, 15140122) using a cell culture incubator maintaining at 37 °C in a 5% CO2 atmosphere. For the PI3K activation experiment, cells were serum-starved in DMEM with high glucose containing 1% FBS for 5 h, then treated with 10 µM 740 Y-P (MedChemExpress, HY-P0175) or 0.1% DMSO for 24 h at 37 °C. For EdU cell proliferation assay, cells were serum-starved in DMEM with high glucose containing 1% FBS for 12 h, then treated with 20 ng/mL EGF (Gibco, PMG8043) or 20 µM LGK974 (MedChemExpress, HY-17545) for 24 h at 37 °C.

Chemical treatment
Tamoxifen (Sigma-Aldrich, T5648) was dissolved in corn oil to a final concentration of 20 mg/ml and used to induce CreER. Tamoxifen was intraperitoneally injected into Epi-iOSKM mice and sex-matched littermate controls once daily for 6 days. To induce OSKM, control and Epi-iOSKM mice were fed 2 mg/ml doxycycline (Dox, Sigma-Aldrich, D9891) in drinking water (supplemented with 5% sucrose) and administered 2 mg/ml Dox dissolved in water by oral gavage every 2 days. For the short-term induction of OSKM, mice were treated with Dox in drinking water for 3 days and orally gavaged with Dox in water on days 0 and 2 after starting Dox treatment. For prolonged induction of OSKM, mice were administered Dox in drinking water for 9 days and orally treated with Dox in water on days 0, 2, 4, 6, and 8. To inhibit PI3K pathway, control and Epi-iOSKM mice were topically treated with 80 nmol of LY294002 (MedChemExpress, HY-10108) in acetone (Sigma-Aldrich, 179124) on the right side of the dorsal skin, and vehicle (acetone) alone on the left side of the dorsal skin once a day on days −1, 0, and 2 of Dox treatment. To inhibit EGFR pathway, control and Epi-iOSKM mice were topically treated with 50 µg of AG1478 (Sigma-Aldrich, 658552) in acetone on the right side of the dorsal skin, and vehicle (acetone) alone on the left side of the dorsal skin twice on days 0 and 2 of Dox treatment. The CCR6 antagonist 1 (MedChemExpress, HY-151435) was dissolved to a final concentration of 125 mg/ml in 1% DMSO in saline and injected intradermally into the dorsal skin of control and Epi-iOSKM mice twice a day for 4 days during Dox treatment. To inhibit HIF-1α, control and Epi-iOSKM mice were topically treated at the wounded site with 10 mg/kg and intraperitoneally injected with 40 mg/kg of PX-478 (MedChemExpress, HY-10231) in saline, 20 min prior to wounding.

Histology and immunostaining
Freshly harvested skin tissues were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, 50-980-487) for 15 min and washed in PBS for at least 3 h at room temperature. For cryo-embedding, skin tissues were put into 30% sucrose/PBS overnight at 4 °C, then embedded in the OCT compound (Sakura Finetek, 4583) and stored at −80 °C until cryo-sectioning step. Cryosections of 14–35 µm in thickness were collected on positively charged glass slides (Globe Scientific Inc., 1358W) and stored at −80 °C until use. For immunostaining, sections were fixed in 4% PFA for 2 min and washed with PBS and 0.1% Triton X-100 in PBS. The slides were then blocked in a blocking buffer (1% bovine serum albumin, 5% donkey serum, and 2% cold-water fish gelatin in 0.3% Triton X-100 in PBS) for 1 h at room temperature. Then the sections were incubated with primary antibodies overnight at 4 °C, and incubated with secondary antibodies for 2–4 h at room temperature. After washing at least three times for 30 min with PBS, the sections were mounted in DAPI Fluoromount-G (SouthenBiotech, 0100-20). For phospho-EGFR staining, sections were permeabilized with 0.3% H2O2 in methanol at −20 °C for 30 min. The following antibodies were used for immunofluorescence in this study at the dilutions indicated: rabbit anti-GFP (Abcam, ab290, 1:500), rat anti-RFP (ChromoTek, 5f8-150, 1:100), rabbit anti-Sox2 (Abcam, ab97959, 1:500), rabbit anti-Oct4 (Abcam, ab181557, 1:200), rabbit anti-Klf4 (Abcam, ab129473, 1:200), rabbit anti-c-Myc (Abcam, ab32072, 1:100), rabbit anti-Cleaved Caspase-3 (Cell Signaling Technology, 9661S, 1:200), rabbit anti-Krt10 (Biolegend, 905404, 1:500), rabbit anti-Krt14 (Biolegend, 905304, 1:500), chicken anti-Krt14 (Biolegend, 906004, 1:500), rabbit anti-Ki-67 (Cell Signaling Technology, 12202S, 1:500), rabbit anti-Krt17 (Abcam, ab53707, 1:500), rabbit anti-Krt6a (Biolegend, 905701, 1:500), rabbit anti-HIF-1α (Cell Signaling Technology, 36169S, 1:100), rabbit anti-phospho-STAT3 (Cell Signaling Technology, 9145S, 1:100), rabbit anti-phospho-AKT (Cell Signaling Technology, 4060S, 1:200), rabbit anti-phospho-EGFR (Cell Signaling Technology, 3777S, 1:200), rat anti-CD3 (Thermo Fisher Scientific, 14-0032-82, 1:100), rat anti-RORγt (Thermo Fisher Scientific, 14-6988-82, 1:100), rabbit anti-Krt18 (Sigma-Aldrich, HPA001605, 1:200), rabbit anti-Nanog (Cell Signaling Technology, 8822S, 1:200), rabbit anti-α-SMA (Abcam, ab5694, 1:500), rat anti-F4/80 (Biorad, MCA497R, 1:200), rat anti-CD31 (Biolegend, 102502, 1:200), rabbit anti-Collagen I (Abcam, ab21286, 1:100), rabbit anti-Collagen III (Abcam, ab7778, 1:50), donkey polyclonal Cy3 anti-Rat IgG antibody (Jackson ImmunoResearch Labs, 712-165-153, 1:400), donkey polyclonal Alexa Fluor 488 anti-Rabbit IgG antibody (Jackson ImmunoResearch Labs, 711-545-152), donkey polyclonal Alexa Fluor 488 anti-Chicken IgG antibody (Jackson ImmunoResearch Labs, 703-545-155), donkey polyclonal Alexa Fluor 647 anti-Rabbit IgG antibody (Jackson ImmunoResearch Labs, 711-605-152), and donkey polyclonal Alexa Fluor 647 anti-Chicken IgG antibody (Jackson ImmunoResearch Labs, 703-605-155). The nucleus was labeled with DAPI. H&E staining was performed using standard methods. Masson’s Trichrome staining was performed using the NovaUltra™ Masson Trichrome Stain kit (IHC World, IW-3006) per the manufacturer’s instructions on the OCT-frozen section. The sections were stained with Weigert’s Iron Hematoxylin solution for 5 min, followed by staining with Biebrich scarlet acid fushin solution for 2 min. These sections were then differentiated in PPAcid solution for 6 min, stained with 16-fold diluted Aniline Blue solution for 1 min, and differentiated with Acetic Acid solution for 2 min. Following dehydration with 95% and 100% ethanol, sections were cleared with Histo-Clear solution and mounted with Histomount solution.

Imaging and image analysis
Fluorescent images were obtained using a Nikon CSU-W1 SoRa spinning disk confocal microscope with a ×20 or ×40 objective (Nikon Instruments Inc.) or an EVOS M7000 microscope with a ×10 objective (Invitrogen). Images from a spinning disk confocal microscope were presented as maximum-intensity z-projection images. Bright-field images were obtained using a Leica Mica imaging system with a ×20 objective (Leica Microsystems), an EVOS M5000 microscope with a ×10 objective (Invitrogen), or a Zeiss Axioscan 7 slide scanner with a ×10 objective (Carl Zeiss Co. Ltd.). Images from Mica were tiled and merged using the Leica software. The images were assembled and processed using Adobe Photoshop (ver. 24.7.4, Adobe, Inc.) and Adobe Illustrator (ver. 27.9.4, Adobe, Inc.). The ImageJ software was used for image analysis and quantification. Trichrome images were used to quantify the dermal collagen ultrastructure. Collagen deposition was measured from the aniline blue-stained area assessed using the Color deconvolution2 plugin in Fiji101,125. To compare its ultrastructure, machine learning analysis was performed using the fiber quantification pipeline in MATLAB126,127. The MATLAB scripts for the fiber quantification workflow are available at the GitHub repository (https://github.com/shamikmascharak/Mascharak-et-al-ENF).

Western blot
HaCaT cells were washed with ice-cold PBS and lysed on ice in 250 µL RIPA lysis and extraction buffer (Thermo Fisher Scientific, 89901) freshly supplemented with a protease inhibitor cocktail (Roche, 04693132001). Protein concentration was determined using the BCA assay (Thermo Fisher Scientific, 23225). Samples were heated in 1× sodium dodecyl sulfate (SDS)-PAGE Loading Buffer (LPS Solution, CBS002) at 70 °C for 10 min, separated by SDS–PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH00010). Membranes were blocked in 5% skim milk in TBS with 0.1% Tween-20 (TBS-T, LPS Solution, CBT007) for 1 h at room temperature, incubated with primary antibodies overnight at 4 °C. After washing with TBS-T, membranes were incubated with the secondary antibodies for 2 h at room temperature and washed again. The following antibodies were used in this study at the indicated dilutions: rabbit anti-EGFR (Cell Signaling Technology, 4267S, 1:1000), rabbit anti-phospho-EGFR (Cell Signaling Technology, 3777S, 1:1000), rabbit anti-AKT (Cell Signaling Technology, 4685S, 1:1000), rabbit anti-phospho-AKT (Cell Signaling Technology, 4060S, 1:1000), rabbit anti-GAPDH (Cell Signaling Technology, 2118S, 1:1000), and goat anti-rabbit IgG HRP conjugate (Bio-Rad 170–6515, 1:5000). The signal was detected using Clarity Western ECL Substrate (Bio-Rad, 1705061) and imaged with an Amersham Imager 680 (GE Health Care). Unprocessed, full-length blots are provided in the Source Data.

In vitro scratch wound healing assay
HaCaT cells were cultured to 95% confluence in 12-well plates, and then the center of the well was scratched using a sterile 1000 µL pipette tip. The scratch areas were measured at 0, 6, 12, 18, and 24 h after scratching and quantified using the ImageJ software. 20 ng/mL EGF (Gibco, PMG8043) and 20 µM LGK974 (MedChemExpress, HY-17545) were treated 12 h before scratching.

Click-iT™ EdU cell proliferation assay
For labeling of newly synthesized DNA by EdU incorporation, HaCaT cells were incubated with 10 µM 5-ethynyl-2′-deoxyuridine (EdU, Sigma-Aldrich, 900584) for 2 h and then processed using the Click-iT™ EdU Cell Proliferation Alexa Fluor™ 488 Imaging Kit (Thermo Fisher Scientific, C10337), per the manufacturer’s protocol.

Splinted full-thickness cutaneous wounding
Punch biopsies were conducted on the center of the backs of anesthetized mice in the 2nd telogen phase of the hair growth cycle (7–9 weeks old). To wound the dorsal back skin, the hair on the dorsal area was shaved using an electric clipper and swabbed with a sterile alcohol preparation pad. A full-thickness wound was created inside each splint using a 6 mm sterile skin biopsy punch (Kai Medical, BP-60F) and surgical scissors. Six interrupted sutures of 6-0 silk (Ailee, SK617) were used to secure a silicone splint, cut from a 0.5 mm thick silicone sheet (Sigma-Aldrich, GBL664581), to the surrounding skin. The wounds and splints were covered with sterile semi-occlusive Tegaderm Film dressing (3M Healthcare, 1624W). Wound progression was documented using standardized photographs.

Trans-epidermal water loss (TEWL) measurement
TEWL of wounded control and Epi-iOSKM skin was analyzed using a GPSkin Barrier Pro-II device (GPOWER Inc., South Korea). TEWL measurements were performed using a 5 mm diameter device probe for about 10 s, and measurements were recorded using the GPskin research program (ver 1.0.0, GPOWER Inc.)128.

STZ and blood glucose assessment
To induce hyperglycemia, male mice were used, as they are more susceptible to STZ-induced diabetes than female mice129–131. Both control and Epi-iOSKM male mice received daily intraperitoneal injections of STZ (Sigma-Aldrich, S0130) dissolved in 50 mM sodium citrate buffer (Sigma-Aldrich, S4641) or vehicle alone for 4 days. Blood glucose levels were measured before injection and 12 days later using a Bayer Contour Plus glucometer (Panasonic Healthcare Co., Japan)132 on tail vein blood samples.

Epidermal cell isolation for scRNA-seq
The dorsal skin tissues of mice were collected and dissociated with the gentleMACS™ Dissociator (Miltenyi Biotec) and Epidermis Dissociation Kit (Miltenyi Biotec, 130-095-928) according to the manufacturer’s instructions. The cell suspension was sequentially filtered through 70 µm (Falcon, 352350) and 40 µm (Falcon, 352340) strainers. Centrifugation was performed at 400 × g for 10 min at 4 °C and the cell pellets were resuspended in PBS containing 5% FBS. The resuspended cells were incubated with Red Blood Cell Lysis Buffer (Roche, 11814389001) for 3 min at room temperature and centrifuged at 400 × g for 10 min at 4 °C. The final cell pellets were resuspended in PBS containing 5% FBS and the resuspended cells were sorted using a CytoFLEX SRT cell sorter (Beckman Coulter) and the CytExpert SRT software (Beckman Coulter). Live singlet cells were identified using DAPI staining (Sigma-Aldrich, D9542) and sorted. Further gating was used to isolate mCherry-positive and mCherry-negative cells. For the scRNA-seq using mCherry-sorted epidermal cells (1st dataset), mCherry-negative cells from three control mice and mCherry-negative and mCherry-positive cells from six Epi-iOSKM mice were sorted. For the scRNA-seq using total epidermal cells from control and Epi-iOSKM mice regardless of OSKM-mCherry expression (2nd dataset), all sorted live cells from one mouse per group were used. All cells within a condition were pooled.

scRNA-seq library preparation
Libraries for scRNA-seq were generated using the Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 for dual indexing including Chromium Next GEM Single Cell 3′ Kit v3.1 (10X Genomics, PN-1000269), Chromium Next GEM Chip G Single Cell 3′ Kit (10X Genomics, PN-1000127), 3′ Feature Barcode Kit (10X Genomics, PN-1000262), Dual Index Kit TT Set A (10X Genomics, PN-1000215), and Dual Index Kit NN Set A (10X Genomics, PN-1000243). For multiplexing, samples were labeled with cell multiplexing oligo (CMO) from the 3′ Cell Plex Kit Set A (10X Genomics, PN-1000261) and pooled in equal ratios. The pooled cell suspension was diluted to 1.6 × 10^6 cells/ml, and 30.9 µl was mixed with reverse transcription (RT) master mix to capture a total of 30,000 cells. To generate the gel bead-in-emulsions (GEMs), the mixture was loaded onto the Chromium Next GEM chip G for microfluidic processing on a Chromium controller (10X Genomics). Following incubation in a thermal cycler, the GEMs were broken, and pooled cDNA was purified using silane magnetic beads. During cDNA size selection using 0.6X SPRIselect beads (Beckman Coulter, B23318), the supernatant and bead pellet fractions were treated independently in order to extract DNA from the CMO feature barcode and cDNA from poly-adenylated mRNA, respectively. To generate cell multiplexing libraries, 75 µl of the supernatant was retained and further purified. Indexing primers (10X Genomics, PN-1000243) and cell multiplexing barcode DNA were then added to the PCR reaction mixture for amplification. For cDNA library generation, bead pellets were purified, and cDNA was eluted. The barcoded cDNAs were amplified using a thermal cycler. Subsequent steps for library construction, including enzymatic fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR, were performed according to the manufacturer’s protocol (10X Genomics, CG000388 RevA). The size and concentration of cDNA and libraries were analyzed using a High Sensitivity DNA ScreenTape Analysis Kit with the TapeStation system (Agilent Technologies). Libraries were pooled and sequenced on a Novaseq S4 platform (Illumina) using a paired-end 100 bp protocol to generate a minimum of 20,000 read pairs per cell for the 3′ gene expression library and 5000 read pairs per cell for the cell multiplexing library.

