Subjects

Abstract

Large cutaneous ulcers are, in severe cases, life threatening1,2. As the global population ages, non-healing ulcers are becoming increasingly common1,2. Treatment currently requires the transplantation of pre-existing epithelial components, such as skin grafts, or therapy using cultured cells2. Here we develop alternative supplies of epidermal coverage for the treatment of these kinds of wounds. We generated expandable epithelial tissues using in vivo reprogramming of wound-resident mesenchymal cells. Transduction of four transcription factors that specify the skin-cell lineage enabled efficient and rapid de novo epithelialization from the surface of cutaneous ulcers in mice. Our findings may provide a new therapeutic avenue for treating skin wounds and could be extended to other disease situations in which tissue homeostasis and repair are impaired.

Main

The epidermis is the outermost layer of the body, helping to maintain organismal homeostasis by protecting against environmental insults and preventing water loss. The multi-layered epidermis is maintained by stem and progenitor cells within the basal layer1 (that is, basal keratinocytes). An important step in repairing damaged skin is the migration of keratinocytes from adjacent epidermis into the wound to promote re-epithelialization3. For large wounds, this process is inefficient. To improve patient outcomes, more rapid and efficient methods for regenerating epidermal coverage must be developed, preferably non-surgical interventions. After recent advances in cellular reprogramming4,5, we reasoned that wound-resident cells could be reprogrammed towards an epidermal progenitor cell fate to generate new epithelial cells, thereby promoting de novo epithelialization from the surface of a cutaneous ulcer.

We initially sought to reprogram mesenchymal cells (human dermal fibroblasts (hDFs) and adipose-derived stromal cells (hADSCs)), as they participate in wound healing3,6. By comparing gene-expression profiles of human keratinocytes and primary hDFs (Extended Data Fig. 1a, b), upstream-promoter analyses (Supplementary Tables 13) and gene-expression reversal analysis (Extended Data Fig. 1c, Supplementary Table 4), we identified 55 transcription factors and 31 microRNAs that are potentially involved in keratinocyte specification (Supplementary Table 5). These candidates were further tested by assessing expression levels after calcium-induced differentiation of keratinocytes (Extended Data Fig. 1d), and by transducing each candidate into hDFs and measuring the levels of the keratinocyte markers, keratin 14 (KRT14) and cadherin 1 (Extended Data Fig. 1e). Selected factors were then co-transduced (via lentivirus) in different combinations into hDFs and the generation of keratinocyte-like cells was assessed (Extended Data Fig. 1f–k, Supplementary Table 6). A combination of 28 transcription factors generated keratinocyte-like cells in vitro. When these cells were grown in 3D organotypic culture, they formed a stratified epithelium (Extended Data Fig. 1l). We therefore named these cells 28TF-induced stratified epithelial progenitors (28TF-iSEPs).

To exclude the possibility that keratinocytes were contaminating the cell-isolation process, we switched to working with hADSCs. To improve transduction efficiency and lower cytotoxicity, we switched to retroviruses (Extended Data Fig. 1n, o). Using this system, we systematically eliminated redundant factors by: (1) excluding factors not integrated into the genome of 28TF-iSEPs (Extended Data Fig. 1m); (2) removing one factor at a time4; and (3) measuring keratinocyte phenotypes (Supplementary Table 7). Transduction of DNP63A (deltaNp63alpha, an isoform of TP63) and GRHL2 reprogrammed hADSCs into cells similar to 28TF-iSEPs (Extended Data Fig. 1p–s). An additional round of screening revealed that: (1) MYC (also known as c-MYC) enhanced reprogramming efficiency, cell proliferation and epithelial stratification, and (2) TFAP2A quickened the emergence of colonies (Supplementary Table 8, Extended Data Fig. 2a–m). Thus, the optimal combination of reprogramming factors was DNP63A, GRHL2, TFAP2A and MYC (DGTM factors). It is important to note that these in vitro-generated epithelia lacked cornification and expressed keratin 13 in the suprabasal layer (characteristic of mucosal epithelium7, foreskin epidermis8, and hyper-proliferative skin9), not keratin 10 (characteristic of adult skin epidermis7) (Extended Data Fig. 2h, m). As epithelia are potently influenced by their niche10, we hypothesized that applying DGTM factors in vivo would more effectively generate skin-like epithelial tissue.

To investigate whether in vivo reprogramming via DGTM factors could induce de novo generation of epithelial tissue from the surface of a skin ulcer, we developed an assay of isolated skin ulcers that simulated the central portion of a large cutaneous ulcer. We surgically removed skin from the back of mice to generate an ulcer and isolated the resulting wound from the surrounding skin using a skin chamber sutured to the deep fascia. This serves to prevent migration of keratinocytes into the wound, and closure of the wound by contraction (Fig. 1a). The absence of epithelial cells within these wounds was confirmed using Krt14cre;Rosa-CAG-loxP-stop-loxP (LSL)-tdTomato (hereafter Krt14cre;LSLtdTomato) mice, in which all cells that express Krt14 sometime during their lifetime are labelled (Extended Data Fig. 3a–h). In the absence of treatment, isolated wounds did not re-epithelialize (Fig. 1b). To test the DGTM factors in vivo, we switched to adeno-associated viruses (AAVs). As AAV cell tropisms differ between serotypes, we determined which AAV could be effective by subcutaneously injecting or directly applying green fluorescent protein (GFP)-expressing AAVs (GFP-AAV) to isolated ulcers. AAVDJ (a serotype of AAV capsid generated by DNA family shuffling11) resulted in the highest levels of GFP (Extended Data Fig. 3i–l). Furthermore, we assessed AAVDJ tissue organ distribution via injection of luciferase-AAVDJs into tail veins (that is, systemically), subcutaneous injection, or inoculation into the wound chamber. Regardless of the delivery method, luciferase expression distal to the injections site was primarily confined to the liver (Extended Data Fig. 3m).

Fig. 1: DGTM factors generate epithelial tissues through in vivo reprogramming.
Fig. 1

a, Schematic of the skin chamber used for separating the ulcer from surrounding skin. b, An ulcer in the chamber on the day of attachment (left) and 28 days later (right). Scale bars, 3 mm. n = 20, all similar results. c, Chambered ulcer 18 days after administration of DGTM-AAV (1.0 × 1012 gene copies). Haematoxylin and eosin (H&E) staining of sections through the generated epithelium. Red dotted lines indicate locations of the sections; yellow arrows indicate the generated epithelium. Black and white scale bars, 3 mm; red scale bars, 500 μm. n = 5, all similar results. d, Number of epithelial colonies 18 days after DGTM-AAV administration (titre indicated). GC, gene copies. e, Left, representative image showing an ulcer 28 days after DGTM-AAV administration. Yellow arrows indicate the periphery of the generated epithelium. Red dotted line indicates the position of the section shown middle top. Right (bottom), H&E staining of the generated epithelium. Right (top), magnified panels showing H&E staining of original skin and the generated epithelium. Black and white scale bars, 3 mm; yellow scale bars, 50 μm. n = 21, all similar results.

Source data

We next administered DGTM-AAVs (DNP63A-AAV, GRHL2-AAV, TFAP2A-AAV, and MYC-AAV) in our in vivo ulcer assay. Within 18 days, we observed epithelia-like tissue inside the chamber (Fig. 1c). Histological analysis of day-18 samples resulting from different titres of AAV (Fig. 1d, Extended Data Fig. 4a), revealed that 5.0 × 1011 gene copies of each factor was the minimal titre needed to efficiently generate epithelial tissue. This titre was used for subsequent in vivo experiments. Twenty-eight days after transducing isolated wounds with DGTM-AAVs, de novo epithelial tissues were histologically very similar to the skin adjacent to the wound edge (Fig. 1e).

To determine the proportion of transduced cells contributing to the healing process, we repeated these experiments using Pdgfracre;R26Rconfetti mice12, in which mesenchymal cells are randomly labelled with GFP, YFP, or RFP, although not all mesenchymal cells were labelled. Each epithelial cluster was derived from a single mesenchymal cell and as these individual clones grew, they intermingled with one another to form a single epithelium. The efficiency of in vivo reprogramming was estimated to be approximately 0.1% (Extended Data Fig. 4b–i).

To assess the role of each DGTM factor in reprogramming mesenchymal cells to epithelial cells, we tested different combinations of DGTM AAVs (for example, D, G, T, but not M). First, a lineage tracing system was introduced using PdgfracreER;LSLtdTomato mice6. Preoperative administration of tamoxifen labelled a broad spectrum of mesenchymal cells (but not epithelial cells) with tdTomato (Fig. 2a). Different combinations of DGTM AAVs were tested in vitro using Pdgfra+ mouse ADSCs (mADSCs) sorted from PdgfracreER;LSLtdTomato mice on the basis of tdTomato fluorescence (Extended Data Fig. 5a–c). After confirmation of in vitro reprogramming of mouse mesenchymal cells towards iSEPs with DGTM-AAVs (Extended Data Fig. 5d–j), different combinations of DGTM factors were tested at relatively lower titre, and the number of epithelial-like colonies counted on day 14 (Extended Data Fig. 5k–n). Epithelial-like colonies arose for all combinations that contained DNP63A (Fig. 2b). All DNP63A-containing combinations resulted in cells similar to primary keratinocytes (Extended Data Fig. 5o, p). Cumulative evidence suggests that the clonogenicity of cultured keratinocytes reflects their stemness, and thus their potential for repairing and maintaining epidermal tissue13,14. We therefore assessed the clonogenicity of epithelial cells generated using each of the DNP63A-containing combinations. On the basis of the expansion of single-cell clones (Extended Data Fig. 5q), clonogenicity was enhanced as the number of transduced factors increased (Fig. 2c). Next, we analysed the efficacy of the eight DNP63A-containing combinations (as well as GTM) in generating epithelial tissue in vivo using isolated skin ulcers, analysing the frequency and size of generated epithelia on day 28. These analyses also suggested an indispensable role for DNP63A in reprogramming (Fig. 2d–f, Extended Data Fig. 5r, s). For both frequency and size of the generated epithelia, GTM factors worked collaboratively with DNP63A to generate epithelial tissues. Notably, the oncogene MYC was dispensable for the de novo generation of epithelial tissues. Considering the in vitro emergence of epithelial cells and clonal ability, in vivo ulcer conditions (such as inflammation) may further constrain the generation and survival of epithelial tissue, thus leaving a relatively limited number of factor combinations that can effectively generate epithelial tissues in vivo.

Fig. 2: DGTM factors work collaboratively to generate epithelial tissues.
Fig. 2

a, Lineage tracing using PdgfracreER;LSLtdTomato mice. A representative image showing tdTomato fluorescence and KRT14 localization in skin from the back of one such mouse. Scale bar, 200 μm. Findings were confirmed in 42 animals used for lineage tracing studies. b, Number of epithelial shaped colonies 14 days after indicated combinations of transcription factors were transduced in vitro. Four cell lines from different animals were tested. Each cell line is represented by a different colour (n = 3 technical replicates). Overlaid dot plot indicates the distribution of the data. c, Surface areas of epithelial colonies derived from single cells. Transcription factor combinations are indicated. Whiskers represent the maximum and minimum, the box represents the range from 25th to 75th percentile and the centre line is the median. Overlaid dot plot indicates the distribution of the data (n = 11 for D, n = 12 for the rest). d, Experimental design schematic with a representative image of a DGM-AAV-treated wound. Red and dotted blue lines indicate area of the chambered ulcer (x) and the area of the generated epithelium (GE) (y), respectively. e, Number of animals with (colour bars) or without (white bars) generated epithelial tissues. f, Surface area of generated epithelial tissues.

Source data

To determine whether DGTM-generated epithelia are maintained over extended periods of time and whether it could promote the healing of large wounds, we subjected mice to multiple rounds of wounding. We used PdgfracreER;LSLtdTomato mice for these tests to ensure that DGTM-generated epithelia originated solely from mesenchymal cells (Fig. 3a, Extended Data Fig. 6a–e). After the generation of epithelial tissue within the isolated ulcer, the skin chamber was replaced with a larger one. Generated tissues laterally expanded within the large chamber (Extended Data Fig. 6f–h). After the large chamber was removed, generated tissue successfully connected to the surrounding epidermis while retaining a stratified epithelial structure (Fig. 3b, Extended Data Fig. 6i). As AAVDJ showed a specific tropism towards liver, we investigated possible histological alterations of the liver, complete blood count, and blood chemistry 3 months after AAV administration, with no evidence of pathology (Extended Data Fig. 6j–l).

Fig. 3: Generated epithelial tissue enables wound healing.
Fig. 3

a, Appearance (top left) and immunohistochemical analysis (top middle) of an ulcer 18 days after DGTM-AAV administration in PdgfracreER;LSLtdTomato mice. Localization of KRT14 and tdTomato are shown. In the photograph, the yellow arrows indicate the periphery of the generated epithelium and the white dotted line indicates the position of the histological section. In the immunohistochemistry image the white dotted outline indicates the position of magnified panels (bottom) and the white solid outline indicates the position of the highly magnified views of the epidermis and generated epithelium (right). White scale bar, 3 mm; black scale bar, 100 μm. Images are representative of the three independent experiments. b, Appearance (top left), stereoscopic analysis (bottom left), H&E staining (top right), and immunohistochemical analysis (bottom right) of skin and subcutaneous tissue including generated epithelium on day 253. Dotted lines in the left panels indicate the position of the sections shown on the right. Stereoscopic analysis revealed the area of generated tissue that is not evident by appearance. Dotted outlines indicate the positions of magnified panels. Scale bars, 5 mm. Similar findings were confirmed in all animals tracked for 6–9 months (n = 5).

Source data

To further assess the regenerative potential of DGTM-generated epithelial tissue, we generated ulcers within a large chamber, but left a small patch of skin intact in the centre of the ulcer. The ability of this skin island to expand and heal the ulcer could then be tested. We tested four types of islands: (1) intact skin, (2) epithelialized skin, (3) cell sheets and (4) DGTM-generated epithelia (Extended Data Fig. 6m–q). On average, epithelialization kinetics of intact skin, epithelialized skin, and DGTM-generated epithelia were similar, whereas the cell sheet group exhibited early contraction of the skin island and delayed epithelialization (Extended Data Fig. 6r, s).

To compare the histological properties of the generated epithelia and differentiated epidermis, samples were collected at day 18, days 28–30 and days 90–110 and analysed via immunohistochemistry. Similar to normal skin, epithelial tissues generated in vivo formed a cornifying envelope and expressed loricrin in the most superficial layers (Fig. 4a). Regarding keratins expressed in the suprabasal layer, generated epithelia could be classified into three types: keratin-10 (K10)+keratin-13 (K13), K10+K13+ and K10K13+ (Fig. 4a, Extended Data Fig. 6t). K10 is expressed in the natural skin epidermis, whereas K13 is expressed in mucosal epithelium7 (Fig. 4a) and hyperproliferative epidermis (for example, fetal skin9) (Extended Data Fig. 6u). Epithelial tissue that first emerged had sporadic K13 expression with or without K10, but this progressively shifted towards a K13K10+ pattern, thereby moving closer to the keratin pattern of natural skin epidermis (Fig. 4b). Thus, if allowed to mature for a sufficient amount of time within the wound niche, DGTM-generated epithelia exhibited histological characteristics of normal skin.

Fig. 4: Histological characterization and functional barrier properties of generated epithelia.
Fig. 4

a, H&E staining and immunohistochemical analysis of different types of generated epithelium, as well as adult skin and oral mucosa. Generated epithelia were obtained at day 104 for Type I (K10+K13) and day 18 for Types II (K10+K13+) and III (K10K13+). Scale bar, 100 μm. Images of skin and oral mucosa are representative of three independent experiments. b, Percentage of Types I–III generated epithelia on day 18, days 20–30 and days 90–110. c, Top, toluidine blue staining assay with a magnified image of the outlined region shown to the right. Middle, cross-section of stained sample with magnified images of the outlined region shown below including toluidine blue and H&E staining. GE, generated epithelia; SS, stained skin; Ul, ulcer; US, unstained skin. Black scale bars, 5 mm; red scale bars, 500 μm. d, Lucifer yellow (LY) dye penetration assay. Middle and bottom panels are fluorescence and H&E staining images, respectively. These helped to identify the border between the original epidermis (OE) and generated epithelium. Scale bars, 200 μm. c, d, Images are representative of three animals. e, TEWL values of generated epithelium, intact skin (IS), and ulcer from ten animals (days 90–110). The overlaid dot plot indicates the distribution of the data. Data are from three or four technical replicates.