scRNA-seq data pre-processing
An identical pipeline was used to pre-process the scRNA-seq data from the 1st dataset with mCherry-sorted cells and the 2nd dataset with total epidermal cells. For all data, generated raw reads were mapped to the mouse reference genome (GRCm39) and demultiplexed based on barcodes specific to each sample using the Cell Ranger multi (v7.0.1, 10X Genomics). Based on quality control metrics calculated by the addPerCellQC function from the scater (v1.26.1) R package133, the cells were considered low-quality if the detected unique molecular identifiers (UMI) were less than 500 or if the mitochondrial RNA content of the total UMI exceeded 20% or 10% for the 1st and 2nd datasets, respectively. Cells were clustered using the quickCluster function from the scran (v1.26.2) R package134. Cell-specific size factors were calculated using the computeSumFactors function from the same package. Raw counts were then normalized by these size factors and log-transformed with a pseudocount of 1. Highly variable genes (HVGs) were identified based on variance modeling computed using the modelGeneVar from the scran R package. The false discovery rate thresholds were 0.05 for the 1st dataset and 0.005 for the 2nd dataset. The top 15 principal components (PCs) were computed based on the HVGs and used for clustering and UMAP dimension reduction for the 1st dataset. The top 20 PCs were used in the case of the 2nd dataset. Cell types were annotated based on cluster marker genes identified using the FindMarkers function from the Seurat (v5.0.1) R package135. IFE cells were identified and subsetted based on expression patterns of canonical gene markers (Krt5, Krt14, Krt10, and Krt1) across cell clusters, then underwent the pre-processing steps described above.

scRNA-seq data analysis
Module scores to analyze the aggregated expression of a gene set were computed using the addModuleScore function from the Seurat R package. The curated gene sets based on the Gene Ontology Biological Process terms were obtained from MSigDB136 and used to evaluate how key biological pathways were regulated across different cell types upon OSKM induction. To examine the regulation of pathways related to hypoxia signaling and cytokine-mediated inflammatory response in epithelial cells under different conditions, module scores were calculated using genesets associated with the REACTOME database terms cellular response to hypoxia and cytokine signaling in immune system. Monocle3 (v1.3.4) was used to assess differentiation in IFE cells. Cells were clustered using the cluster_cells function with UMAP as the dimensionality reduction method. A pseudotime trajectory was constructed with the learn_graph function using default parameters. To establish a biologically relevant root for the pseudotime trajectory, we selected cells with high Krt14 expression and low Krt10 expression, indicative of undifferentiated IFE cells. Using this, we ordered the cells along the inferred pseudotime using the order_cells function. scVelo (v0.2.5)137 was used to analyze dynamic transcriptional changes and infer RNA velocity. The annotated spliced and unspliced reads required for the analysis were obtained using Velocyto (v0.17.17)138. We then computed RNA velocities using the dynamical model implemented in scVelo, which allows for a more flexible, dynamic representation of RNA velocity that accounts for the temporal changes in expression across cell states. This was conducted separately for each condition. Following the computation of RNA velocities, we performed trajectory analysis and connectivity mapping using partition-based graph abstraction (PAGA) also implemented in scVelo. Directed graphs representing the connectivity and progression between transcriptionally distinct clusters were constructed by leveraging the velocity-inferred cell-state transitions. To visualize the RNA velocity results and PAGA trajectories in an embedding that optimally represents trajectory information, we constructed a diffusion map representation that preserves the global topology and directionality of transitions. Diffusion maps were computed using the palantir.utils.run_diffusion_maps function implemented in the Palantir package (v1.3.3)139. We employed the CytoTRACE2 (v1.0.0) R package140 to evaluate cellular potency across IFE cells. Predictions of developmental potential were generated using the cytotrace2 function with the full_model parameter set to TRUE, which enables an ensemble approach that utilizes all available models to maximize robustness and accuracy. For comparison and visualization, we focused on the CytoTrace2_Relative output, which provides a ranked relative order of cells according to their absolute potency scores. The scores are normalized to a range from 0 (most differentiated) to 1 (least differentiated). Pathway activity scores were inferred using the progeny (v1.20.0) R package52 using the top 500 responsive genes for each pathway curated in the package. We used the CellChat (v2.1.2) R package141 to investigate how OSKM induction altered IFE cell communication. Pre-processed data were separated by condition (control versus Epi-iOSKM), processed according to the CellChat pipeline, and then merged again for analysis. Over-expressed ligand-receptor (LR) pairs were identified using the identifyOverExpressedGenes function, followed by the identifyOverExpressedInteractions function for LR interactions curated within the CellChatDB.mouse function. Communication probabilities between interacting cell groups were calculated using the computeCommunProb and computeCommunProbPathway functions, employing the trimean method to compute the average gene expression per cell type. Finally, the rankNet function was used to visualize and compare the relative information flow after merging the processed CellChat objects from each condition. To functionally interpret the gene set upregulated in mChpos IFE cells compared to controls and included in the Gene Ontology Cellular Component term extracellular space, we performed enrichment analysis using the REACTOME database. This was conducted using the gost function from the g:Profiler2 (v0.2.3) R package142. Additionally, we assessed enrichment on the SMART protein domain database curated in the STRING database version 11.5 using the STRINGdb (v2.10.1) R package143. The hypergeometric test was computed with the get_enrichment function, using the Benjamini-Hochberg procedure for multiple testing correction.

Public scRNA-seq data analysis
To compare the molecular changes caused by OSKM induction with those caused by skin wounding, we analyzed publicly available scRNA-seq data (accession code GEO: GSE142471)39 from two wounded and three unwounded mouse skin samples. The individual count matrices were merged and pre-processed using the same pipeline described earlier in the scRNA-seq data pre-processing subsection. Sample details such as origin, condition, and cell type were obtained from the original study39. We specifically focused on basal and spinous cell populations by subsetting such cells from the entire dataset. These subsets were pre-processed following the established methodology. To identify cytokines upregulated upon wounding, we compared gene expression profiles of cells from wounded and unwounded conditions using the FindMarkers function from the Seurat R package. Next, we integrated the public IFE scRNA-seq data with IFE cells from 2nd dataset using an anchor-based integration method144. Integration anchors were identified based on reciprocal PCA using the FindIntegrationAnchors function of the Seurat R package. The two IFE datasets were integrated using the IntegrateData function of the same R package. Finally, a two-dimensional UMAP visualization was generated using the top 10 PCs of the integrated data.

Statistics and reproducibility
Details for statistical analyses, including the number of replicates and the statistical tests used, are presented in the figure legends and method sections where applicable. Statistical analyses were performed by using Prism v. 9.4.1 (Graphpad) and R software (R 4.2.3). Two-tailed Student’s t-test, two-tailed Mann-Whitney test, one-way ANOVA with Tukey’s or Dunnett’s T3 multiple comparison test, or two-way ANOVA with Tukey’s multiple comparison test were applied to analyze the experimental data. Two-tailed Mann-Whitney U test with Bonferroni correction or Benjamini-Hochberg correction was applied to analyze the scRNA-seq data. To analyze the wound closure rates, asymmetric sigmoidal interpolation (5PL, where X is log(concentration)) with two-way repeated measures ANOVA was conducted. Statistical analysis was set at P < 0.05. DEGs were defined as genes with adjusted P-values less than 0.05.