Source data

One of the most important functions of the skin is to serve as a barrier against environmental insults. To assess the outside–in barrier function of DGTM-generated epithelia, a toluidine blue dye penetration assay15 was performed. On day 28, samples including the ulcer, DGTM-generated epithelia and surrounding skin were dissected and their external surfaces were exposed to toluidine blue dye. Similar to the surrounding skin, generated epithelial tissue effectively blocked dye penetration (Fig. 4c). To assess dye penetration after complete epithelialization, we used day-40 PdgfracreER;LSLtdTomato mice in which the skin chamber was removed on day 30. The generated epithelia and surrounding skin exhibited similar abilities to block penetration of lucifer yellow (after 1 h immersion)15 (Fig. 4d). To measure inside–out barrier function, transepidermal water loss (TEWL)15 was compared between generated epithelium (days 90–110), intact skin and a freshly created ulcer on the same animal. DGTM-generated tissues showed TEWL values equivalent to those of intact skin (Fig. 4e).

To assess the clinical relevance of this technique, we characterized the effect of DGTM-AAV on vascularity of the wound bed in our chamber model. DGTM-AAV showed no significant influence on vascularity of the wound bed (Extended Data Fig. 7a–d). Next, we investigated whether in vivo reprogramming could be used to heal an older wound. DGTM-AAVs were applied 7 days after creating the ulcer, and generation of an epithelium was observed in all ten animals tested (18 days after DGTM-AAV application). Notably, generated epithelia tended to be larger than seen with fresh ulcers (compare Extended Data Fig. 7e–g and Extended Data Fig. 4a). To improve the clinical relevance of this potential therapy, we sought to enhance the AAV delivery by administering AAVs via collagen gel. A GFP-AAV solution was mixed with an equal amount of collagen gel and then applied to ulcers in a chamber. The collagen gel increased both GFP expression and residual AAV copy number in ulcer tissue, and reduced AAV copy number in the liver (Extended Data Fig. 7h–k). Thus, collagen gel increased both the potency and specificity of the AAV system.

We reasoned that DGTM-AAV effectiveness could be further improved by adding bioactive molecules, such as FGF216,17 and/or a Rock inhibitor18. To test this, DGTM-AAVs in a collagen gel were applied to an isolated wound. Subsequently, from day 4, wounds were treated with Rock inhibitor and/or FGF2 until day 17. When applied alone, Rock inhibitor and FGF2 both enhanced healing. For wounds treated with both Rock inhibitor and FGF2, the wound surface was almost completely epithelialized within 2–2.5 weeks (Extended Data Fig. 7l–m). Thus, these two factors, in combination with a collagen scaffold, greatly enhanced in vivo reprogramming efficiency, resulting in concomitant de novo epithelialization from different regions within the ulcer leading ultimately to rapid wound healing.

To characterize in vivo reprogrammed cells, iSEPs were sorted from generated epithelia on the basis of tdTomato fluorescence 1, 3 and 6 months after AAV-DGTM administration. To investigate stemness13,14, clonogenic abilities of 3- and 6-month in vivo iSEPs were compared with primary keratinocytes from adult animals and one-day-old pups. In vivo iSEPs consistently showed higher clonogenic abilities than primary keratinocytes (Extended Data Fig. 8a–d). To determine whether this high clonogenic potential resulted from malignant transformation, we cultured in vivo iSEPs in soft agar and confirmed that they lacked anchorage-independent proliferative capacity (Extended Data Fig. 8e–f). To reveal their kinetics in vivo, 3-month in vivo iSEPs were subcutaneously transplanted into immunodeficient mice. For comparison, we also transplanted mouse embryonic stem cells (mES cells) that ubiquitously expressed GFP, HeLa cells that could be detected with a human-specific antibody, and mouse primary keratinocytes from KRT14cre;LSLtdTomato mice. In vivo iSEPs formed smooth, soft, round nodules that were larger than those formed by primary keratinocytes. Histologically, in vivo iSEPs nodules consisted of a large epithelial cyst and a small number of scattered cells within the transplant capsule, consistent with the histological characteristic of primary keratinocytes. Teratogenecity, as observed for mES cells, and transfascial invasive properties, as observed for HeLa cells, were not detected for in vivo iSEPs (Extended Data Fig. 8g–n). As a final test, we generated iSEPs that expressed luciferase and subcutaneously transplantated them into mice. Fifty-six days later, the iSEPs had remained in place, revealing low motility (Extended Data Fig. 8o–q). In summary, in vivo iSEPs formed large epithelial cysts without signs of malignant transformation. They instead behaved like non-malignant epithelial cells with a high proliferative potential.

To molecularly characterize DGTM-reprogrammed cells, RNA sequencing (RNA-seq) analysis was performed on: (1) in vivo iSEPs, (2) in vitro iSEPs (iSEPs generated from Pdgfra+ ADSCs with DGTM-AAV in vitro), (3) Pdgfra+ ADSCs (that is, mesenchymal cells), and (4) primary keratinocytes. When using AAV technologies in dividing cells, the typical goal is to transiently express a transgene in the targeted cells, but AAVs often integrate into the genome19. As our DGTM-AAVs express human versions of the DGTM factors, we could compare levels of AAV-derived gene expression (exogenous/human) to the endogenous/mouse genes. In vitro and in vivo iSEPs had high levels of human DNP63A expression and variable levels of human-GTM factors (Extended Data Fig. 9a). Levels of endogenous Trp63 expression were almost completely suppressed in iSEPs, whereas levels of endogenous GTM factors were similar to those of primary keratinocytes. Consistent expression of exogenous factors in iSEPs at different time points suggested the genomic integration of AAV-derived genes.

Clustering analysis of these RNA-seq data indicated that in vivo iSEPs, in vitro iSEPs, and keratinocytes were similar to one another (for example, they expressed high levels of keratinocyte marker genes), but very distinct from Pdgfra+ ADSCs. Transcriptomic analysis revealed that in vivo iSEPs were more similar to primary keratinocytes than in vitro iSEPs. The top gene ontology terms associated with both primary keratinocytes and in vivo iSEPs involved developmental and/or differentiation processes (Extended Data Fig. 9b–h). We compared a time course of gene expression changes for in vivo iSEPs to Pdgfra+ ADSCs and identified five clusters of genes (Extended Data Fig. 9i–l). The time course of gene expression changes may be influenced by the selective survival of clones with stem-like properties, diminishing effects of the wounding itself and resulting inflammation, and epithelial–mesenchymal interactions8,10,13.

In summary, reprogramming of wound-resident mesenchymal cells enables all regions of the wound to re-epithelialize, relieves the spatial constraints observed during normal healing (Extended Data Fig. 10) and leads to a regenerative functional response in the endogenous skin. Before clinical applications of this potentially transformative, non-surgical technology can be realized, further improvements must be made. These include improving reprogramming efficiency for more prompt epithelialization, and optimizing gene delivery methods to target specific human cell types20. Long-term safety studies must also be performed to evaluate off-target effects and tumorigenic potential, especially becuase DNP63A is a potential oncogene21. The system must also be tested using more clinically relevant animal models of injury. For example, preexisting immunity to AAV reduces transduction efficiencies in humans22. Thus, the inflammatory status of the wound, or other clinical features of the patient may affect reprogramming. Finally, a mechanistic understanding of adult stem cell dynamics and plasticity during wound repair is needed, including the extent of dedifferentiation and the inherent heterogeneity of the reprogrammed cells. Our observations constitute an initial proof of principle for functional in vivo regeneration of not only specific and individual cell types, but also of a three dimensional functional tissue in mice. This knowledge might not only be useful for enhancing skin repair, but could also serve to guide in vivo regenerative strategies in other human pathological situations in which tissue or organ homeostasis and repair are impaired.

Methods

Human iSEPs generation

Dermal fibroblasts or adipose-derived stromal cells were seeded at 3,000–5,000 cells/cm2 in 6-, 12- and 24-well cell culture plates. The next day, retroviral supernatant of DGBM factors was obtained and mixed with complete DMEM medium in a ratio of 4:1 v/v. Polybrene (Sigma) was added at a final concentration of 8 μg/ml. The plate was centrifuged twice at 800g for 30 min at 33 °C, then washed with PBS and changed to fresh medium. The medium was changed on days 1, 2 and 4. Cells were reseeded onto mitomycin C-treated 3T3-J2 feeder cells with F medium containing Y27632 as well as vitamin C at a final concentration of 50 μg/ml (Fisher). The medium was changed daily. Typically, epithelial shaped colonies could be identified after 7 to 9 days. When the colonies were ~1,000 cells23, the cells were passaged by removing the feeder cells and non-converted cells with an initial 1 to 2 min of trypsinization and PBS wash, and a further 5 to 10 min of trypsinization to dissociate the cells within the clones. Passaged cells were maintained in the same way as primary keratinocytes.

Mouse in vitro iSEPs generation

Adipose-derived stromal cells were seeded at 20,000 cells per well in 24-well culture plates. The next day, AAVs were mixed with complete DMEM medium. The medium was changed on days 1, 2 and 4. The medium was changed to F medium containing Y27632 and vitamin C from day 4 or 5. The medium was changed daily. After the emergence of colonies, cells were treated with the same protocols as human iSEPs.

Organotypic culture

Organotypic cultures were prepared using a previously reported method with some modifications10. Epithelial cells were cultured in 3D, at the air–liquid interface, on top of a dermal equivalent. Dermal equivalents were constructed by casting 1.0 × 106 BJ cells (ATCC CRL-2522) per 6 ml pyruvate (-) complete DMEM medium and 3 ml of bovine dermis-derived atelocollagen, I-PC50 (KOKEN) into a 60-mm Petri dish. The solution was allowed to form a gel and contract for 7 days. Final concentrations of collagen and fibroblasts were 1.7 mg/ml and 1.2 × 105 cells/ml, respectively. After loading the dermal equivalents onto an aluminium mount using nylon mesh, an acrylic ring (10 mm in diameter) was set on top and the cells were seeded at more than 4 × 105 cells/cm2 inside the ring with Y27632 containing F-medium. Skin equivalent cultures were maintained in a 60-mm Petri dish or in one well of a six-well plate. For the first 6 days, the concentration of Ca2+ inside and outside of the ring was gradually increased to 1.8 mM. On day 7, the volume of the medium inside the ring was reduced to the level of the epithelial cell sheet, so that the epithelial cells were grown at the air–liquid interface. The specimens were harvested on day 14 for histological and immunohistochemical examinations. The schedule for changing the medium during 3D organotypic culture is summarized in Supplementary Table 9.

Clonogenicity assay

The clonogenic ability of primary keratinocytes and epithelial cells generated using DGTM-AAVs was assessed by measuring the proliferative ability of single cell clones. First, the cells were inoculated in 96-well plates at a density of 1 cell per well after being passed through a 70-μm filter. For primary keratinocytes, 3–4 plates were required to obtain around 10 expandable clones, whereas for iSEPs, 1 plate is sufficient. Seven days later, only the wells containing a visually identifiable single colony were passaged to 12-well plates. Seven days later, the plates were stained with rhodanile blue (Alfa Aesar), photographed using a Gel Doc XR+ Imager (Bio-Rad), and analysed for the percentage areas of colonies for each well using ImageJ and Photoshop (Adobe).

Soft-agar colony formation assay

Soft-agar colony formation assay was performed as described elsewhere24,25. In brief, 2 ml of 0.5% base layer SeaPlaque agarose (Lonza) in DMEM containing 10% FBS, Keratinocyte feeder medium (KFM) or 3T3-J2 cell-conditioned keratinocyte feeder medium (CKFM) was plated in each well of six-well plates, and 5,000 cells suspended in 1.5 ml 0.5% SeaPlaque agarose (Lonza) in the same medium were placed over the base layer. Medium was replaced every 3 days. Three weeks later the clones were counted and photographed.

Tumorigenicity and teratogenicity assay

In our protocol approved by IACUC, a maximum tumour diameter of <20 mm was permitted and none of the experiments exceeded these limits. mES cells established from C57BL/6-TfN(act-EGFP) mice26 were a gift from F. Sugiyama. HeLa cells were obtained from ATCC (ATCC CCL-2). Primary keratinocytes were cultured from the back skin of Krt14cre;LSLtdTomato mice. In vivo iSEPs were sorted from primary culture of generated epithelium 3 months after the administration of DGTM-AAVs in PdgfracreER;LSLtdTomato mice. Cells culture medium (5.0 × 106 in 100 μm) were mixed with an equal amount of Matrigel (BD Biosciences) and subcutaneously injected into the back of NOD/SCID IL-2Rnull (NSG) mice. Twenty-eight days later, after hair removal, the size of the nodule was estimated with the equation; tumour volume (mm3) = (major axis) × (minor axis)2/2. Each nodule was collected with overlying skin and histologically investigated. For the assessment of motility and metastatic profile of iSEPs, luciferase-expressing cells were prepared by transduction of MSCV Luciferase PGK-hygro retrovirus (a gift from S. Lowe (Addgene plasmid 18782)) and drug selection. Fifty-six days after injection, the systemic distribution of the cells was investigated by the detection of luciferase.

Animals

C57BL/6 mice, NSG mice, PdgfracreER mice6, Pdgfracre mice27, Krt14cre mice28, Fsp1cre mice29, LSLtdTomato mice6, R26Rconfetti mice12, and RosamT/mG mice30 were purchased from the Jackson laboratory.

Mice of both genders were used in this study. All animal procedures were approved by the IACUC committee of the Salk Institute for Biological Studies or the Animal Research Committee of Kyorin University.

Lineage tracing animal

PdgfracreER;LSLtdTomato, PdgfracreER;RosamT/mG, Pdgfracre;LSLtdTomato, Pdgfracre;RosamT/mG, and Fsp1cre;LSLtdTomato mice were used for labelling a broad spectrum of mesenchymal cells to screen for the leakage of signal expression to the epidermis. All strains show aberrant epidermal labelling, but we noticed that a portion of PdgfracreER;LSLtdTomato mice was free from epidermal leakage. In other words, epidermal leakage with PdgfracreER;LSLtdTomato mice was specific to individual animals and at different anatomical locations. For a more stringent lineage-tracing system and to eliminate animals with epidermal leakage, we used PdgfracreER;LSLtdTomato mice. This was accomplished via a two-step selection process: (1) histological investigation of ear biopsies and (2) investigation with primary keratinocytes cultures of skin removed to generate the wound. All lineage-tracing animals were prepared by intraperitoneal injection of 200 μg/day tamoxifen (postnatal day (P)28–32), histological investigation of ear biopsies (P33), and investigation of primary keratinocytes cultures from the skin portion obtained during attachment of the chamber (P35).

Attachment of the chamber and rubber ring

To evaluate the de novo generation of epithelial tissues from the bottom of cutaneous ulcers, we aimed to induce epithelial tissues in skin ulcers that were isolated from the surrounding skin by a skin chamber. To avoid possible contamination from existing epithelial tissues, we performed these assessments using Krt14cre;LSLtdTomato mice. In these mice, cells developmentally expressing Krt14 are labelled. With the surgical removal of skin and panniculus calnosus, tdTomato signals were detected at the site of the subcutaneously located glands, such as the thyroid and mammary glands. Accordingly, to avoid these structures, two anatomical locations (interscapular area and lateral thoracic area) were chosen as optimal sites for chamber attachment.