The sample size was not predetermined using any statistical method, and no data were excluded from the analyses. Experimental and control mice were determined by genotyping and were randomly selected for the experiments. Data collection and analysis were not performed blind to the conditions of the experiments.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors aim to explore partial reprogramming of skin epidermal cells using OSKM mice in which Yamanaka Transcription factors are expressed by epidermal cells. The authors report intriguing findings, including ectopic expression of keratin 17 and upregulation of upstream regulators such as phosphorylated AKT and EGFR. These may suggest an altered cell state in OSKM-expressing epidermal cells as well as secondary effects on other cells including neighboring keratinocytes, dermal cells, endothelial cells and T cells. Importantly, the authors demonstrate a significant enhancement of re-epithelialization in OSKM mice under both normal and diabetic conditions. The authors analyzed many distinct aspects of the phenotype in OSKM mice, making it a nice descriptive paper.\n1. Their data do not convincingly demonstrate a loss of epidermal lineage identity. Traditionally, reprogramming via OSKM refers to the erasure of somatic cell identity and acquisition of pluripotency, enabling differentiation into multiple lineages. In the current study, it is unclear to what extent the cells undergo this transition. If the epidermal cells retain their original lineage characteristics, labeling the observed cellular changes as “reprogramming” may be misleading. Given that OSKM factors have diverse physiological functions depending on tissue context, the observed effects might result from another cellular responses rather than de-differentiation per se. Even at the expense of other experiments examining peripheral mechanisms in the paper, this fundamental issue needs to be much more robustly investigated with various methods.\n2. Basically, the most important aspect of this study is to determine whether epidermal cells are really undifferentiated in this model under homeostasis and upon wounding. It is unclear whether K17 expression and K10 pattern distribution indicate de-differentiation of epithelial cells. Why epithelial cells with OSKM expression can still undergo terminal differentiation in the epidermis?\n3. Can the authors describe how epithelial changes influence collagen composition? Is it known that epidermal differentiation promotes collagen I deposition? Given that the scarring depends upon dermal healing, more in depth mechanistic exploration would be helpful.\n4. Hair follicle cells significantly contribute to skin physiology and wound healing. How are hair follicles altered in OSKM mice? Would the phenotype in OSKM mice partly due to the changes in hair follicle cells?", "reviewer_2": "Kwak et al. demonstrate that transient expression of reprogramming factors in a subset of keratinocytes promotes skin repair through cell-autonomous and non-cell-autonomous mechanisms. The study is interesting, and the data are generally convincing.\nThe finding that partial reprogramming of cells promotes tissue repair is not completely novel and has been shown in other tissues. However, this is the first study that shows the effect of partial keratinocyte reprogramming on epidermal homeostasis and wound repair. In particular, the finding that reprogramming promotes repair by cell-autonomous and non-autonomous mechanisms is interesting. However, the study has also several weaknesses and raises some questions (see specific comments below).\nGeneral points:\n1.) The authors show interesting phenotypes after transient expression of OSKM in keratinocytes in mice. This is likely a consequence of partial reprogramming, but it cannot be excluded that the phenotype is simply a consequence of the expression of one of these transcription factors. Can the authors exclude this possibility?\n2.) The translational relevance of the study is not very strong. It is unlikely that partial reprogramming will be used for the promotion of acute wound healing. The authors show the effect in diabetic mice, but this is a model for delayed acute healing, but not nor chronic wounds. In a chronic wound, the risk of transformation is likely to be high, and the effect of reprogramming may be different than in an acute wound.\nSpecific points:\n3.) Line 97: K14-CreER mice do not induce specific deletion in epidermal keratinocytes. The K14 promoter is also active in hair follicle keratinocytes and basal epithelial cells of other stratified epithelia. This sentence should be modified. In particular, it is unclear if and to what extent reprogrammed hair follicle keratinocytes contribute to the observed effects.\n4.) The K14-CreER mice are known to be “leaky”. The authors should include GFP and mCherry fluorescence pictures of mice that were not treated with Dox.\n5.) In Fig. 1b the authors should add a high magnification hematoxylin/eosin staining of the skin. This is important to judge the overall histological phenotype of the mice.\n6.) It is not clear what percentage of basal and suprabasal keratinocytes express OSKM. This information should be provided.\n7.) Expression of Klf4, c-Myc and Oct-4 in the epidermis should also be confirmed (at least at the RNA level). This information should be available from the scRNA-seq data.\n8.) The scRNA-seq data suggest alterations in apoptosis. Does reprogramming affect apoptosis of mCherry+ and mCherry- keratinocytes?\n9.) The reversibility of the reprogramming effect is particularly important. Are the mCherry+ cells completely lost at day 10 after Dox withdrawal? If yes, do they undergo apoptosis or are they lost by terminal differentiation?\n10.) Fig. 3g and h: PI3K inhibition is likely to affect epidermal cell proliferation in general. The authors should add control mice treated with the PI3K inhibitor for comparison. Same for the EGFR inhibitor in Fig. 4.\n11.) The result in Fig. 3g requires quantification.\n12.) Fig. 3j: It has not been shown that PI3K directly regulates keratin 6/16/17. This may be simply a consequence of the increased proliferation.\n13.) Line 292: NRG (neuregulin) does not activate the EGFR – it activates ERBB3 and ERBB4.\n14.) Fig. 4h: Please confirm the efficient EGFR inhibition by immunofluorescence staining for pEGFR.\n15.) The model proposed in Fig. 4j is interesting, but it is only based on immunofluorescence stainings. It is important to test if PI3K activation in cultured keratinocytes promotes EGFR activation.\n16.) Line 321: IL1 and VEGF do not directly regulate keratinocyte proliferation. This sentence should be modified.\n17.) SFRP1 is a secreted protein, which should act on mCherry- and on mCherry+ cells. How do the authors explain a selective effect on mCherry- cells if SFRP1 is indeed responsible?\n18.) Supplementary Fig. 5e: An important control is missing: LGK974 alone (same for the migration experiment). It seems likely that this compound already inhibits cell proliferation in control cells. The very weak effect of EGF on proliferation is surprising.\n19.) Line 374: Epidermal T cells in mice are almost exclusively gd T cells. These cells are not recruited upon injury. They only populate the skin once during embryonic development (see publications from W. Havran and A. Hayday). The increase in these cells is likely a consequence of increased T cell proliferation, which should be tested. Alternatively, there may be an abnormal recruitment of ab T cells to the epidermis, which seems likely because there are more RORgt cells. Is there an increase in epidermal and dermal ab or gd T cells or both?\n20.) Lines 390-392: The effect of the CCR6 antagonist on the number of epidermal RORgt cells is not significant.\n21.) Page 16, first paragraph: It is not surprising that no tumors were observed after 10 days of Dox treatment, because epidermal skin tumors develop slowly. It may well be that tumors would develop at a later time point (perhaps even after discontinuation of Dox treatment). This possibility should at least be discussed. In any case, it is clear from these results that long-term OSKM induction in keratinocytes is detrimental.\n22.) The effect of partial reprogramming on wound closure is minor (Supplementary Figure S7A). This is not too surprising, because wound healing in healthy mice is highly optimized. The data on the length of the epithelial tongue and the transepidermal water loss are more convincing. It is essential to show histological stainings of cross-sections from wounds at different time points. Is the area of granulation tissue also affected?\n23.) It is unclear if mCherry+ or mCherry- keratinocytes preferentially contribute to reepithelialization. This is mechanistically important and should be checked at different time points.\n24.) The analysis of immune cells is restricted to T cells. However, macrophages are much more important players in wound healing. Is there a difference in the number of macrophages?\n25.) Line 468: Fig. 6i,j and Supplementary Fig. 7a do not show that wound healing is delayed in the streptozotocin-treated mice. This should be shown in one figure. Again, histological cross-sections from the wounds at different stages should be shown. This is important to fully judge the phenotypic alterations.\n26.) Line 494: It is correct that some reduction in the extent of angiogenesis can reduce scarring in mice. However, this is an indirect effect, which is not very strong. The extent of scarring strongly depends on myofibroblast differentiation. The authors should check if this is affected in the OSKM mice. It is mechanistically unclear how OSKM expression suppresses scarring. It may also be a consequence of the enhanced reepithelialization.\n27.) Title of Fig. 1: The reprogramming does not drive epidermal dedifferentiation – it prevents differentiation (the K14 promoter targets non-differentiated basal cells)."}, "author_response": "We appreciate the reviewer’s thoughtful assessment of our work and helpful suggestions, which have allowed us to further improve the quality and impact of our work. Q1. Their data do not convincingly demonstrate a loss of epidermal lineage identity. Traditionally, reprogramming via OSKM refers to the erasure of somatic cell identity and acquisition of pluripotency, enabling differentiation into multiple lineages. In the current study, it is unclear to what extent the cells undergo this transition. If the epidermal cells retain their original lineage characteristics, labeling the observed cellular changes as “reprogramming” may be misleading. Given that OSKM factors have diverse physiological functions depending on tissue context, the observed effects might result from another cellular responses rather than de-differentiation per se. Even at the expense of other experiments examining peripheral mechanisms in the paper, this fundamental issue needs to be much more robustly investigated with various methods. We appreciate the reviewer for this thoughtful suggestion, which we’ve addressed in our revised manuscript. The reviewer noted that the complete reprogramming of cells through OSKM expression typically involves the loss of the original cell identity and the acquisition of pluripotency. In contrast, our data indicate that “partial” epidermal reprogramming induces a transient and reversible plastic state, without progression to pluripotency. To avoid confusion, we have thoroughly revised the manuscript to clearly distinguish partial reprogramming from complete reprogramming. (A) In vivo partial reprogramming refers to the induction of cellular plasticity without reaching pluripotency Kurian et al. (Nat Methods, 2013) and Thier et al. (Cell Stem Cell, 2012) reported that transient in vitro expression of OSKM induces a dedifferentiated progenitor-like state rather than pluripotent stem cells. Specifically, Kurian et al. generated mesodermal progenitor cells from human fibroblasts, while Thier et al. generated neural stem cells, both demonstrating partial reprogramming without conferring full pluripotency1,2. Following these findings, Ocampo et al. (Cell, 2016) and Browder et al. (Nat Aging, 2022) demonstrated that short-term OSKM induction in vivo ameliorates age-associated hallmarks and prevents the loss of cell identity, tumorigenesis, and functional decline, thereby extending lifespan in a mouse model of accelerated aging3,4. Ocampo et al. emphasized that “reprogramming proceeds in a stepwise manner allowing for the induction of partial reprogramming without the complete loss of cellular identity by short exposure to the Yamanaka factors”. More recently, Wang et al. (Nat Commun, 2021) reported that in vivo partial reprogramming of myofibers promotes muscle regeneration, demonstrating that in vivo partial reprogramming, while preserving the original identity, can be physiologically beneficial5. Furthermore, Chen et al. (Science, 2021) reported partial reprogramming of cardiomyocytes (CMs), noting that “transient expression of OSKM in CMs stimulates proliferation without loss of CM identity”, and demonstrated this approach promotes cardiac repair. They referred to this process as “partial reprogramming” or “reversible reprogramming”6. Similarly, Kim et al. (Sci Adv, 2023) showed short-term OSKM expression enables intestinal regeneration by transient dedifferentiation without loss of their original identity7. Yucel et al. (Nat Commun, 2024) summarized that “partial reprogramming aims to maintain cellular identity while achieving cellular rejuvenation, whereas full iPSC reprogramming aims to facilitate cellular dedifferentiation”8. Collectively, these studies demonstrate that the concept of “partial reprogramming” is distinct from the traditional “complete reprogramming”, as it does not progress to pluripotency. Our work follows this established definition of partial reprogramming. (B) Our data demonstrates partial epidermal reprogramming, which induces epidermal lineage-restricted cellular plasticity with regenerative features We confirmed that OSKM expression in epidermal cells for 3 or 9 days did not induce Nanog expression (Supplementary Fig. 2a and 12b), indicating that complete reprogramming did not occur. By contrast, long-term OSKM induction for 2 months in epidermal keratinocytes resulted in Nanog expression and teratoma formation in vivo (Rebuttal Fig. 1), showing that the epidermal cells can acquire pluripotency under long-term induction. These findings suggest that partially reprogrammed cells induced by short-term (3 day) OSKM expression include intermediate reprogrammed populations that remain on the complete reprogramming trajectory. Nanog was not induced under short-term epidermal OSKM expression for 3 or 9 days (Supplementary Fig. 2a and 12b) Nanog was induced under long-term epidermal OSKM expression for 2 months (Rebuttal Fig. 1) Although pluripotency was not induced, our multiple lines of data support the acquisition of cellular plasticity and a decrease in differentiated keratinocyte characteristics following transient OSKM expression. Cytoscape2 analysis revealed increased cell potency after 3-day OSKM expression (Supplementary Fig. 7e). Expression of Krt17, Krt16, and Krt6a, representative hallmarks for epidermal plasticity9-11, was upregulated in the partially reprogrammed epidermis (Fig. 2g-i, n; Supplementary Fig. 6d). In addition, epidermal differentiation markers including Krt10 were reduced in suprabasal keratinocytes (Fig. 1f-h; Supplementary Fig. 4c,d). Pseudotime and RNA velocity analyses of our scRNA-seq data further showed that suprabasal keratinocytes in Epi-iOSKM epidermis displayed reduced differentiation signatures and exhibited cell fate trajectories opposite to normal epidermal differentiation (Fig. 1i,l-n; Supplementary Fig. 4f-h,k-n). In addition, multiple datasets indicated increased proliferation in the epidermis (Fig. 1j,k; Supplementary Fig. 4i,j). To more clearly illustrate cellular transition into more plastic state with reduced differentiation characteristics, we incorporated additional scRNA-seq analyses in the revised manuscript (new Fig. 1m,n; new Supplementary Fig. 4d,g,h,l-n; see also responses to reviewer 1, question #2). Partial epidermal reprogramming confers cellular plasticity (Supplementary Fig. 7e; Fig. 2n) Partial epidermal reprogramming attenuates epidermal differentiation characteristics (Fig. 1i, l) Partial epidermal reprogramming induces cell proliferation (Fig. 1k) Additionally, we found that after 3 days of short-term partial reprogramming, epidermal cells preserved their original identity (Supplementary Fig. 7i-k) while acquiring plasticity with regenerative features (Fig 2; Supplementary Fig. 7e). This state was reversible (Supplementary Fig. 7f-h), indicating that epidermal keratinocytes subjected to transient OSKM expression in vivo were partially reprogrammed within their epidermal cell lineages. Taken together, these results provide molecular and functional evidence that transient OSKM expression partially dedifferentiates epidermal keratinocytes with preserving their lineage identity (see also responses to reviewer 1, question #2), consistent with the widely accepted definition of partial reprogramming as loss of differentiation markers and increased proliferation without acquisition of pluripotency. (C) Injury-responsive-like conversion and enhanced healing outcomes in Epi-iOSKM skin result from partial reprogramming-induced epidermal plasticity Importantly, the restricted cellular plasticity within epidermal cell lineage upon ectopic OSKM expression recapitulated the natural partial dedifferentiation process during wound repair (Fig. 2). Integrative analysis of our scRNA-seq data with reference datasets showed that partially reprogrammed cells and wounded cells share common cellular characteristics (Fig. 2b-e). For example, not only Krt17 and Krt6 were induced, but also stress responses such as HIF-1α activation were induced in partially reprogrammed cells and their neighbors, similar to the wound repair process (Fig. 2f-m). Plastic cells induced by wounding propagate injury responses to their surroundings for rapid restoration of the tissue12,13. Similarly, plastic IFE cells induced by short OSKM induction extended partial reprogramming effects to their neighbors and niches through hijacking EGFR signaling and CCL20-CCR6 axis that crucially function during wound repair (Fig. 4,5). These collective changes improved skin wound repair in Epi-iOSKM mice (Fig. 6, new Fig. 8). Notably, our new data revealed that epidermis-wide HIF-1α activation upon mosaic partial reprogramming, one of the recapitulative phenotypes of healing characteristics14, was a key modulator of enhanced repair in Epi-iOSKM mice (new Fig. 7i-n, see also responses to reviewer 1, question #3). Together, these findings suggest that mosaic partial epidermal reprogramming, by conferring plasticity within epidermal cell lineages, induces regenerative features not only in themselves but also in their neighbors and niches to enhance skin repair. Partial reprogramming-induced epidermal plasticity confers regenerative characteristics to themselves and their neighbors/niches (Fig. 9a, images are reproduced with permission from Bioinsight under the CC BY license) Ultimately, the major phenotypes observed in Epi-iOSKM epidermis—increased proliferation, loss of differentiated characteristics, acquisition of regenerative characteristics, and the extended effects of OSKM-expressing cells on their neighbors and niches—represent direct and indirect outcomes of partial reprogramming, which confers cellular plasticity within epidermal lineages. As a result, the Epi-iOSKM epidermis collectively exhibited injury-responsive-like characteristics (Fig. 2; Supplementary Fig. 7), resembling a state in which healing has already been initiated before wound occurrence. This led us to hypothesize that partial epidermal reprogramming would facilitate wound repair. Consistent with this, we observed accelerated re-epithelialization and reduced scarring. In summary, partial reprogramming confers plasticity to epidermal cells within the lineage, enabling them to mimic repair processes, which proved broadly beneficial for cutaneous healing. (D) Corrections and clarifications in the revised manuscript As noted at the outset, and supported by both our own data and reference studies, the observations in this study reflect the effects of “partial reprogramming”, not “reprogramming” in the traditional concept of full reprogramming. However, in the original manuscript, the two terms were occasionally used interchangeably, which may have led to confusion. To address this, we have systematically revised the terminology throughout the manuscript. For example: ● epidermal reprogramming  partial epidermal reprogramming ● reprogrammed cells/epidermis  partially reprogrammed cells/epidermis In line with this correction, we also revised the paper title to: Mosaic partial epidermal reprogramming remodels neighbors and niches to refine skin homeostasis and repair. The titles of Fig. 3 and Fig. 4 were also revised as follows: ● Fig. 3: Partially reprogrammed epidermal cells recapitulate repair states via PI3K activation. ● Fig. 4: Non-cell-autonomous effects in the partially reprogrammed epidermis via EGFR activation. In addition, we have added an illustration in the Introduction to clearly explain the concept of partial reprogramming, as shown below: “Cellular reprogramming by Oct-4, Sox2, Klf4, and c-Myc (OSKM), known as the Yamanaka factors, has emerged as a valuable tool for the manipulation of cell fate. Originally, OSKM were used only in vitro because their forced expression in vivo leads to teratoma formation due to the production of induced pluripotent stem cells. However, it was found that transient and reversible OSKM expression could induce “partial reprogramming”, which confers cellular plasticity while retaining the original cell identity and functionality. This process did not induce pluripotency, indicating that in vivo partial reprogramming could prevent teratoma formation. Partial reprogramming in mice rejuvenated progeria or physiological aging. Furthermore, recent studies have revealed that in vivo partial reprogramming promotes tissue repair in various organs, such as the muscle, liver, and intestine. Another study observed a reduction in fibrosis and scarring after injury.” Taken together, the more detailed description of reference studies, our original and newly provided data, along with clarification of ambiguous wording, and the addition of explanations of the key concept in the revised manuscript collectively demonstrate that our study is the first to report both cell-autonomous and non-autonomous effects of in vivo “partial reprogramming” of epidermal cells on skin homeostasis and repair. Q2. Basically, the most important aspect of this study is to determine whether epidermal cells are really undifferentiated in this model under homeostasis and upon wounding. It is unclear whether K17 expression and K10 pattern distribution indicate de-differentiation of epithelial cells. Why epithelial cells with OSKM expression can still undergo terminal differentiation in the epidermis? Thank you for this important comment. We do not claim that epidermal cells become “undifferentiated” in the sense of losing epidermal lineage identity or acquiring pluripotency. Throughout the revised manuscript, we use “partial reprogramming” in the established meaning: a transient, reversible increase in cellular plasticity within the epidermal cell lineage, without progression to pluripotency. Please see also responses to question #1. Krt17 induction and Krt10 pattern distribution represent hallmarks of this partial dedifferentiation of epidermal cells with preserving their own lineage, contrasting with full dedifferentiation into iPSCs. Importantly, these marker alterations are only part of the broader phenotypes that support this process. In addition to them, extensive original and additional data demonstrate epidermal lineage-restricted dedifferentiation: 1. No pluripotency program under short-term OSKM expression: Short-term OSKM induction (3 or 9 days) did not induce Nanog expression (Supplementary Fig. 2a, 7i, 12b), and cells retained epidermal lineage markers (Krt5 and Trp63, Supplementary Fig. 7j,k) while resuming the normal differentiation cycle after Dox withdrawal (Supplementary Fig. 7f-h). 2. Differentiation characteristics are attenuated, not abolished, and reversible: Krt10, a marker for epidermal differentiation, in suprabasal cells was reduced (not absent) under short-term OSKM expression and recovered after Dox withdrawal (Fig. 1f-h; Supplementary Fig. 7h), consistent with a reversible plastic state rather than lineage erasure. Cleaved caspase-3 staining was negative during and after induction (new Supplementary Fig. 2c,d), indicating recovery occurred without apoptotic loss. In addition to the marker gene expression, pseudotime analysis with scRNA-seq showed that comprehensive epidermal differentiation extent was declined (Fig. 1i; new Supplementary Fig. 4g,h). We also observe the similar pattern of decrease in another suprabasal marker Dsg1a in the new manuscript (new Supplementary Fig. 4d). Partial reprogramming reduces epidermal differentiation markers (Fig. 1g; new Supplementary Fig. 4d) and correlated pseudotime values (Fig. 1i; new Supplementary Fig. 4g,h) 3. Cell property reversion toward an earlier state (Supra(Krt10+)  Basal(Krt14+)): scRNA-seq RNA velocity analysis in the previous manuscript exhibited that cell fate trajectories from Supra to Basal cells, suggesting a reversal of epidermal differentiation upon partial epidermal reprogramming (Fig. 1l). In the new manuscript, we additionally examined the marker gene expression and identified that fate transition trends of Epi-iOSKM IFE cells were correlated with decreases in differentiation markers and increases in epidermal stem cell markers. Specifically, the trends were from Krt10/Dsg1a-high to Krt10/Dsg1a-low and Krt14/Itga6-high states, exhibiting the opposite trend from the normal epidermal differentiation. We have added this result as new Fig. 1m and new Supplementary Fig. 4l-n. Cell fate transition from Supra to Basal cells under partial reprogramming (Fig. 1l) Cell fate transition from differentiated to stem-like IFE cells under partial reprogramming (new Fig. 1m; new Supplementary Fig. 4l-n) 4. Repair-like, lineage-restricted plasticity: Integrative scRNA-seq analysis with public reference wound datasets revealed that Epi-iOSKM IFE cells acquire the epidermal plasticity that deviates toward a wound-responsive state, not toward a pluripotency program (Fig. 2b-e). Upregulation of Krt17/Krt6 and stress-response pathways such as HIF-1α and STAT3 is a hallmark of activated/wound-edge keratinocytes, not of full dedifferentiation (Fig. 2f-m). Together, these findings explain why OSKM-expressing IFE cells can still undergo terminal differentiation in vivo: partial reprogramming confers lineage-restricted, reversible plasticity that primes cells for repair without extinguishing the epidermal differentiation network. After OSKM withdrawal, epidermal architecture and marker expression normalize, and no pluripotency markers are detected. We have clarified this definition and supporting evidence in the Introduction and Results, and we now consistently use the term “partial reprogramming” throughout to avoid confusion. Q3. Can the authors describe how epithelial changes influence collagen composition? Is it known that epidermal differentiation promotes collagen I deposition? Given that the scarring depends upon dermal healing, more in depth mechanistic exploration would be helpful. We appreciate this comment, which prompted us to further investigate a mechanistic link between partial epidermal reprogramming and enhanced dermal healing. Through additional experiments, we identified epidermal HIF-1α activation as a key factor modulating enhanced dermal healing in Epi-iOSKM skin (new Fig. 7i-n; new Supplementary Fig. 15). Re-epithelialization can influence collagen deposition in multiple ways, but due to the complexity of the wound healing process, its effects cannot be generalized and may vary depending on context. For example, accelerated re-epithelialization can reduce scar formation by shortening the overall repair duration and preventing excessive healing15-17, but in other cases, rapid epithelial closure can instead promote increased scarring by driving overactive healing processes18-20. In the Epi-iOSKM skin, accelerated re-epithelialization was accompanied by reduced scarring, indicating enhanced repair in both epidermis and dermis. Because mosaic partial reprogramming of the epidermis was stopped at 1 day post-wounding (dpw)—a very early stage of the healing process—these long-lasting beneficial phenotypes likely represent extended effects from a temporal perspective as well as from a spatial perspective. In the original manuscript, we reported that HIF-1α activity is more elevated in Epi-iOSKM epidermis at 3 and 7 dpw, even after the partial reprogramming had already ceased, unlike in unwounded skin (Fig. 7b-e). This suggests that HIF-1α may mediate the prolonged effects of reversible reprogramming. Notably, HIF-1α was activated in the Epi-iOSKM epidermis in an atypical pattern: more increased activation was detected in the old epidermis that had undergone partial reprogramming, rather than in the newly regenerated epithelium (Fig. 7b-d). Interestingly, angiogenesis patterns during wound repair were significantly altered in Epi-iOSKM skin according to the altered pattern of HIF-1α activation in the epidermis upon partial reprogramming (Fig. 7f-h), suggesting that HIF-1α may also spatially extend the effects of partial epidermal reprogramming to the dermal repair process. Partial epidermal reprogramming increases HIF-1α activity and alters neovascularization patterns during wound repair (Fig. 7a-h) To test this, we examined the role of epidermal HIF-1α activation in the wound healing process of Epi-iOSKM mice. We found that HIF-1α is essential not only for promoting re-epithelialization but also for reducing scar formation (new Fig. 7i-n; new Supplementary Fig. 15a-e). Together, these findings demonstrate that partial epidermal reprogramming promotes dermal collagen remodeling and scar attenuation through activation of epidermal HIF-1α. The related data and explanation in the revised manuscript are as follows: “Previous studies have reported that forced activation of HIF-1α accelerates re-epithelialization. In our model, HIF-1α activation induced by partial reprogramming was sustained after wounding (Fig. 7c), suggesting it may continue to promote re-epithelialization even after transient reprogramming has ceased. Moreover, the altered patterns of HIF-1α activation correlated with reorganized angiogenesis (Fig. 7b-h), potentially influencing dermal repair. Notably, appropriate regulation of neovascularization during wound repair is known to reduce scar formation. When a wound occurs in Epi-iOSKM mice, HIF-1α is activated twice in the epidermis—first by partial reprogramming and again by the wound itself. To disrupt the synergistic effect of these two stimuli, we transiently inhibited HIF-1α activity by treating the HIF-1α inhibitor PX-478 once at the time of wounding (Fig. 7i). HIF-1α activity decreased at 1 dpw but recovered by 10 dpw as expected (Supplementary Fig. 15a,b). Interestingly, transient HIF-1α inhibition during the early repair phase reversed the accelerated re-epithelialization in Epi-iOSKM mice, bringing it to levels comparable to controls (Fig. 7j,k). Moreover, temporal inhibition of HIF-1α markedly reduced angiogenesis. Without inhibition, control mice displayed widespread angiogenesis across the wound bed, whereas Epi-iOSKM mice showed angiogenesis concentrated near the original wound site (Fig. 7f-h). Following PX-478 treatment, however, both groups exhibited a pronounced reduction in angiogenesis, with low CD31 expression at newly formed dermis near both the old wound edges and the regenerated epidermis (Fig. 7j,I; Supplementary Fig. 15c,d). These findings indicate that HIF-1α is a key regulator of neovascularization patterns during wound repair in both control and Epi-iOSKM mice. We next examined whether HIF-1α, which is essential for the alterations in re-epithelization and angiogenesis in Epi-iOSKM mice, also contributes to scar reduction. At 20 dpw, dermal thickness in the scarred area of PX-478 treated Epi-iOSKM mice was significantly thicker than that that of untreated Epi-iOSKM mice and was comparable to that of PX-478 treated control mice (Fig. 7m). Consistently, the alterations in collagen I deposition observed in Epi-iOSKM skin were restored by PX-478 treatment (Supplementary Fig. 15e). In addition, the reduced proportion of collagen III+ area observed in Epi-iOSKM mice was reversed by HIF-1α inhibition during partial epidermal reprogramming (Fig. 7n), indicating that elevated HIF-1α levels are essential for the reduction in scarring. Collectively, increased HIF-1α activation upon partial epidermal reprogramming—particularly the atypical pattern observed in the old epidermis near the initial wound site rather than in the newly generated epithelium (Fig. 7b-d)— substantially contributes to the widespread changes that promote wound repair.” HIF-1α activation is essential for enhancing wound healing and attenuating scar formation in Epi-iOSKM skin (new Fig. 7i-n; new Supplementary Fig. 15a-e) These results illustrate how epidermal changes can influence dermal healing. HIF-1α, a responder to stressful environments, is rapidly activated after wounding and coordinates multiple repair processes12,21. When the control group was transiently treated with by a HIF-1α inhibitor, re-epithelialization was not altered, but dermal healing process was affected (new Fig. 7j-l), suggesting that the primary role of HIF-1α during the early phase of wound healing is to coordinate dermal niche activation. Thus, the alterations in HIF-1α activity may broadly affect healing processes. Indeed, spatial and temporal regulation of HIF-1α is known to exert complex, context-dependent effects on healing, which can be either beneficial or detrimental14,21,22. The Epi-iOSKM epidermis exhibited the elevation of HIF-1α activity as an unusual pattern: the signal was increased in the old epithelium that had undergone partial reprogramming, rather than in the newly regenerated epithelium (Fig. 7b-d). HIF-1α activation in wounded Epi-iOSKM epidermis (Fig. 7b-d) This alteration substantially remodeled wound repair mechanisms, including re-epithelialization, angiogenesis, and scarring (Fig. 9b). Given the complexity of the changes, it is challenging to establish direct causality among the different repair processes. Nevertheless, our results suggest the potential that accelerated re-epithelialization, accompanied by reduced neovascularization near the rapidly regenerating epithelium, contributes to decreased scar formation. Importantly, HIF-1α emerges as a key regulator coordinating these processes. Taken together, partial epidermal reprogramming, which induces healing characteristics, activated HIF-1α even prior to wounding (Fig. 2l), and wounding itself also activated HIF-1α. Consequently, the Epi-iOSKM epidermis during healing exhibited an atypical pattern of HIF-1α activation, which appears to promote coordinated repair of both the epidermis and dermis. Q4. Hair follicle cells significantly contribute to skin physiology and wound healing. How are hair follicles altered in OSKM mice? Would the phenotype in OSKM mice partly due to the changes in hair follicle cells? Thank you for the valuable comments. We agree that hair follicular cells can contribute to skin physiology and the wound healing process23,24. However, in our Epi-iOSKM mice, hair follicle cells exhibited markedly low OSKM induction efficiency and showed minimal phenotypic changes, suggesting that they contributed little to the observed outcomes. Although the Krt14 promoter does not induce an entirely specific induction, both our observations and previous reports indicate that the relative efficiency of gene induction is substantially higher in the interfollicular epidermis (IFE) than in hair follicles when using Krt14-driven CreER systems (Jax #005107)25. In our model with the administered tamoxifen (TAM) and DOX dosages, we consistently observed that TAM-induced GFP expression and the subsequent mCherry expression were much more robust in IFE cells than in the hair follicle cells (new Supplementary Fig. 1b,c). Consistently, transient OSKM expressions induced under the Krt14 promoter did not alter the proliferative capacity of hair follicle cells (Rebuttal Fig. 2), whereas cell proliferation was markedly increased in IFE cells (Fig. 1j,k). Fluorescence induction in hair follicles of Epi-iOSKM mice was detected only at very low levels compared to that in the interfollicular epidermis (new Supplementary Fig. 1b,c) No detectable changes were observed in hair follicles of Epi-iOSKM mice (Rebuttal Fig. 2) Despite the low efficiency of OSKM-mCherry expression in hair follicle cells, we examined whether hair follicle cells influence the phenotypes observed in the Epi-iOSKM skin. As expected, the expression of EGF ligands (Tgfa, Epgn, and Areg), which can induce non-cell-autonomous effects in mChneg IFE keratinocytes, as well as immune cytokines (Tnf, Ccl8, Cxcl10, Ccl20, Il1b, and Cxcl16), which can induce T cell recruitment and activation, were not significantly altered in Epi-iOSKM hair follicle cells (Rebuttal Fig. 3). These results suggest that hair follicles had little influence on the phenotypes we reported in our study. Hair follicles of Epi-iOSKM mice show no detectable alterations (Rebuttal Fig. 3) Consistently, K14-CreER mice driven by the human K14 promoter have been widely used in studies specifically examining the characteristics of the IFE26-28. Moreover, Zhang et al. (Cell Cycle, 2010) showed that among several Krt14-CreER strains, the line used in our study (Jax #005107) exhibits relatively low sensitivity to tamoxifen and highly inefficient recombination within hair follicles, whereas K14-CreER2 (Jax #038390) is a more suitable model for studying both hair follicles and the epidermis25. In addition, because we induced OSKM expression using the K14-CreER line (Jax #005107) in combination with the Dox-inducible system, the observed activity may have been concentrated in the more efficient IFE. Taken together, while some recombination in hair follicle cells is expected, both our data and prior reports demonstrate that the human K14 promoter functions most strongly and consistently in the epidermis. Thus, in the context of our study, the induced genetic changes predominantly reflect biology in the epidermis rather than in hair follicles (please see also responses to Reviewer 2, question #3). Inefficient recombination was observed within hair follicles when using the same K14-CreER driver in our system (Zhang et al., Cell Cycle, 2010)25 Reviewer #2 (Remarks to the Author): Kwak et al. demonstrate that transient expression of reprogramming factors in a subset of keratinocytes promotes skin repair through cell-autonomous and non-cell-autonomous mechanisms. The study is interesting, and the data are generally convincing. The finding that partial