Chambers were made up by cutting 1.5-ml Eppendorf tubes. A section was smoothened and broadened by warming with a flame. Two layers of holes were made using 22-gauge needles warmed using a flame. After tanning the internal surface by electric rasp, the chambers were autoclaved. Under general anaesthesia with isoflurane, the site of the chamber attachment was shaved and sterilized. Afterwards, a circle of skin and sub-cutaneous tissue of 1-cm diameter was removed beneath the panniculus carnosus, the chamber was sutured down to the deep fascia with 4–6 horizontal mattress sutures using 4-0 Ethilon (Ethicon Inc.). Then, 200 μl of Cell Matrix Type-1A collagen (Nitta Gelatin) was poured into the gap between the external surface of the chamber and the surrounding skin flap to ensure sealing of the chamber. After suturing the chamber to the skin flap with 4 horizontal mattress sutures, AAVs were administrated and the lid was closed. The edge of the skin flap was glued to the chamber when necessary. Large chambers were made by cutting 5-ml syringes (Becton, Dickinson and Company) using a metal knife warmed with a flame. Basic procedures for manufacturing and fixation were the same as used for the small chamber, except collagen sealing was not used. For the lid, a rubber disk from a syringe was used with a hole in the centre. Fixative rubber rings were made using rubber disks from 5- or 10-ml syringes. After making a hole in the central part, the ring was sutured to the skin and deep fascia to ensure fixation.

Maintenance of the wound

The wound in the chamber is prone to be contaminated because of the use of artificial implant and the closed nature of the wound. When chambers were first attached, in principle, the lids were kept closed to avoid contamination. When chambers were to be changed to larger ones, the lids were kept open for several days in advance, to dry up the wound and prevent carryover of contamination. After changing to the larger chamber, the wound was washed with PBS and photographed every two days under inhalation anaesthesia.

Skin island in chamber assay

An ulcer is generated within a large chamber, but a small patch of skin is left intact in the centre of this ulcer. Four animal groups are defined for comparison; (1) intact skin group, (2) epithelialized skin group, (3) cell sheet groups, and (4) generated epithelium group. For the intact skin group, an island of primary skin was created within a large chamber. For the epithelialized skin group, an ulcer was created and allowed to heal via epithelialization from the surrounding skin (a rubber ring was attached to prevent wound contraction). After epithelialization, the rubber ring was removed and an island of this epithelialized skin was created within a large chamber. For the cell sheet group, epithelial cell sheets were prepared by primary culture of keratinocytes from skin biopsies from each animal. Cell sheets were transplanted onto the backs of these mice via two operations: transplantation of the sheet beneath a silicone sheet, and removal of the overlying skin. An island of this cell sheet-generated epithelium was then created within a large chamber. We attempted to test pure grafted areas, but we found that skin purely composed of a cell-sheet transplant could not survive within a chamber for more than 4 weeks after transplantation. Therefore, we allowed the transplants to contract for another 1–2 weeks before subjecting the cell sheets to the skin island assay. For the DGTM-generated epithelium group, an ulcer was generated using PdgfracreER;LSLtdTomato mice and treated via DGTM-AAV administration. After removal of the chamber and confirmation of complete epithelialization, an island of DGTM-generated epithelium was created. For this group, we histologically confirmed that the tested skin island was composed of mesenchymal-reprogrammed epithelium by tdTomato fluorescence after completion of the assay. In each group, on experimental day 0, the skin island was created by circumferential removal of the surrounding skin and the large chamber was attached to make the skin island in an ulcer. Epithelialized areas from the skin island were measured by imaging analysis every 2 days until experimental day 14. For the DGTM-generated epithelium group, after completion of the study period, the tested skin island is subjected to histological analysis to test tdTomato expression in the epithelial layer and confirm that tested skin islands are entirely composed of mesenchymal-derived generated epithelium.

Estimation of nucleated cell number in deep fascia

Deep fascia was dissected out of the chamber, just after the attachment of chamber. Tissues beneath fascia were removed under dissection microscope. Dissected deep fascia was stained with DAPI and mounted on slide glass. The numbers of nucleated cells were counted in ten randomly selected visual fields in 3D images obtained using confocal microscopy. The area of deep fascia was measured in images obtained using a slide scanner. Using the areas and number of nucleated cells, total numbers of nucleated cells were calculated.

Estimation of efficiency of in vivo reprogramming

The efficiency of in vivo reprogramming was estimated by dividing the numbers of clones that contributed to each skin-chamber epithelium in Pdgfracre;R26Rconfetti mice by the numbers of estimated nucleated cells present in the deep fascia at the time of virus administration.

Blood test

Blood samples were obtained from animals that underwent DGTM-AAVs administration and multiple wounding procedures (PdgfracreER;LSLtdTomato mice) 3 months after the administration of DGTM-AAVs. Control samples were obtained from age (±2 weeks)-, sex- and genotype-matched non-operated animals. Samples were subjected to test haematology and chemistry assessment at the UCSD murine haematology and coagulation core laboratory.

Vascularity assay

Four groups of animals (n = 5) underwent chamber attachment and administration of VEGF165a-AAV (1.0 × 1011 gene copies (GC) per animal), DGTM-AAVs (1.0 × 1011 GC for each factor per animal), GFPNLS-AAVs (4.0 × 1011 GC per animal) or PBS to the inside of the chamber. The volume of solutions are adjusted to 100 μl in all groups. Seven days after application, chambers were collected and immunohistologically investigated for microvessel densities31. Sections are stained with H&E and for CD31 and subjected to imaging analysis. Density of vessels is quantified by the proportion of areas of vessels in 6–8 sections of the central portion of the ulcer for each animal. Tissues above and below the fascia are analysed.

Sample collection for RNA-seq analysis

For comparison of primary keratinocytes, iSEPs, and mesenchymal cells, three sets of (1) primary keratinocytes, (2) Pdgfra+ ADSCs, (3) in vitro iSEPs, and (4) in vivo iSEPs were prepared from three PdgfracreER;LSLtdTomato mice. On postnatal day 35, under general anaesthesia, groin adipose tissues and back skin was collected before chamber attachment and DGTM-AAV inoculation. From skin and adipose tissue, primary keratinocytes (1) and ADSCs were isolated and cultured, respectively. Pdgfra+ ADSCs (2) were sorted on the basis of tdTomato signal using a FACS Vantage SE DIVA. Using Pdgfra+ ADSCs, in vitro iSEPs (3) were generated with DGTM-AAVs. Mice underwent a stepwise operation to obtain generated epithelial tissues. On postnatal days 125–145, mice were euthanized, and generated epithelium tissues were collected. In vivo iSEPs were isolated with primary keratinocytes from surrounding skin. Pdgfra+ in vivo iSEPs (4) were sorted from contaminating primary keratinocytes on the basis of tdTomato signal using a FACS. Other in vivo iSEPs (1 month (1M), n = 3, 3M, n = 2, 6M, n = 5) were obtained from different animals with the same procedures described above. Total RNA was purified from each sample and subjected to RNA-seq analysis.

Toluidine blue penetration assay

Tissue samples were collected in a way that included generated epithelium, ulcer, and the surrounding skin. Samples were dehydrated by incubations (1 min each) in 25%, 50%, and 75% methanol in PBS with a further 1 min in 100% methanol. Then rehydrated with the same series of methanol solutions (1 min incubations) and washed in PBS. Soaking the whole sample caused staining from the backside of the sample and affected the results. Therefore, we attached the tube on the surface of the sample to partially include the surrounding skin, and added 0.1% toluidine blue O/PBS into the tube for 1 min before washing32. Samples were photographed. The frozen cross-section was also photographed. After sectioning, both non-stained sections and serial H&E sections were prepared to analyse the detailed location of toluidine blue stain.

Lucifer yellow dye penetration assay

Under general anaesthesia, mice were restrained in Petri dishes with generated tissues in contact with 1 mM Lucifer yellow in PBS (pH 7.4) at room temperature. After 1 h of incubation, mice were euthanized and tissues were dissected, frozen and sectioned at a thickness of 5 μm33. The sections were counterstained with 10 μg/ml DAPI and then analysed by fluorescence microscopy.

Transepidermal water loss (TEWL) measurement

On the day of measurement, hair was carefully shaved from the testing area in mice under general anaesthesia using isoflurane. Special effort was taken not to damage the skin. TEWL was measured using a VapoMeter (Delfin Technologies) with nail adapters at the area of generated epithelium and intact skin. Because TEWL measurements can be influenced by factors such as room and body temperatures and depth of anaesthesia, at least three rounds of measurements were taken. Subsequently, a portion of the skin was surgically removed to generate an ulcer (1-cm diameter) and the TEWL value was measured more than three times.