reprogramming of cells promotes tissue repair is not completely novel and has been shown in other tissues. However, this is the first study that shows the effect of partial keratinocyte reprogramming on epidermal homeostasis and wound repair. In particular, the finding that reprogramming promotes repair by cell-autonomous and non-autonomous mechanisms is interesting. However, the study has also several weaknesses and raises some questions (see specific comments below). We appreciate the reviewer’s positive assessment and helpful suggestions, which helped us to further enhance the quality and impact of our study. General points: Q1. The authors show interesting phenotypes after transient expression of OSKM in keratinocytes in mice. This is likely a consequence of partial reprogramming, but it cannot be excluded that the phenotype is simply a consequence of the expression of one of these transcription factors. Can the authors exclude this possibility? Thank you for the thoughtful comments and suggestions. Investigating the contribution of individual transcription factors (TFs) to cellular reprogramming or partial reprogramming is indeed an important question, particularly for identifying the minimal TF combination that can confer plasticity. However, as this work represents the first investigation of mosaic partial reprogramming in the skin epidermis, our primary objective was to comprehensively characterize its phenotypes across multiple skin cell types during homeostasis and repair, including the complex interplays between partially reprogrammed cells and their neighbors/niches, rather than to dissect the role of each factor. While defining the contribution of each factor is meaningful for identifying molecular mechanisms of partial reprogramming and optimal utilization conditions, we believe our work, which has deeply and broadly elucidated the fact that their combination induces intriguing and valuable phenotypes, is already sufficiently comprehensive. Moreover, recent studies support that the effects of OSKM are largely mediated through their combined action. (A) Importance of the collective effects of OSKM in reprogramming Recent studies on partial and full reprogramming consistently indicate that no single Yamanaka factor alone can account for the broad phenotypes of reprogramming. To be specific, Sheng et al. (Nat Commun, 2018) reported that Oct4 is not essential for epigenetic rejuvenation under partial reprogramming29; likewise, Onder et al. (Nature, 2012) showed that Klf4 is not necessary for achieving reprogramming30. Furthermore, Sox2 plays a crucial role in full reprogramming as part of multimeric complexes with Oct4 and Klf4, rather than functioning alone31. Lu et al. (Nature, 2020) found that c-Myc is dispensable for in vivo partial reprogramming in some contexts32. Yucel et al. (Nat Commun, 2024) emphasized that epigenetic reprogramming is not attained through overexpression of a single factor, but rather through synergistic interactions among multiple factors and their downstream networks8. Taken together, these reports support that the phenotypes we observed are unlikely to be explained by a single factor, but rather by the combined action of OSKM. (B) c-Myc is not a main contributor to the induction of healing features in the Epi-iOSKM epidermis Although definitive verification of each factor’s role in the epidermis, an approach that would require extensive resources and future studies, we performed exploratory experiments within the feasible scope of our laboratory. We introduced viral constructs carrying Oct4, Sox2, Klf4, or c-Myc sequences individually into the skin dermis of mice, with the intention of assessing whether any single factor could induce regenerative features, the most crucial phenotypes observed in the Epi-iOSKM epidermis. However, as previously reported, the delivery efficiency to the epidermis was extremely low33,34, limiting meaningful analyses. As an alternative approach, we conducted the inhibition of a specific factor during partial epidermal reprogramming. In particular, we focused on c-Myc, which has been reported in certain other tissues as a non-essential factor for in vivo partial reprogramming while synergistic effects among OSK are still necessary32. To test whether c-Myc is dispensable in our model, we treated control and Epi-iOSKM skin with the c-Myc inhibitor 10058-F435. We found that Krt17/6a induction and HIF-1α activation were maintained in the Epi-iOSKM+10058-F4 epidermis (Rebuttal Fig. 4). These results indicate that c-Myc is not essential for the healing features induced by partial epidermal reprogramming. Although we cannot entirely exclude the contribution of c-Myc in mosaic partial epidermal reprogramming effects, our results demonstrated that the healing features can also be induced by the combination of Oct4, Sox2, and Klf4. Induction of healing features in Epi-iOSKM epidermis is maintained under c-Myc inhibition (Rebuttal Fig. 4) Although delineating the precise contribution of each OSKM factor will be an important avenue for future studies, our experimental results—together with recent reports—provide evidence that the phenotypes in Epi-iOSKM mice arise from the cooperative and synergistic actions of reprogramming factors in the epidermis. Q2. The translational relevance of the study is not very strong. It is unlikely that partial reprogramming will be used for the promotion of acute wound healing. The authors show the effect in diabetic mice, but this is a model for delayed acute healing, but not nor chronic wounds. In a chronic wound, the risk of transformation is likely to be high, and the effect of reprogramming may be different than in an acute wound. Thank you for the comments. They allowed us to refine the potential practical applications of our work and, with additional experimental data, to better address the translational relevance of partial epidermal reprogramming to promote wound healing. (A) Clinical implications of our data demonstrating enhanced healing in acute wounds The reviewer noted that our data are restricted to normal and delayed acute wounds, rather than chronic wounds. However, we believe our findings, which clearly demonstrate improved acute wound healing, are also of practical clinical relevance. Most human wounds are acute, but delayed healing can lead to secondary complications such as infection, which in turn may progress to chronic wounds16,17. Therefore, it is essential to treat acute wounds at risk of delayed healing promptly to prevent progression to chronicity. This is particularly important in surgical wounds, where delays in repair can cause more severe secondary damage. Accelerating acute healing is valuable in a broader clinical context, as it improves overall recovery and reduces the risk of long-term complications. In our study, we employed a silicone-splinted excisional model to minimize contraction and promote re-epithelialization-dominant closure36, thereby enhancing the translational relevance to human wound healing. Importantly, beyond the direct application of partial reprogramming, our data also provide mechanistic insights for developing new therapeutics for injury repair. We have shown that transient activation of epidermal cells at early stages of healing can be beneficial for tissue repair. This finding offers new avenues for developing pharmaceuticals aimed at enhancing wound healing, as discussed in the revised manuscript: “From a translational perspective, our findings possess the potential that not only OSKM expression but also other ways that results in epidermal plasticity in a mosaic and transient manner could enhance wound healing in a similar manner”. Particularly, we have demonstrated that partial reprogramming of the epidermis also contributes to enhanced dermal healing. This represents a novel and meaningful phenotype, as we have discussed in the manuscript: “This suggests that epithelial stem cell progenies act as a key player regulating the overall skin repair process, unlike most previous studies that focused on how these cells were regulated by their niches”. In particular, our new data demonstrate that increased epithelial HIF-1α activation is essential for enhancing not only epithelial but also dermal healing (new Fig. 7i-n; new Supplementary Fig. 15). This finding provides specific insights into how transient changes in the epidermis during the early stages of wound repair can facilitate dermal restoration and reduce scarring (please see also responses to Reviewer 1, question #3, and Reviewer 2, question #26). Reduced scarring is further associated with complete regeneration, which is characterized by the absence of scar formation and the induction of hair follicle neogenesis24,37,38; so, our findings hold significant potential to serve as a foundational basis for extensive practical applications. Collectively, we believe that the direct application of partial epidermal reprogramming may be feasible in certain contexts. Beyond this, our findings—highlighting the important roles of epithelium in orchestrating wound healing—should be valuable for researchers studying tissue repair and regeneration. Exploring other factors that can recapitulate the effects of partial reprogramming by targeting epidermal cells would be a promising avenue for future studies with translational potential. (B) Clinical implications of our data demonstrating enhanced healing in diabetic wounds with defective repair Although our findings already hold clinical value, as the reviewer noted, including data on chronic wounds would further strengthen the translational relevance. However, establishing a truly chronic, non-healing wound model in mice remains highly challenging. Numerous mouse studies attempting to improve chronic wound healing have established wound conditions using various factors known to promote wound chronicity such as diabetes, oxidative stress, or infection. However, most attempts produced delayed wound healing rather than non-healing wounds39-43. There is no rodent model that fully replicates human chronic wounds that do not heal39,44-46. Nevertheless, fortunately, frequently utilized defect healing models in laboratories, such as the hyperglycemia mice, still provide highly translational relevance by enabling the study of potential therapeutics targeting key drivers of severe impairments in wound healing39-43,47. Consistent with this rationale, we employed the streptozotocin (STZ)-induced hyperglycemia mouse model (new Fig. 8; new Supplementary Fig. 16), the most widely used impaired repair model, to investigate the effects of partial epidermal reprogramming on defective wound healing. In addition to our original data, and in response to question #25 regarding validation of this model, we further characterized the diabetic wound healing process (new Fig. 8; new Supplementary Fig. 16). We then identified several previously known wound healing disorder phenotypes induced by hyperglycemia48-50 in our model. Healing was not only delayed (revised Fig. 8b,d,e), but the proliferative ability of epithelial cells was also markedly reduced in STZ-treated mice (new Fig. 8c). Furthermore, the angiogenesis and dermal healing were significantly impaired (new Fig. 8d,f; new Supplementary Fig. 16d). These new results demonstrate that our diabetic model exhibits not merely delayed healing, but the defective healing with pathophysiological alterations. In this defect model, partial epidermal reprogramming exhibited broad beneficial effects. Wound closure rates increased (revised Fig. 8b), and re-epithelialization was accelerated (revised Fig. 8d,e). Moreover, the proportion of proliferating epidermal keratinocytes were increased at 3 days post-wounding (dpw) (new Fig. 8c). In wildtype mice with no diabetes, epidermal proliferation was affected by partial reprogramming only under homeostatic conditions, but not during repair (new Supplementary Fig. 14b), suggesting partial epidermal reprogramming effects are distinct in diabetic mice compared with those in wildtype mice. Further, distinctive effects of partial epidermal reprogramming on diabetic models were observed also in dermal healing. Unlike in wildtype mice, where partial epidermal reprogramming only altered the pattern of angiogenesis, in the diabetic model where angiogenesis was sharply reduced, partial epidermal reprogramming restored the diminished angiogenesis (new Fig. 8d,f). Reduced granulation tissue area by STZ treatment was also rescued by partial epidermal reprogramming (new Supplementary Fig. 16d). These results suggest that while the effects of partial epidermal reprogramming act differently in a diabetes model exhibiting dysfunctional repair processes, it ultimately improves wound healing in a positive direction even in disease models. Improvement of diabetic wound healing by partial epidermal reprogramming (new Fig. 8) Collectively, together with our observations that STZ treatment results in impaired wound healing with several pathogenic phenotypes, and literature indicating that STZ treatment is one of the most reliable approaches to generate impaired healing in rodents44,45,51, we believe our model sufficiently captures key aspects of pathological wound repair relevant to clinical translation. Moreover, our previous and newly added findings, subjected in new Fig. 8, clearly demonstrate the efficacy of partial epidermal reprogramming in this disease model to enhance wound repair, further supporting the clinical applicability of our findings. Specific points: Q3. Line 97: K14-CreER mice do not induce specific deletion in epidermal keratinocytes. The K14 promoter is also active in hair follicle keratinocytes and basal epithelial cells of other stratified epithelia. This sentence should be modified. In particular, it is unclear if and to what extent reprogrammed hair follicle keratinocytes contribute to the observed effects. Thank you for the suggestion and comments. We agree that Krt14 activity is not restricted solely to the interfollicular epidermis, as it is also expressed in hair follicles and some other stratified squamous epithelia. Accordingly, we revised the sentence to remove the word “specific”. Although the Krt14 promoter does not induce an entirely specific induction, both our observations and previous reports indicate that the relative efficiency of gene induction is substantially higher in the interfollicular epidermis than in hair follicles when using Krt14-driven CreER systems. In our model with the administered tamoxifen (TAM) and DOX dosages, we consistently observed that TAM-induced GFP expression, and the subsequent mCherry expression, were much more robust in keratinocytes of the interfollicular epidermis compared to those in the hair follicles (new Supplementary Fig. 1b,c). Consistently, transient OSKM expressions induced under the Krt14 promoter did not alter the proliferative capacity of hair follicle cells (Rebuttal Fig. 2), unlike the interfollicular epidermis, where cell proliferation was markedly increased (Fig. 1j,k). Fluorescence induction in hair follicles of Epi-iOSKM mice was detected only at very low levels compared to the interfollicular epidermis (new Supplementary Fig. 1b,c) No detectable changes were observed in hair follicles of Epi-iOSKM mice (Rebuttal Fig. 2) Despite the low efficiency of OSKM-mCherry expression in hair follicular keratinocytes, we examined whether hair follicle cells influence the phenotypes observed in the Epi-iOSKM skin. As expected, the expression of EGF ligands (Tgfa, Epgn, and Areg), which can induce non-cell-autonomous effects in mChneg interfollicular epidermal keratinocytes, as well as immune cytokines (Tnf, Ccl8, Cxcl10, Ccl20, Il1b, and Cxcl16), which can induce T cell recruitment and activation, were not significantly altered in Epi-iOSKM hair follicle keratinocytes (Rebuttal Fig. 3). These findings suggest that hair follicles likely had little influence on the phenotypes we reported in our study. Hair follicles of Epi-iOSKM mice show no detectable alterations (Rebuttal Fig. 3) Consistently, K14-CreER mice under control of the human K14 promoter have been widely used in studies specifically examining the characteristics of the IFE cells26-28. Moreover, Zhang et al. (Cell Cycle, 2010) reported that even among several Krt14-CreER strains, the line used in our study (Jax #005107) exhibits relatively low sensitivity to tamoxifen and highly inefficient recombination within hair follicles. They recommended another mouse called K14-CreER2 (Jax #038390) as a better model for studying hair follicle stem cells25. Additionally, because we induced gene expression from K14-CreER using the Dox-inducible system, the observed activity may have been concentrated in the more efficient interfollicular epidermis. Taken together, while some recombination in hair follicle basal cells is expected, the K14 promoter functions most strongly and consistently in the epidermis. Thus, in the context of our study, the induced genetic changes predominantly reflect biology in the epidermis rather than in hair follicles. Inefficient recombination was observed within hair follicles when using the same K14-CreER driver in our system (Zhang et al., Cell Cycle, 2010)25 Q4. The K14-CreER mice are known to be “leaky”. The authors should include GFP and mCherry fluorescence pictures of mice that were not treated with Dox. Thank you for this question, which allows us to include GFP and mCherry fluorescence data without Tam/Dox treatment. We have newly added these data in Supplementary Fig. 1d, confirming chemically induced target gene expression. K14-CreER mice may exhibit a very low background level of CreER activity, as has been occasionally reported52. However, in our experiments, no GFP expression was observed prior to Tam induction, and no mCherry expression was detected prior to Dox treatment (new Supplementary Fig. 1d). Consistently, previous studies using this K14-CreER line (Jax #005107) also reported the absence of leakiness in the absence of tamoxifen53. Thus, any leaky activity was negligible in our system and did not affect the reliability or specificity of our results. No detectable leakiness in the absence of tamoxifen and doxycycline in our system (new Supplementary Fig. 1d) Q5. In Fig. 1b the authors should add a high magnification hematoxylin/eosin staining of the skin. This is important to judge the overall histological phenotype of the mice. Thank you for the comments and suggestions. In addition to the results in Fig. 1b, we carefully examined whether short-term OSKM expression induces histological alterations in the skin using H&E staining. Our data show that three days of OSKM induction in epidermal keratinocytes did not cause detectable abnormalities in overall skin or epidermal architecture (new Supplementary Fig. 1e). Consistently, the epidermal thickness of Epi-iOSKM mice was comparable to that of control mice (new Supplementary Fig. 1f). Representative high-magnification H&E staining images of Epi-iOSKM skin (new Supplementary Fig. 1e,f) Q6. It is not clear what percentage of basal and suprabasal keratinocytes express OSKM. This information should be provided. Thank you for the comments. We obtained reliable quantitative data on the mosaic ratio in both the basal and suprabasal compartments of the Epi-iOSKM epidermis. Overall, the proportion of Sox2+ cells in the Epi-iOSKM epidermis was 23.10 ± 3.39%. Further quantitative analysis showed that Sox2+ cells accounted for 21.91 ± 3.97% in the Krt14+ basal layer and 25.55 ± 3.43% in the Krt14- suprabasal layer, indicating that OSKM induction efficiency was comparable between basal and suprabasal keratinocytes. This new data is provided in the new Supplementary Fig. 1j. Proportion of OSKM-induced cells in basal and suprabasal compartments of the Epi-iOSKM epidermis (new Supplementary Fig. 1j) Q7. Expression of Klf4, c-Myc and Oct-4 in the epidermis should also be confirmed (at least at the RNA level). This information should be available from the scRNA-seq data. Thank you for this comment. Our system incorporates the TetO-OSKM-mCherry transgene (vector map provided below), in which mCherry expression reliably marks OSKM induction, consistent with previous reports54. In the original manuscript, we verified co-expression of Sox2 and mCherry for the induction of TetO-OSKM-mCherry transgene (Fig. 1c). To further strengthen this point, we performed new immunostaining analyses and confirmed the induction of Oct4, Klf4, and c-Myc in mChpos cells of the Epi-iOSKM epidermis upon Dox treatment. These new data are included in new Supplementary Fig. 1g-i of the revised manuscript. Immunostaining showing Oct4 expression in mChpos cells (new Supplementary Fig. 1g) Immunostaining showing Klf4 expression in mChpos cells (new Supplementary Fig. 1h) Immunostaining showing c-Myc expression in mChpos cells (new Supplementary Fig. 1i) Q8. The scRNA-seq data suggest alterations in apoptosis. Does reprogramming affect apoptosis of mCherry+ and mCherry- keratinocytes? Thank you for this comment. To examine whether partial reprogramming induces apoptosis in mChpos or mChneg keratinocytes, we performed immunostaining for cleaved caspase-3. No significant signal was detected, indicating that partial epidermal reprogramming does not induce apoptosis in the Epi-iOSKM epidermis. These data have been added as the new Supplementary Fig. 2c. Immunostaining for cleaved caspase-3 revealed no detectable apoptosis upon partial epidermal reprogramming (new Supplementary Fig. 2c) We further investigated why the ontology term “programmed cell death” was changed upon partial reprogramming (Fig. 2a; Supplementary Fig. 5c) by analyzing the leading-edge differentially expressed genes (DEGs) that contributed most to this enrichment. Genes related to inflammation and epidermal differentiation, including Ntrk1, Cxcl10, Ccl2, Slc40a1, and Il1b 27,55-58, were upregulated. This suggests that cell death itself is not induced; rather, apoptosis-associated genes—many of which are also linked to regenerative processes—are increased. Another gene, Tmigd1, one of the most critical contributors to the “programmed cell death” enrichment, was upregulated upon partial epidermal reprogramming; however, this gene is known to inhibit apoptosis59,60, further supporting our cleaved caspase-3 staining data showing that partial reprogramming does not promote apoptotic cell death in either mChpos or mChneg keratinocytes. Q9. The reversibility of the reprogramming effect is particularly important. Are the mCherry+ cells completely lost at day 10 after Dox withdrawal? If yes, do they undergo apoptosis or are they lost by terminal differentiation? Thank you for the comments. Our mouse model utilizes a Dox-inducible gene expression system (Fig. 1a), in which Oct4, Sox2, Klf4, Myc, and mCherry are expressed only during the period of Dox administration54 (please see our response to question #4 and #7). In this Tet-On system, doxycycline binds to rtTA, inducing a conformational change that increases its affinity for the tetO binding site, as previously described61. This activation was validated in our model by the expression of mCherry and OSKM (Fig. 1b,c; new Supplementary Fig. 1d,g-j). Upon Dox withdrawal, ectopic gene expression ceases. Indeed, mCherry signals were almost absent three days after Dox withdrawal (Supplementary Fig. 2b), and no cell death was detected (new Supplementary Fig. 2c,d). Consequently, in our system, mCherry+ cells were not observed 10 days after Dox withdrawal; however, it cannot be determined whether the cells that expressed OSKM-mCherry during Dox treatment remained or were lost by that time. (Fig. 1a) Loss of mCherry signal after Dox withdrawal without cell death (new Supplementary Fig. 2b,d) Our data support the conclusion that partially reprogrammed cells undergo normal differentiation within the epidermal lineage. They acquired regenerative cellular characteristics (Fig. 2) and enabled them to return to the normal epidermal differentiation program. This is likely because three days of Dox treatment endowed the cells with plasticity and regenerative characteristics without causing escape from the epidermal lineage. Consistently, after partial epidermal reprogramming, none of the cells expressed the full reprogramming marker Nanog, whereas most of the cells expressed epidermal cell lineage markers such as Krt5 and Trp63 (Supplementary Fig. 7i-k). Partial epidermal reprogramming does not drive cells to escape from the epidermal lineage (Supplementary Fig. 7i-k) Partial reprogramming confers epidermal lineage-preserved plasticity (Fig. 2n) Fig. 2n illustrates our partial reprogramming model, in which cellular plasticity is conferred within epidermal cell lineages. IFE cells that acquired plasticity through partial reprogramming were converted into a state resembling IFE cells observed during a natural wound healing situation (Fig. 2). In typical wound healing, such wound-responsive cells return to the original state of normal IFE cells after healing62,63, and the partially reprogrammed cells in our study likely followed a similar trajectory. Consistently, after 10 days of Dox withdrawal, the Epi-iOSKM epidermis had returned to its normal state. The induction of Krt17/6a was reversed (Supplementary Fig. 7f,g), demonstrating that the effects of partial reprogramming are reversible. Most cells expressed Krt14 or Krt10, markers of basal and suprabasal epidermis, respectively, and the proportions of cells expressing these markers were comparable to those in normal epidermis (Supplementary Fig. 7h). This recovery occurred without cell death through apoptosis: Immunostaining for cleaved caspase-3 revealed no detectable signal in the Epi-iOSKM epidermis during Dox treatment (Supplementary Fig. 2c; see also our response to reviewer 2, question #8) or after Dox withdrawal (Supplementary Fig. 2d; Rebuttal Fig. 5). These findings suggest that the Epi-iOSKM epidermis returned to its original state without apoptotic cell death. Collectively, the partially reprogrammed cells likely re-acquired normal epidermal cell identity. Considering that the average cycle for mouse epidermal stem cells to fully differentiate is approximately 10 days64,65, by 10 days after discontinuation of Dox, it can be expected that some cells were likely differentiating within the normal epidermal cell lineages, while others had already been shed through terminal differentiation. Taken together, the complete reversibility of the changes highlights the potential safety of partial epidermal reprogramming for therapeutic applications, as it avoids permanent alteration of cell identity. Partial epidermal reprogramming is reversible (Supplementary Fig. 7f-h) No apoptosis occurs after withdrawal of partial reprogramming (Rebuttal Fig. 5) Q10. Fig. 3g and h: PI3K inhibition is likely to affect epidermal cell proliferation in general. The authors should add control mice treated with the PI3K inhibitor for comparison. Same for the EGFR inhibitor in Fig. 4. Thank you for the comments, which allowed us to strengthen our data by comparing the effects of PI3K and EGFR inhibition on Epi-iOSKM epidermis with those on control epidermis. We revised Figs. 3h and 4h, which originally showed the reversal of Krt17 expression following PI3K or EGFR inhibitor treatment, by adding new control groups in which the inhibitors were applied to the skin of control mice. These results demonstrated that PI3K and EGFR inhibition reduced the Krt17 level in the Epi-iOSKM epidermis to levels similar to those in the control epidermis. (revised Figs. 3h and 4h) In control epidermis, the inhibitor effects were not significant. As the reviewer noted, PI3K and EGFR pathways could influence Krt17 expression in control epidermis, either directly or indirectly, for example, by regulating cell proliferation9,66. However, as shown in new Figs. 3h and 4h, endogenous Krt17 levels are already low in normal epidermis. Moreover, PI3K and EGFR activity is also low in normal epidermis, as shown in Figs. 3f and 4f. Consistent with this, previous studies also reported that baseline p-EGFR and p-AKT signals are generally low in the epidermis and often near the detection limit67-69. Therefore, inhibitor treatments in control epidermis, which already exhibited low Krt17 expression and low PI3K/EGFR activity, would not have dramatic effects, as observed in our experiments. In conclusion, PI3K and EGFR pathways are essential for Krt17 expression specifically in the Epi-iOSKM epidermis. For Figs. 3g and 4g, which examined HIF-1α activity, we also added data comparing the effects of inhibitor treatment on control epidermis in Supplementary Figs. 8c and 9e, respectively. Similar to the results for Krt17, inhibitors had no significant effects in control mice, confirming that PI3K and EGFR inhibition reduced HIF-1α activity only in the Epi-iOSKM epidermis. (revised Supplementary Fig. 8c and 9e) Q11. The result in Fig. 3g requires quantification. Thank you for the comments. We have added the quantification data of Fig. 3g in new Supplementary Fig. 8c, which confirms the reduction observed upon PI3K inhibition. Please see also our response to question #10. Q12. Fig. 3j: It has not been shown that PI3K directly regulates keratin 6/16/17. This may be simply a consequence of the increased proliferation. Thank you for this comment. We agree that our data do not demonstrate a direct regulation of Krt6a/16/17 by PI3K. Accordingly, we have revised the model in Fig. 3j to avoid implying direct causality. (revised Fig. 3j) Functionally, PI3K inhibition with LY294002 reversed Krt17 and Krt6a upregulation in Epi-iOSKM epidermis (Fig. 3h; Supplementary Fig. 8d), indicating PI3K activation is essential for Krt17/6a induction upon partial epidermal reprogramming. Although we cannot determine whether PI3K regulates Krt17/6a directly or indirectly, our data demonstrate that OSKM expression in epidermal keratinocytes activates the PI3K pathway, and this activation led to Krt17/6a upregulation as depicted in Fig. 3j. A point that was not fully elucidated in Fig. 3j is the relationship between the phenotypes observed in Epi-iOSKM epidermis, such as increased proliferation, Krt17/6a expression, and HIF-1α activation. Actually, these are closely related: HIF-1α activation can induce cell proliferation70,71; Krt17 expression also can induce cell proliferation11,72; Krt17 expression level is correlated with enhanced proliferation9,10; and Krt17 expression level increases under hypoxia/HIF-1α activation conditions73-75. In addition, EGFR activation via autocrine effects can also result in HIF-1α and Krt17 induction. Collectively, the outcomes of partial reprogramming, mirroring injury responses, can influence each other. To disentangle these effects, we utilized single cell transcriptomics, which distinguishes proliferating cells from non-proliferating basal/suprabasal cells, allowing us to discern correlations between increased proliferation and other phenomena such as Krt17/6a induction. Importantly, in Epi-iOSKM epidermis, our scRNA-seq data revealed that KRT17/6a was upregulated in non-proliferating Basal and Supra mChpos cells (Fig. 2g). This suggests that the PI3K pathway may contribute to Krt17/6a induction in two ways: (1) by expanding the pool of proliferating cells that intrinsically express high levels of Krt17, and (2) by elevating Krt17/6a expression even in non-proliferating basal and suprabasal cells. This highlights that the observed induction cannot be explained solely by enhanced proliferation. In summary, while our data demonstrate that Krt17 induction in Epi-iOSKM epidermis is not simply a consequence of increased proliferation, the complex correlations among proliferation, HIF-1α, and keratin expression cannot be fully disentangled at present. To avoid misinterpretation, we revised Fig. 3j so that the updated illustration no longer implies a direct link, thereby minimizing potential confusion. Q13. Line 292: NRG (neuregulin) does not activate the EGFR – it activates ERBB3 and ERBB4. Thank you for the comments. We agree with the reviewer that neuregulins (NRGs) are ligands for ERBB3 and ERBB4, but not direct ligands for EGFR (ERBB1)76. However, ERBB3 lacks intrinsic kinase activity and requires heterodimerization with other ERBB family members, such as EGFR or ERBB2, to transduce signals77. Indeed, EGFR/ERBB3 heterodimers can be activated in a complementary manner, with EGF binding to EGFR and NRG binding to ERBB3, thereby inducing full complex activation78,79. In this context, the upregulation of NRG observed in our data likely contributes to EGFR/ERBB3 complex activation, rather than direct EGFR activation. We have revised the text to clarify this point as follows: “Notably, signaling pathways associated with the ECM, such as FN1 and KLK, those activating EGFR, such as EPGN and EGF, and those promoting EGFR/ERBB3 complex signaling, such as NRG, were significantly activated in the Epi-iOSKM epidermis.” Q14. Fig. 4h: Please confirm the efficient EGFR inhibition by immunofluorescence staining for pEGFR. Thank you for the comments. We have added immunofluorescence staining data demonstrating effective inhibition of EGFR phosphorylation by AG1478, now provided in new Supplementary Fig. 9d. Effective inhibition of EGFR activation in Epi-iOSKM epidermis by AG1478 (new Supplementary Fig. 9d) Q15. The model proposed in Fig. 4j is interesting, but it is only based on immunofluorescence stainings. It is important to test if PI3K activation in cultured keratinocytes promotes EGFR activation. Thank you for the comments. We performed in vitro experiments using HaCaT human keratinocyte cell lines. Treatment with the PI3K activator 740 Y-P for 24 hours increased phospho-AKT levels, a hallmark for PI3K pathway downstream activation. Interestingly, the phospho-EGFR level was elevated together. We added this data as new Supplementary Fig. 9f. It is known that EGFR can function as upstream of the PI3K pathway. However, PI3K reciprocally affects EGFR activity, with context-dependent outcomes. Among several related reports, there are also some studies showing that PI3K activation induces EGFR activation, consistently with our results: for example, PI3K-AKT activation is required for UVB-induced EGFR activation in human keratinocytes80; PIK3CA gain-of-function mutations induce AREG-EGFR-ERK signaling81; and PI3K causes reactive oxygen species82,83, which can activate EGFR phosphorylation in keratinocytes84. Collectively, our original data and the new data prompted by this comment, emphasize that PI3K activation is a crucial intermediate step in partial epidermal reprogramming. PI3K activation induces EGFR activation in HaCaT cells (new Supplementary Fig. 9f) Q16. Line 321: IL1 and VEGF do not directly regulate keratinocyte proliferation. This sentence should be modified. Thank you for the comments. While IL-1 may not have direct mitogenic effects on keratinocytes, in vivo, it can promote keratinocyte proliferation through indirect pathways85. For example, IL-1β activation in epidermal keratinocytes has been reported to induce hyperplasia86. Although IL-1β itself does not act directly, it can indirectly stimulate keratinocyte proliferation by activating immune cells and fibroblasts87,88. VEGF has also been reported to promote keratinocyte proliferation. VEGF signaling via keratinocyte-expressed VEGFR1 can enhance keratinocyte proliferation89. In addition, correlations between VEGF and/or VEGFR1 expression and increased proliferation have been reported in wound healing and inflammatory pathological processes90,91. To avoid over-interpretation, we have revised the sentence in the manuscript as follows: “The cellular pathways identified in the Epi-iOSKM epidermis were associated with increased proliferation; EGF signaling, a key modulator for keratinocyte proliferation, as well as IL1 and VEGF signaling that can indirectly contribute, were significantly enriched.” Q17. SFRP1 is a secreted protein, which should act on mCherry- and on mCherry+ cells. How do the authors explain a selective effect on mCherry- cells if SFRP1 is indeed responsible? Thank you for the comments. We proposed that Sfrp1 overexpression and Wnt4/6/16 reduction contributed to WNT pathway downregulation in mChneg cells of Epi-iOSKM epidermis. As the reviewer noted, secreted Sfrp1 could affect both mChpos and mChneg cells; therefore, the observed differences suggest the involvement of an additional regulator. We identified PI3K pathway activation specifically in mChpos cells (Fig. 3e,f). This can activate the WNT-β-catenin pathway by inhibiting the β-catenin repressor GSK-3β92,93. Thus, mChpos cells are simultaneously affected by activation of PI3K pathway and gene expression changes to inhibit WNT ligands activity, respectively. In contrast, mChneg cells are affected only by gene expression changes, such as Wnt4, Wnt6, Wnt16 and RSPO3 downregulation and Sfrp1 upregulation, which can downregulate WNT-β-catenin pathway (Supplementary Fig. 10b,c). Accordingly, WNT-β-catenin pathway downregulation was observed exclusively in mChneg cells of Epi-iOSKM epidermis (Supplementary Fig. 10a). We have discussed this model, which may illustrate the mechanisms underlying cellular pathway alterations in the Epi-iOSKM epidermis in the revised manuscript as follows: “Despite these changes in gene expression suppressing the WNT pathway, we observed that the activity of WNT pathway was not altered in mChpos cells (Supplementary Fig. 10a). This may be the involvement of mChpos cells-specific PI3K pathway activation. PI3K-AKT activation is known to activate WNT-β-catenin pathway by inhibiting GSK-3β, a repressor of β-catenin.” Proposed model illustrating mechanisms underlying WNT pathway activity regulation in mChpos and mChneg cells (Rebuttal Fig. 6) Q18. Supplementary Fig. 5e: Am important control is missing: LGK alone (same for the migration experiment). It seems likely that this compound already inhibits cell proliferation in control cells. The very weak effect of EGF on proliferation is surprising. Thank you for the comments. We have now included LGK alone control in our in vitro experiments. The revised data are provided in Supplementary Fig. 10e,f (original Supplementary Fig. 5e,f). The weak effect of EGF observed in our earlier results is likely due to the inherently high proliferative capacity of the cell lines and the presence of growth factors in the culture media. To address this, we performed the proliferation assay under starvation conditions, which revealed a significantly greater increase in proliferation upon EGF treatment. In vitro EdU cell proliferation assay including LGK alone control (revised Supplementary Fig. 10e) In vitro scratch wound healing assay including LGK alone control (revised Supplementary Fig. 10f) Despite the well-known proliferative effects of the WNT pathway, the LGK alone group did not show a significant reduction in proliferation compared to control cell. This indicates that the inhibitory effect of LGK is particularly pronounced in keratinocytes whose proliferative ability is elevated by EGF. This aligns with prior studies showing that the effects of WNT inhibition depend on cell state or experimental conditions94. Specifically, it is consistent with earlier reports that porcupine inhibitors including LGK974 suppress the excessive proliferation of oncogenic mutant cells and tumor growth without affecting the