Statistical analysis

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability

Data from the microarray, RNA-seq and miRNA microarray analyses have been deposited in the Gene Expression Omnibus (GEO) and ArrayExpress under accession numbers GSE85803, GSE106419 and E-MTAB-5055, respectively. All other relevant data that support the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

This work was supported by MEXT KAKENHI Grant numbers JP26293381(Grant-in-Aid for Scientific Research (B) to M.K.), JP23689073 (Grant-in-Aid for Young Scientists (A) to M.K.), JP21689046 (Grant-in-Aid for Young Scientists (A) to M.K.), Kyorin University research promotion award to M.K. (2013), JSPS Overseas Research Fellowships (2015–17) to M.K., and the Uehara Memorial Foundation Research Fellowship for Research Abroad (2017–18) to M.K. M.K. thanks H. Green for support materials. T.H. thanks F. Sugiyama for support materials. M.N.S. is supported by NIH-NCI CCSG: P30 014195 and The Leona M. and Harry B. Helmsley Charitable Trust. Work in the laboratory of J.C.I.B. was supported by the G. Harold and Leila Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust, The Moxie Foundation, The Evergreen Foundation, Fundacion Dr. Pedro Guillen and Universidad Católica San Antonio de Murcia (UCAM).

Reviewer information

Nature thanks S. Akita, V. Horsley, A. Lombardo and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Affiliations

  1. The Salk Institute for Biological Studies, La Jolla, CA, USA

    • Masakazu Kurita
    • , Toshikazu Araoka
    • , Tomoaki Hishida
    • , David D. O’Keefe
    • , Yuta Takahashi
    • , Akihisa Sakamoto
    • , Masahiro Sakurai
    • , Keiichiro Suzuki
    • , Jun Wu
    • , Mako Yamamoto
    • , Reyna Hernandez-Benitez
    • , Alejandro Ocampo
    • , Pradeep Reddy
    •  & Juan Carlos Izpisua Belmonte
  2. Department of Plastic Surgery, Kyorin University School of Medicine, Tokyo, Japan

    • Masakazu Kurita
    • , Hitomi Eto
    •  & Kiyonori Harii
  3. Universidad Católica San Antonio de Murcia (UCAM), Campus de los Jerónimos, Guadalupe, Spain

    • Toshikazu Araoka
    • , Akihisa Sakamoto
    • , Masahiro Sakurai
    •  & Estrella Núñez Delicado
  4. The Razavi Newman Integrative Genomics & Bioinformatics Core, The Salk Institute for Biological Studies, La Jolla, CA, USA

    • Maxim Nikolaievich Shokhirev
  5. King Abdullah University of Science & Technology (KAUST), Thuwal, Saudi Arabia

    • Pierre Magistretti

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Contributions

M.K., K.H. and J.C.I.B. conceived and designed the experiments. M.K. conceptualized the study and designed and performed most of the experiments and analysis. T.A., T.H., M.S., M.Y. and R.H.-B. prepared the animals and embryos. T.A. and M.Y. helped with histological imaging. K.S., Y.T. and A.S. helped with preparation of plasmid constructs and AAVs. Y.T. and H.E. helped with 3D organotypic cultures. T.H. helped with tumorigenic assay and luciferase detection. M.N.S. analysed the RNA-seq datasets. M.K., D.D.O., J.W., A.O., P.R. and J.C.I.B. prepared the figures and wrote the manuscript. P.M., E.N.D., K.H. and J.C.I.B. coordinated and oversaw the study.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Juan Carlos Izpisua Belmonte.

Extended data figures and tables

  1. Extended Data Fig. 1 Selection of factors for reprogramming of human mesenchymal cells into iSEPs.

    a, Gene-expression analyses from DNA microarrays of human primary keratinocytes and primary hDFs. Pink or red indicates higher expression levels, green indicates lower expression levels. b, MicroRNA microarray analyses from three pairs of primary human keratinocytes and primary hDFs obtained from different tissues of a single subject. c, Reversal participation analysis of a candidate gene set (19 genes) for keratinocyte specification. d, Changes in the expression levels of candidate transcription factors after calcium-induced terminal differentiation of keratinocytes. e, Changes in the expression levels of keratinocyte markers after transduction of each candidate gene as assessed by quantitative PCR (qPCR). f, Schematic of the experimental design for the generation of iSEPs from primary hDFs. g, Morphological analysis of primary hDFs and human primary keratinocytes. Arrows indicate keratinocyte colonies on feeder cells. Scale bars, 200 μm. Similar results were observed five times for both cell types. h, Representative bright field images showing the morphology of colonies obtained during the selection of factors for the generation of iSEPs. Combinations of factors were optimized on the basis of colony morphology. Scale bars, 200 μm. i, Representative bright field images (left and middle) showing the morphology of colonies at passage 0 (P0) and passage 3 (P3) obtained after the transduction of 28 factors. The KRT14-RFP vector was transduced with the 28 factors (right). Red scale bars, 500 μm; yellow scale bars, 200 μm. j, RT–PCR analyses of keratinocyte markers. h, i, j, Images are from one experiment. k, Growth curve of primary keratinocytes and 28TF-iSEPs on feeder cells. Data are mean of technical triplicates. l, Representative images showing H&E staining of human skin and 3D organotypic culture of 28TF-iSEPs-hDF. Scale bars, 100 μm. Similar results were observed in two organotypic cultures. m, Transgene-specific PCR analyses of genomic DNA of 28TF-iSEPs. Each plasmid was used for comparative controls. Images are from one experiment. n, Schematic of the experiment for comparative assessment of transduction efficiency and cytotoxicity between concentrated enhanced GFP (eGFP)-expressing retroviruses and lentiviruses. o, Higher eGFP expression could be obtained with lower cytotoxicity with retroviruses than lentiviruses. Consistent findings were observed in three technical replicates for microscopic findings and flow cytometric analyses. Results of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay represent the mean of three technical replicates. Overlaid dot plots indicate the distribution of the data. p, Schematic of the experiment for generation of iSEPs-ADSCs. q, Representative bright field images showing colony morphologies during factor reduction. Scale bars, 200 μm. r, Transgene-specific PCR analyses of genomic DNA. Plasmids were used as controls. Integration of plasmid-derived sequences is described. q, r, Images are from one experiment. s, Representative images showing the morphological analysis and H&E staining of 3D organotypic culture of iSEPs-ADSCs generated by the transduction of DNP63A and GRHL2. White scale bars, 200 μm; black scale bar, 100 μm. Similar results were confirmed in two independent experiments. j, m, r, For gel source data, see Supplementary Fig. 1. o, For gating strategy example, see Supplementary Fig. 2. Source data

  2. Extended Data Fig. 2 Optimization of factors for reprogramming of hADSCs into iSEPs.

    a, Bright field images showing the representative morphology of colonies that emerged during generation of iSEPs after transduction of 67 combinations of factors tested in addition to the minimum factors DNP63A and GRHL2. Scale bars, 1,000 μm. b, Representative H&E staining images of 3D organotypic cultures of iSEPs induced with 35 combinations of factors. Scale bars, 100 μm. c, Rhodanile staining of cells generated with selected combinations of factors 14 days after transduction. d, Schematic representation of the experiment for testing the combinations of DG and non-MYC factors. e, Representative bright field images showing cell morphologies of generated iSEPs. Yellow scale bars, 1,000 μm; red scale bars, 500 μm. f, Quantification of iSEPs generated with different combinations of factors on day 22. Values represent the mean of three technical replicates. Overlaid dot plots indicate the distribution of the data. g, Growth curves of iSEPs and human primary keratinocytes. Cells with GRHL1 could not be isolated. Data are average of triplicates. h, H&E staining and immunohistochemical analysis of 3D organotypic cultures. Scale bars, 100 μm. i, Schematic representation of the experiment for testing the combinations of DGM+1 factors. j, Representative bright field images showing cell morphologies of generated iSEPs. Yellow scale bars, 1,000 μm; red scale bars, 500 μm. k, Quantification of iSEPs generated with different combinations of factors on day 19. Values represent mean of three technical replicates. Overlaid dot plots indicate the distribution of the data. l, Growth curves of iSEPs and human primary keratinocytes (hPK1 and hPK4), and the cumulative population doublings of iSEPs on day 60. Data are the mean of triplicates. m, H&E staining and immunohistochemical analysis of 3D organotypic cultures. Scale bars, 100 μm. a, b, h, m, Findings were confirmed in two technical replicates. c, e, j, Findings were confirmed in two independent experiments. Source data