proliferation of normal adult stem cells95,96. Together, the added control supports that WNT pathway inhibition could more effectively counteract the promotion of proliferative capacity by EGFR activation in the Epi-iOSKM epidermis. This further demonstrates that mCherry-neg cells in the Epi-iOSKM epidermis, simultaneously affected by upregulated EGFR signaling and downregulated WNT signaling, acquire properties favorable for wound healing without increased proliferation. Q19. Line 374: Epidermal T cells in mice are almost exclusively gd T cells. These cells are not recruited upon injury. They only populate the skin once during embryonic development (see publications from W. Havran and A. Hayday). The increase in these cells is likely a consequence of increased T cell proliferation, which should be tested. Alternatively, there may be an abnormal recruitment of ab T cells to the epidermis, which seems likely because there are more RORgt cells. Is there an increase in epidermal and dermal ab or gd T cells or both? Thank you for the comments and suggestions. We have supplemented our data to clarify the origin of the increased T cells observed in the skin of Epi-iOSKM mice. The increase in epidermal T cells upon partial epidermal reprogramming could result either from the proliferation of resident epidermal T cells or from the migration of T cells from other sites. In the original manuscript, we showed that increased ectopic RORgt cells—normally absent in the steady-state epidermis—in Epi-iOSKM skin had migrated from elsewhere, but we didn’t assess how epidermal T cell proliferation was affected. To test this, we analyzed the proportion of Ki67-positive proliferating cells among epidermal T cells. Both scRNA-seq and immunohistochemistry analyses demonstrated that partial epidermal reprogramming does not alter epidermal T cell proliferation. In the scRNA-seq data, the proportions of Ki67-positive cells among T cells were 6.25% (6/96) in controls and 6.45% (14/217) in Epi-iOSKM epidermis, indicating comparable levels between the two conditions (new Supplementary Fig. 11c). Consistently, immunohistochemistry revealed no difference in the proportion of CD3 and Ki67 double-positive cells between control and Epi-iOSKM epidermis (new Supplementary Fig. 11d). These results indicate that the increased T cells in Epi-iOSKM epidermis result from migration rather than local proliferation. The related data have been added in the new Supplementary Fig. 11c,d. T cell proliferation remains changed in Epi-iOSKM epidermis (new Supplementary Fig. 11c,d) As the reviewer noted, most T cells in the murine epidermis are γδ T cells—particularly, Vγ3+ T cells—that originate during embryonic development97. During injury repair, however, additional subsets of γδ T cells are further recruited to the epidermis; most of these are γδ T17 cells that express RORγt98-100. RORγt+ T cells can be αβ Th17 cells or γδ T17cells, and among these, RORγt+ γδ T17 cells are recruited to the epidermis and dermis via the CCL20-CCR6 axis100-102 during wound healing. Consistently, partial epidermal reprogramming recruited RORγt+ γδ T cells to the epidermis. It was demonstrated that partial epidermal reprogramming recruited RORγt+ cells by CCL20-CCR6 axis similarly with healing skin in the previous version of the manuscript (Fig. 5f). To further validate this, we examined the expression of marker genes for αβ and γδ T cells in our epidermal scRNA-seq data. Trdc, which encodes the delta chain of the T cell receptor and serves as a marker for γδ T cells, was highly expressed at comparable levels in both control and Epi-iOSKM skin (Rebuttal Fig. 7). In contrast, Trac, which encodes the alpha chain of the T cell receptor and is a marker for αβ T cells, was nearly absent in both groups. These results indicate that the infiltrating epidermal T cells are predominantly γδ T cells (Rebuttal Fig. 7). No increase in αβ T cell populations in the Epi-iOSKM epidermis (Rebuttal Fig. 7) We did not determine whether the recruited T cells in the dermis were αβ or γδ T cells, as this would require additional time and resources. Nevertheless, it is plausible that the dermis harbors similar T cell subsets to those observed in the epidermis, since CCL20–CCR6–mediated recruitment of RORγt⁺ cells likely occurs throughout the skin without a strict distinction between epidermal and dermal compartments (Fig. 5f, please see also response to reviewer 2, question #20). Q20. Lines 390-392: The effect of the CCR6 antagonist on the number of epidermal RORgt cells is not significant. Thank you for the comments. In the dermis, the increase in the number of RORγt+ cells was completely abrogated by CCR6 antagonist treatment. In the previous manuscript (lines 390-392), our point was that although the effect of the antagonist was less pronounced in the epidermis than in the dermis, the significant difference in the number of RORγt+ cells between control and Epi-iOSKM epidermis was also eliminated under CCR6 antagonist treatment (Rebuttal Fig. 8). Thus, it cannot be excluded that the mechanisms via CCR6 also operate in the epidermis. Previous Fig. 5f in the original manuscript (Rebuttal Fig. 8) In the revised manuscript, we now provide newly quantified data that more reliably demonstrate the contribution of the CCL20-CCR6 axis (revised Fig. 5f). In normal skin, RORγt+ cells are absent in the epidermis and present only in small numbers in the dermis (the first image of revised Fig. 5f, left). Upon activation of the CCL20-CCR6 axis via epidermal CCL20 expression, RORγt+ cells migrate toward the epidermis, leading to an overall increase in RORγt+ cells in both epidermis and dermis, with more accumulation in the adjacent dermis100-102. Because the CCL20-CCR6 axis recruits RORγt+ cells to both compartments through the same mechanism, the most accurate way to evaluate its function is to quantify cells across the whole skin, rather than separating epidermis and dermis. Consistently, our revised Fig. 5f, which quantifies total skin RORgt+ cells, shows a significant antagonist effect. As RORγt+ cells are far more abundant in the dermis than in the epidermis (revised Fig 5f, left), this result is in line with our previous quantifications showing a clear antagonist effect in the dermis. In the original manuscript, we analyzed epidermis and dermis separately to align with total T cell quantification in Fig. 5c. However, due to the very low number of RORγt+ cells in the epidermis, such comparisons are less reliable. Thus, the integrated quantification presented here provides a more accurate assessment of CCL20-CCR6 axis function and supports its key role in recruiting RORγt+ cells, as described in the revised manuscript. CCR6 antagonist rescues the recruitment of RORgt+ cells in the Epi-iOSKM skin (revised Fig. 5f) Q21. Page 16, first paragraph: It is not surprising that no tumors were observed after 10 days of Dox treatment, because epidermal skin tumors develop slowly. It may well be that tumors would develop at a later time point (perhaps even after discontinuation of Dox treatment). This possibility should at least be discussed. In any case, it is clear from these results that long-term OSKM induction in keratinocytes is detrimental. We appreciate this comment, as it prompted us to further examine the safety validation of our Epi-iOSKM model. Research on in vivo partial reprogramming, which inherently carries certain risks6,103, requires rigorous safety validation. While previous studies have reported adverse outcomes from whole-body OSKM expression in mice104,105, our results show that short-term OSKM induction in keratinocytes (3 days) did not cause any deleterious phenotypes. In line with the reviewer’s suggestion, we have now added detailed histological analyses demonstrating the absence of histological abnormalities in this condition as new Supplementary Fig. 1e,f (please see also response to reviewer 2, question #5). To further assess potential risks, we extended Dox treatment to 9 days and observed inflammatory phenotypes (Fig. 5h-j). Notably, after 10 days of Dox withdrawal, these inflammatory phenotypes persisted. The results of this experiment were described in the revised manuscript as follows: “Moreover, following 10 days of Dox withdrawal after a 9-day administration period, the mice exhibited other pathologies, such as dry skin, scaly ears, and hair loss, along with abnormal skin histology characterized by excessive thickening of the stratum corneum and an increased number of CD3+ T cells in both the epidermis and dermis (Supplementary Fig. 12g-i).” Skin inflammation pathologies induced by 9-day prolonged OSKM expression in the epidermis persisted for 10 days (Supplementary Fig. 12g-i) To address the reviewer’s comments regarding whether prolonged OSKM induction in keratinocytes for 9 days might lead to cancer or other severe long-term outcomes after a long time has passed, we examined Epi-iOSKM skin phenotypes 30 days after DOX withdrawal following 9 days of OSKM induction. Unexpectedly, our new findings suggested the reversibility of the induced phenotypes by 9-day induction. The epidermis recovered from hyperplasia (new Supplementary Fig. 12j,k) and the pluripotency marker Nanog was not expressed (new Supplementary Fig. 12l). These findings indicate that prolonged OSKM expression for 9 days did not cause progressive epidermal pathology over time. We speculate that this reversibility may be due to the cells that expressed OSKM did not reach a pluripotent state. The phenotypes observed after 9 days of OSKM induction were consistent with inflammatory changes associated with epidermal hyperplasia (Fig. 5h-j), rather than neoplasm formation (Supplementary Fig. 12b,c). In many instances, inflammation caused by epithelial perturbations without systemic immune compromise can be reversible within weeks to months, even when initially severe106. Our findings suggest that the Epi-iOSKM phenotype may follow a similar, reversible situation. Recovery from the prolonged OSKM expression effects (new Supplementary Fig. 12j-l) Although partial epidermal reprogramming is not fatal, unlike in vivo partial reprogramming in other tissues, it is evident that the longer continuous OSKM induction will eventually lead to deleterious effects. Therefore, we assessed the period of time required for such fatal outcomes, including tumorigenesis or death. In other tissues, OSKM induction caused death within only a few days103,105; however, even after long-term partial reprogramming of epidermal keratinocytes for a month, none of the six treated mice died. We further extended the Dox treatment durations. When the Dox treatment period lasted for more than six weeks, mice began to die. Using the surviving mice, we examined the results of 60 days of OSKM expression and observed the formation of tumors. Cells in the tumor highly expressed Nanog, suggesting teratoma formation (Rebuttal Fig. 1). Nanog induction and teratoma formation after 2 months of OSKM expression (Rebuttal Fig. 1) In summary, in vivo OSKM expression in epidermal keratinocytes for three days was beneficial for wound healing without adverse effects; for nine days, it induced skin inflammation that is recovered over time; and for two months, it caused both teratoma formation and mortality. Q22. The effect of partial reprogramming on wound closure is minor (Supplementary Figure S7A). This is not too surprising, because wound healing in healthy mice is highly optimized. The data on the length of the epithelial tongue and the transepidermal water loss are more convincing. It is essential to show histological stainings of cross-sections from wounds at different time points. Is the area of granulation tissue also affected? Thank you for the comments and suggestions. In our original analysis, we focused on the analysis of repairing wound-edge sections, consistent with recent notable studies in this field12,98, enabling a detailed assessment of cell- and tissue-level dynamics with diverse markers. We have now added cross-sectional images as requested, which, together with our original analysis, provide a broader characterization of the healing process. In particular, we assessed granulation tissue formation from H&E images of whole-wound cross-sections at both early (3 dpw) and middle (10 dpw) stages of wound healing. We added these data as new Supplementary Fig. 13b,c. Because we employed a splinted wound model and covered it with a semi-occlusive transparent film to maximize the effects of re-epithelialization, full-thickness wounds heal without scab formation, as previously reported107. In this model, granulation tissue thickness was increased in Epi-iOSKM mice compared to controls at both 3 and 10 dpw (new Supplementary Fig. 13b,c). In the original manuscript, we also reported that the scar thickness reduced after wound closure (Fig. 6d), and collagen composition was more rapidly recovered in Epi-iOSKM mice (Fig. 6e,f; Supplementary Fig. 13e-i). The new and previous results show that granulation tissue formation is promoted during healing, and the tissue rapidly returns to its original state after healing, respectively, further supporting that partial epidermal reprogramming enhances dermal healing. Increased granulation tissue formation in wounded Epi-iOSKM skin (new Supplementary Fig. 13b,c) Q23. It is unclear if mCherry+ or mCherry- keratinocytes preferentially contribute to reepithelialization. This is mechanistically important and should be checked at different time points. Thank you for the comments and suggestions. It would be valuable to apply a lineage-tracing tool to directly determine how mChpos and mChneg cells contribute to re-epithelialization, which appears to be the basis of the reviewer’s suggestion. However, our current system does not allow such analysis. In our model, mCherry expression is limited to the period of Dox administration and ceases upon withdrawal (please see also response to reviewer 2, question #9). Consequently, after stopping Dox induction at 1 dpw, mCherry fluorescence cannot distinguish partially reprogrammed cells (mChpos) from their neighbors (mChneg), and indeed no mCherry signals were detected at 7 dpw. Although GFP fluorescence was observed in a substantial portion of the newly regenerative epithelium, this GFP+ population includes both mChpos and mChneg cells, indicating only that both contribute to re-epithelialization (Rebuttal Fig. 9). Immunofluorescence staining of GFP and mCherry in migrating epithelial tongues (Rebuttal Fig. 9) Despite this technical limitation, we confirmed that both mChpos and mChneg cells contribute to re-epithelialization, without an apparent preferential role, as evidenced by the key mechanisms underlying accelerated healing. In unwounded skin, the most prominent difference between the two populations in the Epi-iOSKM epidermis was the presence of increased proliferation in mChpos cells (Fig. 1j,k; Supplementary Fig. 4i), while other phenotypes associated with regenerative characteristics were observed in both populations (mChpos and mChneg cells) (Fig. 2). We then examined epithelial cell proliferation during repair at 3 dpw, but found no significant differences between control and Epi-iOSKM skin (new Supplementary Fig. 14b), indicating that mChpos cell-specific proliferation during DOX treatment alone cannot account for accelerated re-epithelialization. No significant difference in epidermal cell proliferation were observed between control and Epi-iOSKM skin at 3 dpw (new Supplementary Fig. 14b) In the original manuscript, we found that HIF-1α activity is increased in the healing epithelium even after discontinuation of Dox treatment (Fig. 7b,c), suggesting that sustained HIF-1α activation could be a potential driver of enhanced re-epithelialization. In additional experiments, we confirmed that elevated HIF-1α activity is required for the accelerated re-epithelialization observed in Epi-iOSKM skin. We have added this data as new Fig. 7i-k and new Supplementary Fig. 15a,b. The relevant details described in the revised manuscript are as follows: “When a wound occurs in Epi-iOSKM mice, HIF-1α is activated twice in the epidermis—first by partial reprogramming and again by the wound itself. To disrupt the synergistic effect of these two stimuli, we transiently inhibited HIF-1α activity by treating the HIF-1α inhibitor PX-478 once at the time of wounding (Fig. 7i). HIF-1α activity decreased at 1 dpw but recovered by 10 dpw as expected (Supplementary Fig. 15a,b). Interestingly, transient HIF-1α inhibition during the early repair phase reversed the accelerated re-epithelialization in Epi-iOSKM mice, bringing it to levels comparable to controls (Fig. 7j,k).” Experimental scheme for HIF-1α inhibition with wounding (new Fig. 7i) Effective inhibition of HIF-1α by PX-478 at 1 dpw with recovery at 10 dpw (new Supplementary Fig. 15a,b) HIF-1α activation is necessary for the enhanced re-epithelialization in Epi-iOSKM mice (new Fig. 7j,k) Increased activation of HIF-1α, essential for promoting re-epithelialization, was observed at comparable levels in both mChpos and mChneg cells of Epi-iOSKM epidermis (Fig. 2l and 7b), indicating that both cell populations contribute similarly to the accelerated re-epithelialization. Q24. The analysis of immune cells is restricted to T cells. However, macrophages are much more important players in wound healing. Is there a difference in the number of macrophages? Thank you for the helpful comments and suggestions. We assessed the macrophage proportions at the early stages of wound healing (3 and 7 dpw) and found no significant differences between control and Epi-iOSKM skin. These results have been included in the revised manuscript as new Supplementary Fig. 14e. No significant difference in the number of macrophages was observed between control and Epi-iOSKM skin at 3 and 7 dpw (new Supplementary Fig. 14e) As the reviewer noted, macrophages are important players in wound repair. They are usually activated by recognizing microenvironmental changes immediately after injury, for example through pattern recognition receptors108. Once activated, macrophages orchestrate various healing steps, including proliferation and migration of epithelial cells109,110. In contrast, T cells are primarily recruited and activated under the influence of epithelial cells and myeloid cells following the inflammatory phase99,111. Thus, it is reasonable that partial reprogramming of epidermal cells would have a greater effect on T cells than on macrophages. Consistent with this, our data show that most immune cytokines significantly upregulated by partial epidermal reprogramming, including Ccl20, Ccl8, Cxcl10, and Cxcl16 (Fig. 5e), are chemokines that primarily recruit T cells rather than macrophages99,112-114. Taken together, these findings suggest that T cells, rather than macrophages, are the main mediators shaping the immune niche in Epi-iOSKM skin. Q25. Line 468: Fig. 6i,j and Supplementary Fig. 7a do not show that wound healing is delayed in the streptozotocin-treated mice. This should be shown in one figure. Again, histological cross-sections from the wounds at different stages should be shown. This is important to fully judge the phenotypic alterations. Thank you for the suggestions. By reorganizing the experimental control group setup, we enabled simultaneous examination of the inhibitory effects caused by STZ and the recovery effects induced by partial epidermal reprogramming within one figure. Improvement of diabetic wound healing through partial epidermal reprogramming (new Fig. 8) Wound closure data are provided in the revised Fig. 8b, and re-epithelialization data are provided in the revised Fig. 8d,e, each encompassing the three experimental conditions (normal control mice, diabetic control mice, diabetic Epi-iOSKM mice). These results clearly demonstrate that wound healing is delayed in STZ-treated disease models. Furthermore, we found reduced epidermal cell proliferation and angiogenesis during the diabetic healing process (new Fig. 8c,d,f), indicating that this model represents not merely delayed healing, but a disease model that disrupts multiple repair processes. Moreover, our data show that these defective phenotypes, both previously recognized and newly identified, were recovered through partial epidermal reprogramming (please see also responses to Reviewer 2, question #2). We also provide H&E staining images of wound cross-sections at 3 dpw and 11 dpw with the same configuration as the data above: normal control mice, diabetic control mice, and diabetic Epi-iOSKM mice. This enabled an assessment of the overall histological alterations. Especially, the granulation tissue thickness was decreased in STZ-treated models than wildtypes as previously reported49,50 at 11 dpw, and this was restored by partial epidermal reprogramming (new Supplementary Fig. 16c,d). Recovery of granulation tissue formation by partial epidermal reprogramming in diabetic impaired wounds (new Supplementary Fig. 16c,d) Taken together, our reorganized and newly provided data clearly demonstrate that STZ treatment impairs multiple aspects of wound repair, and that partial epidermal reprogramming can effectively rescue these defects. Q26. Line494: It is correct that some reduction in the extent of angiogenesis can reduce scarring in mice. However, this is an indirect effects, which is not very strong. The extent of scarring strongly depends on myofibroblast differentiation. The authors should check if this is affected in the OSKM mice. It is mechanistically unclear how OSKM expression suppresses scarring. It may also be a consequence of the enhanced reepithelialization. Thank you for the comments, which give us the opportunity to further examine the mechanisms underlying reduced scarring in Epi-iOSKM mice. Through the analyses, we identified HIF-1α as a key regulator modulating the enhancement of both epidermal and dermal repair in Epi-iOSKM mice. As the reviewer noted, myofibroblasts can be contributors to regulating scar formation after wounding. To assess their presence, we performed immunostaining for αSMA, most commonly used marker of myofibroblasts115, in healing dermal region of control and Epi-iOSKM skin at 10 dpw. However, we did not detect significant differences in αSMA-positive myofibroblast abundance between two groups (new Supplementary Fig. 14d). No significant differences in the number of αSMA+ myofibroblasts were observed between control and Epi-iOSKM skin at 10 dpw (new Supplementary Fig. 14d) These results suggest that other mechanisms are involved. We therefore focused on HIF-1α as a potential candidate. The rationale for focusing on HIF-1α is described in detail in the revised manuscript as follows: “Previous studies have reported that forced activation of HIF-1α accelerates re-epithelialization. In our model, HIF-1α activation induced by partial reprogramming was sustained after wounding (Fig. 7c), suggesting it may continue to promote re-epithelialization even after transient reprogramming has ceased. Moreover, the altered patterns of HIF-1α activation correlated with reorganized angiogenesis (Fig. 7b-h), potentially influencing dermal repair. Notably, appropriate regulation of neovascularization during wound repair is known to reduce scar formation.” Indeed, HIF-1α activation in the epidermis was required for the reduced scarring in Epi-iOSKM mice. To test this, we transiently inhibited HIF-1α during the early phase of wound healing (new Fig. 7i), which reversed the accelerated re-epithelialization observed in Epi-iOSKM skin (new Fig. 7j,k; see also responses to reviewer 2, question #23). Experimental scheme for HIF-1α inhibition with wounding (new Fig. 7i) In addition to re-epithelialization, angiogenesis was also affected by HIF-1α inhibition. We have included these new data in Fig. 7j,l and Supplementary Fig. 15c,d of the revised manuscript with the corresponding description as follows: “Moreover, temporal inhibition of HIF-1α markedly reduced angiogenesis. Without inhibition, control mice displayed widespread angiogenesis across the wound bed, whereas Epi-iOSKM mice showed angiogenesis concentrated near the original wound site (Fig. 7f-h). Following PX-478 treatment, however, both groups exhibited a pronounced reduction in angiogenesis, with low CD31 expression at newly formed dermis near both the initial wound sites and leading edges (Fig. 7j,I; Supplementary Fig. 15c,d). These findings indicate that HIF-1α is a key regulator of neovascularization patterns during wound repair in both control and Epi-iOSKM mice.” Disturbed angiogenesis by HIF-1α inhibition during wound healing (new Fig. 7j,l; new Supplementary Fig. 15c,d) Finally, we found that the scarring itself was also influenced by HIF-1α, as shown in new Fig. 7m,n; new Supplementary Fig. 15e: “We next examined whether HIF-1α, which is essential for the alterations in re-epithelization and angiogenesis in Epi-iOSKM mice, also contributes to scar reduction. At 20 dpw, dermal thickness in the scarred area of PX-478 treated Epi-iOSKM mice was significantly thicker than that that of untreated Epi-iOSKM mice and was comparable to that of PX-478 treated control mice (Fig. 7m). Consistently, the alterations in collagen I deposition observed in Epi-iOSKM skin were restored by PX-478 treatment (Supplementary Fig. 15e). In addition, the reduced proportion of collagen III+ area observed in Epi-iOSKM mice was reversed by HIF-1α inhibition during partial epidermal reprogramming (Fig. 7n), indicating that elevated HIF-1α levels are essential for the reduction in scarring. Collectively, increased HIF-1α activation upon partial epidermal reprogramming—particularly the atypical pattern observed in the old epidermis near the initial wound site rather than in the newly generated epithelium (Fig. 7b-d)— substantially contributes to the widespread changes that promote wound repair.” Reversion of reduced scarring in Epi-iOSKM skin by HIF-1α inhibition (new Fig. 7m,n; new Supplementary Fig. 15e) These results illustrate how epidermal changes can influence dermal healing. HIF-1α, a responder to stressful environments, is rapidly activated after wounding and coordinates multiple repair processes12,21. When the control group was transiently treated with by a HIF-1α inhibitor, re-epithelialization was not altered, but dermal healing process was affected (Fig. 7j-n), suggesting that the primary role of HIF-1α during the early phase of wound healing is to coordinate dermal niche activation. Thus, the alterations in HIF-1α activity may broadly affect healing processes. Indeed, spatial and temporal regulation of HIF-1α is known to exert complex, context-dependent effects on healing, which can be either beneficial or detrimental 14,21,22. The Epi-iOSKM epidermis exhibited the elevation of HIF-1α activity as an unusual pattern: the signal was increased in the old epithelium that had undergone partial reprogramming, rather than in the newly regenerated epithelium (Fig. 7b-d). HIF-1α activation in wounded Epi-iOSKM epidermis (Fig. 7b-d) This alteration substantially remodeled wound repair mechanisms, including re-epithelialization, angiogenesis, and scarring (Fig. 9b). Given the complexity of the changes, it is challenging to establish direct causality among the different repair processes. Nevertheless, our results suggest the potential that accelerated re-epithelialization, accompanied by reduced neovascularization near the rapidly regenerating epithelium, contributes to decreased scar formation. Importantly, HIF-1α emerges as a key regulator coordinating these processes. Taken together, partial epidermal reprogramming, which induces healing characteristics, activated HIF-1α even prior to wounding (Fig. 2l), and wounding itself also activated HIF-1α. Consequently, the Epi-iOSKM epidermis during healing exhibited an atypical pattern of HIF-1α activation, which appears to promote coordinated repair of both the epidermis and dermis. Q27. Title of Fig. 1: The reprogramming does not drive epidermal dedifferentiation – it prevents differentiation (the K14 promoter targets non-differentiated basal cells). Thank you for the comments. The loss of differentiated characteristics in the epidermis can result from a block in the differentiation of basal cells or from the dedifferentiation of suprabasal cells. Among these two possibilities, our data strongly indicate that suprabasal keratinocytes undergo dedifferentiation upon partial epidermal reprogramming (Please see also responses to Reviewer 1, question #2). Firstly, our system using the Krt14-CreER; LoxP-STOP-LoxP-rtTA-EGFP mouse line can induce rtTA-EGFP expression and following OSKM-mCherry expression in both basal and suprabasal epidermal keratinocytes. This is shown in UMAP plots from scRNA-seq data of Epi-iOSKM mChpos cells (Fig. 1d; Supplementary Fig. 3d), and in staining results with quantifications of the proportion of Sox2+ cells, conducted according to the suggestion in Reviewer 2 question #6 (new Supplementary Fig. 1j). Both results demonstrate mChpos cells exist both in basal and suprabasal layer. In addition, fluorescent images in Fig. 1b and new Supplementary Fig. 1d show GFP expression in whole epidermal layer, and most of our fluorescent images show mCherry expression in suprabasal keratinocytes (Fig. 1b,c,h,k, etc.). OSKM induction in both basal and suprabasal layers of Epi-iOSKM epidermis (new Supplementary Fig. 1j) GFP and mCherry expression in both basal and suprabasal layers of Epi-iOSKM epidermis (new Supplementary Fig. 1d) This occurs because our system has two steps for ectopic gene expression: rtTA-EGFP expressions in Krt14+ cells following tamoxifen injection, and OSKM-mCherry expressions in their progenies under Dox administration. Two steps to induce OSKM-mCherry expression in our system (Fig. 1a) We note that the excision of LoxP sites by CreER activation upon tamoxifen administration is irreversible during physiological differentiation. Krt14 promoter targets non-differentiated basal cells, but these cells differentiate into the suprabasal keratinocytes by maintaining the genetic modifications acquired when they were basal epidermal cells. We injected tamoxifen for six days, took a day off, and administrated Dox for three days. Thus, OSKM induction occurred for a maximum of seven to ten days after CreER activation upon tamoxifen treatment. This is enough time to rtTA-expressing basal cells differentiate into suprabasal cells, by considering that it takes 4-5 days for keratinocytes to differentiate from the basal layer to the suprabasal layer, and 8-10 days for them to undergo terminal differentiation and be shed from the epidermis64,65. In conclusion, our system can induce OSKM expression in all epidermal keratinocytes differentiated from Krt14+ epidermal stem cells. We have shown a loss of differentiated characteristics in the Epi-iOSKM epidermis, including reductions in differentiation marker expression and correlated pseudotime values (Fig. 1f-i; Supplementary Fig. 4c,e,f, new Supplementary Fig. 4d,g,h). We also identified that partial epidermal reprogramming induces plasticity within epidermal cell lineages (Fig. 2; Supplementary Fig. 8). In addition to these, our original and new data from RNA velocity analyses clearly demonstrate that these findings reflect dedifferentiation, not the perturbation of differentiation. Specifically, mChpos and mChneg cells of the Epi-iOSKM epidermis exhibited cell fate transitions from Supra to Basal IFE cell, showing an opposite pattern to the control cells, which displayed a differentiation pathway from Basal to Supra cells (Fig. 1l). Moreover, we provide new consistent data with marker gene expressions. In control cells, the trajectory is positively correlated with the increase of epidermal differentiation marker Krt10 and Dsg1a expression, and the decrease of epidermal stem cell marker Krt14 and Itga6 expression. However, Epi-iOSKM cells exhibited the opposite pattern. The partially reprogrammed cells and their neighbors were transited from epidermal differentiation marker (Krt10, Dsg1a)-highly expressing cells to epidermal stem cell marker (Krt14, Itga6)-highly expressing cells, representing a reversal of the normal differentiation process (new Fig.1m; new Supplementary Fig. 4l-n). These data demonstrate the partial dedifferentiation of epidermal keratinocytes within their lineages in cell-autonomous and non-cell-autonomous manners. Cell fate transition from Supra to Basal cells under partial reprogramming (Fig. 1l) Cell fate transition from differentiated to stem-like IFE cells under partial reprogramming (new Fig. 1m; new Supplementary Fig. 4l-n) References 1 Kurian, L. et al. 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The HIF1a inhibitor studies strongly suggest that HIF1a is required for wound angiogenesis (as expected from the literature) and also reduces dermal thickness. However, this was seen in control and in iOSKM mice. Therefore, the authors cannot conclude that HIF1a inhibition SPECIFICALLY reverses the iOSKM effect. The data on collagen III positive area suggest a specific effect of HIF1a inhibition on the iOSKM effect, but the variability of the data is high. I believe that this point requires further clarification in the manuscript. There are probably additional mechanisms that contribute to the effect of iOSKM on the dermis/granulation tissue.\nIt is difficult to explain how enhanced reepithelialization inhibits angiogenesis. Usually, it is the opposite, because keratinocytes are an important source of VEGF. This point should also be discussed.\nTaken together, the (non-cell-autonomous) mechanisms underlying the alterations in dermal repair remain speculative, and therefore, this part of the study remains descriptive. This limitation should be mentioned in the discussion.\nThe questions of reviewer 2 have been appropriately addressed."}, "author_response": "We sincerely thank Reviewer 2 for the careful and thorough evaluation of the revised manuscript, including the reassessment of the comments originally raised by Reviewer 1. We are pleased that the reviewer finds the manuscript to be substantially improved and that most concerns have been satisfactorily addressed. Below, we provide detailed responses to the remaining points, particularly those related to the interpretation of the HIF-1α experiments and dermal repair mechanisms. Specificity of HIF-1α inhibition in Epi-iOSKM skin We agree with the reviewer’s assessment and have revised the manuscript to clarify this point. Our data indicate that HIF-1α inhibition affects dermal repair in both control and Epi-iOSKM skin, demonstrating that HIF-1α functions as a general regulator of wound healing rather than a factor acting exclusively under partial reprogramming conditions. Accordingly, we have revised our conclusions and now describe HIF-1α as an essential mediator of enhanced dermal repair in Epi-iOSKM skin, but not a reprogramming-specific factor. While HIF-1α inhibition reversed the enhanced dermal healing phenotypes observed in Epi-iOSKM skin, similar effects were also observed in control skin (Fig. 7k–n). This point is now clearly stated in the revised Discussion. Relationship between enhanced re-epithelialization and altered angiogenesis We appreciate this insightful comment and have expanded the Discussion to directly address this apparent paradox. In the revised manuscript, we propose that the altered angiogenesis pattern in Epi-iOSKM skin may be explained not by reduced pro-angiogenic signaling itself, but by the spatial distribution of HIF-1α activation. Specifically, HIF-1α activation was predominantly detected in the pre-existing epidermis undergoing partial reprogramming, rather than in the newly regenerated epidermis at the leading edge (Fig. 7b–d). Given that HIF-1α regulates angiogenic and chemotactic signaling pathways1,2, stronger signaling near the initial wound site could bias neovascularization toward this region and limit vessel extension toward the migrating epidermal front. We now clearly state that this explanation remains hypothetical and that a direct causal link has not yet been demonstrated. Speculative and descriptive nature of non-cell-autonomous dermal mechanisms We agree with this assessment and have revised the Discussion accordingly. We now explicitly state that, although our study identifies HIF-1α as one of the key mediators linking epidermal partial reprogramming to altered dermal repair, the underlying non-cell-autonomous mechanisms remain incompletely defined. We emphasize that this aspect of the study is descriptive and hypothesis-generating, and we outline future studies aimed at dissecting how spatially and temporally restricted activation of epidermal stress-response pathways influences dermal repair and scar formation. In response to the reviewer’s comments, we added the following clarification to the Discussion section: Mosaic partial reprogramming in IFE cells promoted not only epidermal repair but also dermal repair. In particular, accelerated re-epithelialization was accompanied by altered angiogenesis patterns and reduced scar formation. Mechanistically, we identified epidermal HIF-1α activation as a key factor contributing to enhanced dermal healing in Epi-iOSKM skin. However, both accelerated re-epithelialization and HIF-1α activation are generally associated with increased angiogenesis, contrary to the angiogenic phenotype observed in Epi-iOSKM skin. One possible explanation for this unexpected angiogenesis pattern is the spatially biased hyperactivation of HIF-1α. Specifically, HIF-1α signaling was increased in the old epithelium that had undergone partial reprogramming, rather than in the newly regenerated epithelium (Fig. 7b-d). Because HIF-1α regulates angiogenic and chemotactic signaling pathways1,2, relatively stronger HIF-1α induction near the initial wound site compared to the leading edge may contribute to a spatial bias in neovascularization, with enhanced vascularization in the initial wound-adjacent dermis and reduced vessel extension toward the leading edge. Although this model is plausible, a direct causal relationship has not been demonstrated. To assess the contribution of HIF-1α to the repair phenotypes of Epi-iOSKM skin, we inhibited HIF-1α activity, which reversed the enhanced dermal healing phenotype. However, similar effects were also observed in control skin (Fig. 7k-n), indicating that HIF-1α does not function exclusively in the context of partial reprogramming. Indeed, HIF-1α is a stress-responsive transcription factor that is rapidly activated after wounding and coordinates multiple repair processes3,4. Taken together, our data identify HIF-1α as one of the key factors linking the effects of partial epidermal reprogramming to altered dermal healing. However, the precise mechanisms by which the transient activation of IFE cells leads to reduced scar formation remain to be elucidated. References 1 Pugh, C. W. & Ratcliffe, P. J. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med 9, 677-684 (2003). 2 Forsythe, J. A. et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16, 4604-4613 (1996). 3 Liu, S. et al. A tissue injury sensing and repair pathway distinct from host pathogen defense. Cell 186, 2127-2143 e2122 (2023). 4 Li, G. et al. A small molecule HIF-1alpha stabilizer that accelerates diabetic wound healing. Nat Commun 12, 3363 (2021)."}]

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