  3. Extended Data Fig. 3 Defining the procedure of in vivo experiments.

    a, Lineage tracing using Krt14cre;LSLtdTomato mice. b, H&E staining and immunohistochemical analysis of ear biopsies from Krt14cre;LSLtdTomato mice. c, Exterior and stereoscopic view of Krt14cre;LSLtdTomato mice. d, Appearance and stereoscopic view of Krt14cre;LSLtdTomato mice after resection of skin. White arrows indicate signals from glands such as the thyroid and mammary glands. e, Appearance and stereoscopic view of resected mammary gland. f, Appearance and H&E staining of resected axillary skin and subcutaneous tissues including the mammary gland. g, Stereoscopic and immunohistochemical analysis of resected skin and subcutaneous tissues including the mammary gland. h, Appearance and stereoscopic analysis after chamber attachment. tdTomato signals were not detected inside the chamber when attached at the interscapular area and lateral thoracic area, whereas signals were detected when attached close to the axilla. The procedure was optimized not to include the mammary gland within the wound chamber. i, Schematic representation of the subcutaneous injection of AAVs into the back skin of mice. Eighteen serotypes of AAVs expressing eGFP under the CAG promoter were tested. j, Fluorescence images showing GFP signals of the injected skin and subcutaneous tissue 1 week after the injection of eGFP-expressing AAVs of different serotypes. AAVDJ resulted in the highest levels of GFP fluorescence. Scale bar, 200 μm. k, Schematic representation of the inoculation of AAVs in a skin chamber on the back of a mouse. Tested AAVs included: (1) AAVDJ with or without the addition of surfactant, (2) AAVs previously identified as optimal for skin and wounds (AAV2 and AAV5), and (3) new AAV serotypes (AAV9 and AAV10). Highly concentrated retroviruses were used as a control. l, H&E and fluorescence analyses of ulcers 1 week after the inoculation of eGFP-expressing viruses. AAVDJ without surfactant yielded by far the highest gene transduction efficiency. Scale bar, 200 μm. m, In vivo luminescent images 1 week after the administration of luciferase-expressing AAVs through tail vein injection, interscapular subcutaneous injection or inoculation into a chamber on the back. Systemic directivity of AAVDJ is mainly focused to the liver. B, brain; Ce, caecum; Co, colon; Es + St, oesophagus and stomach; Ey, eye; F, fat; H, heart; K + Ad, kidney and adrenal gland; L, liver; Lu, lung; Ov + U, ovary and uterus; Pa, pancreas; SI, small intestine; Sk, skin; Sp, spleen. Similar findings were obtained in five (b), three (cg, l) and two (h, j, m) independent experiments.

  4. Extended Data Fig. 4 Generation of epithelial tissue after DGTM-AAV administration in vivo.

    a, H&E analysis of epithelial tissue 18 days after DGTM-AAV administration. Both high magnification (left) and low magnification images (right) are shown. Viral titres are indicated on the left (×1010 GC/animal). Black scale bars, 500 μm; red scale bars, 2 mm. Data from 14 out of 25 animals treated with different titres of DGTM-AAV as shown (summarized data is shown in Fig. 1d). b, Lineage tracing using Pdgfracre;R26Rconfetti mice. c, A representative image showing GFP, YFP, RFP fluorescence and Pdgfra staining of the subcutaneous tissue isolated from the back skin of a Pdgfracre;R26Rconfetti mouse. Mesenchymal cells were differentially labelled with GFP, YFP, RFP or not labelled. Similar findings were confirmed in five animals. c, Representative visual, stereoscopic and histological images showing single-cell-derived epithelial cell clusters 14 days after DGTM-AAVs administration. Similar findings were confirmed in two animals (out of three animals treated with the same procedure). de, Representative visual, stereoscopic, and histological images showing participation of YFP-labelled and non-labelled epithelial cell clusters to a single epithelial tissue on day 14 (d) and day 18 (e). Similar findings were confirmed in all three animals treated with the same procedure for each day. f, Representative visual and stereoscopic images of three mice (left) showing that multicolour-labelled cell clusters contribute to epithelial tissues covering the ulcer surface inside the chamber. Histological images (right) of one animal showing the participation of GFP, YFP, RFP and unlabelled cells to generated epithelial tissue. Similar findings were confirmed in all three animals. g, For the estimation of cell number in deep fascia at the time of surgery, deep fascia was dissected out just after the attachment of the chamber. h, Stereoscopic images showing a horizontal image of the deep fascia after DAPI staining (left). Imaging analysis for the counting of nucleated cells (right). g, h, All three samples were processed with similar findings. i, Estimated cell numbers in the deep fascia just after the attachment of chamber for three animals. Data represents mean ± s.d. of from ten stereoscopic images. Overlaid dot plots indicate the distribution of the data. ch, White scale bar, 5 mm; black scale bars, 1 mm; red scale bars, 200 μm; yellow and magenta scale bars, 100 μm. External diameter of the chamber, 12.5 mm. Source data

  5. Extended Data Fig. 5 Reprogramming of mouse mesenchymal cells with different combinations of DGTM-AAVs.

    a, Mesenchymal cells were sorted out from mouse adipose-derived stromal cell fractions by PdgfracreER-driven tdTomato signals with the proportion of 63.2 ± 16.0% (n = 12, established from different animals). For gating strategy example, see Supplementary Fig. 2. Scale bar, 200 μm. b, Fluorescence images of the cells 3 days after addition of GFPNLS-AAV with indicated titre in vitro. GFPNLS expression increases with increase of titre. Scale bars, 200 μm. Similar findings were confirmed in two experiments. c, Results of MTT cell-viability assay of the cells 3 days after the addition of GFPNLS-AAV at the indicated titre in vitro. Overlaid dot plots indicate the distribution of the data (n = 6, technical replicates). d, Schematic of the experimental design. e, Time course stereoscopic and fluoroscopic analysis of the emergence of an iSEPs colony. Yellow scale bar, 5 mm; red scale bar, 1 mm; white scale bar, 200 μm. f, Immunocytochemical analysis of the iSEPs colony in e. e, f, Similar findings were confirmed in two sets of eight wells of samples. g, Time course immunocytochemical analysis of iSEPs colony emergence. iSEPs colonies are strongly positive for Krt14 and Pdgfra on initial emergence. With time, Pdgfra signal intensity decreases. Scale bars, 200 μm. Similar findings were confirmed in a set of eight wells of samples. h, Appearance (left) and stereoscopic analysis (right) of transplanted cell sheet. tdTomato signals indicates the area of cell sheet survival. Yellow scale bars, 1.0 cm. i, Findings of H&E and immunohistological analysis of transplanted cell sheet. Arrows indicate the position of magnified findings in j. Orange scale bars, 2 mm. j, H&E and immunohistological analysis of transplanted cell sheet. White scale bars, 100 μm. h, i, j, Similar findings were confirmed in five animals in two sets of experiments. k, Schematic representation of the experimental design at the lower titre of virus. l, DGTM-AAVs transduced wells. Arrows indicates epithelial shaped colonies. The dotted outline is shown magnified on the right. White scale bar, 5 mm; yellow scale bars, 1 mm. m, Immunocytochemical analysis of epithelial shaped colonies. White scale bars, 1 mm. l, m, Similar findings were confirmed in two sets of three wells (six-well plate). n, Time course numbers of epithelial colonies in eight wells (24-well plate) treated with DGTM-AAVs. Similar findings were confirmed in two series of experiments. o, Immunocytochemical analysis of epithelial cells obtained after transduction of different combinations of factors into Pdgfra+ mADSCs. Scale bar, 50 μm. Images are representative of one experiment. p, Proliferation of epithelial cells obtained after transduction of different combinations of factors into Pdgfra+ mADSCs. Data are mean of triplicates. q, Schematic representation of clonogenicity assessment by expansion of single-cell-derived clones. r, Analysis of ulcers 28 days after administration of nine combinations of AAV and no virus control (ten animals for each group, including samples shown in Fig. 1b, e). Green arrows indicate epithelial tissues confirmed by histological analysis. External diameter of chamber, 12.5 mm. Data are summarized in Fig. 2c–e. Findings were confirmed in a set of experiment. s, Epithelial tissue generated with DTM-, DGM-, DGT-, and DM-AAVs in vivo. Arrows indicate generated epithelial tissue. Black and white scale bars, 2 mm; yellow scale bars, 200 μm. Similar findings were confirmed in three (DTM), six (DGM), two (DGT) or one (DM) animals (out of ten animals for each group). Source data

  6. Extended Data Fig. 6 DGTM-AAVs enable generation of epithelium with the ability to cover ulcers.

    a, Appearance (top left) and immunohistochemical analysis (top middle) of ulcers, 28 days after the administration of DGTM-AAV in PdgfracreER;LSLtdTomato mice, analysed for KRT14 and tdTomato expression. Yellow arrows indicate the periphery of the generated epithelium. The white dotted line indicates the approximate position of the histological section. The white dotted outline indicates position of the magnified panels (bottom). The solid white box indicates the position of the magnified panels of epidermis and generated epithelium (right). White scale bars, 3 mm; black scale bar, 100 μm. Similar findings were confirmed in three animals. b, Appearance of the ulcer before (top left) and after (bottom left) biopsy of generated epithelium 28 days after administration of DGTM-AAV in PdgfracreER;LSLtdTomato mice and immunohistochemical analysis (top middle) of biopsied generated epithelium. Intensity of the tdTomato signal in the biopsied sample is higher than in the chamber (see a) because fixation without the chamber allowed the sample to contract. Yellow arrows indicate the position of the biopsy. The white dotted outline indicates the position of the magnified panels (lower middle). The white solid outline indicates the position of the magnified panels of generated epithelium (far right). White scale bars, 3 mm; black scale bar, 100 μm. Similar findings were confirmed in five biopsies. c, Experimental design of flow cytometric analysis of generated epithelium. d, Representative appearance of an ulcer 18 days after the administration of DGTM-AAV in PdgfracreER;LSLtdTomato mice (left) and primary cultured cells on feeder (middle) and no feeder condition (right). Yellow arrows indicate generated epithelium subjected to flow cytometric analysis. White scale bar, 3 mm; yellow scale bars, 200 μm. Similar findings were confirmed in three animals. e, Flow cytometric analysis of three primary cultured cells from the surface of an ulcer 18 days after administration of DGTM-AAV in PdgfracreER;LSLtdTomato mice (day 18 iSEPs). Primary keratinocytes from the back skin of Krt14cre;LSLtdTomato mice (top) and wild-type mice (bottom) were prepared for positive and negative controls, respectively. For gating strategy example, see Supplementary Fig. 2. f, Schematic of stepwise operations performed with representative images. A wound was created and treated with DGTM-AAVs. After the generation of epithelial tissue, the initial skin chamber was removed and replaced with a larger one (twice the area of the small chamber). After the large chamber was removed, early contraction of the generated epithelium was prevented by a rubber ring. White scale bars, 5 mm. g, Chronological changes in surface area of generated epithelia in the large chamber. Coloured lines represent animals treated with DGTM-AAV (n = 10). The black line represents animals without AAV (n = 5). h, Representative images showing the gross appearance of ulcers in large chambers at different time points. White scale bars, 5 mm. Similar findings were confirmed in ten animals for DGTM-AAV+ group and five animals for the no-virus group. i, Appearance (top left), stereoscopic analysis (bottom left), H&E staining (top right) and immunohistochemical analysis (bottom right) of skin and subcutaneous tissue including generated epithelium on day 93. Yellow arrows indicate the periphery of the generated epithelium. Dotted lines in the left panels indicate the approximate position of the histological sections shown on the right. Stereoscopic analysis revealed areas of generated tissues that are not clear by appearance. Dotted outlines indicate position of magnified panels. Scale bars, 5 mm. Similar findings were confirmed in all animals monitored over days 90–110 (n = 10). j, A representative H&E staining of liver tissue 3 months after the administration of DGTM-AAVs for induction of lineage conversion. Scale bar, 1.0 cm. Similar findings were confirmed in five animals. k, Complete blood count analysis after administration of AAV-DGTM (n = 4 for experimental and control groups). l, Blood chemistry analysis after administration of DGTM-AAVs (n = 3 for experimental and control groups). k, l, Data are mean ± s.d.; statistical differences are analysed with a two-sided Student’s t-test. m, Schematic of the skin island in chamber assay performed with representative images (intact skin group). n, Schematic of wounding, epithelialization and the skin island in chamber assay performed with representative images (epithelialized skin group). o, Schematic of skin biopsy, primary culture, cell-sheet transplantation and the skin island in chamber assay performed with representative images (cell sheet group). p, Schematic of chamber attachment and DGTM-AAV administration, chamber detachment, and the skin island in chamber assay performed with representative images (generated epithelium group). q, Appearances of skin island in chamber assay performed for pure cell sheet transplanted areas. Skin, purely composed of a cell sheet disappeared with time within the ulcer. Similar findings were confirmed in two animals. With these findings, we allowed the transplants to contract for another 1–2 weeks before subjecting the cell sheets to the skin island assay in the cell sheet group. r, Chronological changes in surface area of epithelial tissues after skin island creation and chamber attachment. Mean of four groups (left top), mean (thick line) with respective animals (thin line) for intact skin (top middle, n = 8), epithelialized skin (top right, n = 8), cell sheet (bottom left, n = 6), and generated epithelium (bottom right, n = 8) groups. s, Appearances of skin island in animals with maximum and minimum epithelialized areas for each group on day 14 of the skin island in chamber assay. The abilities of generated epithelium to laterally expand within an ulcer were more variable than seen with controls. m–q, s, External diameter of chamber, 14.3 mm. t, H&E staining and immunohistochemical analyses (K10 and K13) of generated tissues by DGTM-AAV at different time points (day 18, n = 18; days 28–30, n = 20; days 90–11, n = 10). Samples collected from the same generated epithelial tissues by partial biopsy (days 28–30) and thorough histological investigation (days 90–110) are indicated with asterisks (bottom two rows of the middle and left columns). In these two animals, K13 expression was confirmed in a biopsied sample at day 28, but after 3 months, K13 expression had been extinguished. Results are summarized in Fig. 4b. u, H&E staining and immunohistochemical analysis of mouse fetal skin at different gestational ages. Mouse fetal skin was transiently positive for K13. Similar findings were confirmed in three embryos from two different mothers for each gestational age. Source data

  7. Extended Data Fig. 7 In vivo reprogramming in a clinically relevant context.

    a, Schematic of investigation of influences of DGTM-AAVs administration on the vascularity of the wound bed. b, Appearance (top left) and immunohistochemical analysis of microvessel densities (right top and bottom) of an ulcer 7 days after the administration of DGTM-AAVs for CD31. Black and white boxes indicate the position of the magnified panels (lower). Yellow and red dotted lines indicate tissues above and below the deep fascia, respectively. Microvessel density was histologically evaluated for tissues above and below deep fascia. Scale bar, 1 mm. c, d, Analysis of microvessel densities for tissues above (c) and below (d) deep fascia in five animals for each group. Between six and eight sections were analysed for each animal. Mean microvessel densities are presented. Overlaid dot plots indicate the distribution of the data. Differences between groups were analysed with one-way ANOVA with a Tukey’s multiple comparison test. DGTM-AAVs showed no influences on vascularity. e, Schematic of the experimental design for the investigation of the efficiency of in vivo reprogramming during the generation of epithelial tissues in old wounds (7 days after the creation of the ulcer). f, Representative appearances of an ulcer at each time point (on day of ulcer creation (day 0), upon administration of DGTM-AAVs (day 7) and 18 days after administration of DGTM-AAVs (day 25)). Scale bar, 3 mm. g, Appearances of ulcers 18 days after administration of DGTM-AAVs in ten animals (including one shown in f) and histological images of small generated epithelial tissues that are unidentifiable by appearance. Yellow arrows indicate visible generated epithelial tissues. Red arrows indicate the position of generated tissues in magnified panels. Red scale bars, 200 μm. h, Schematic of the experimental design for the investigation of efficiency of GFPNLS-AAV administration with or without collagen gel on ulcer. i, Stereoscopic analysis of the centre of an ulcer and the surface of the liver. Different exposure times were used for imaging of ulcer and liver (fluorescence of the ulcer is far stronger than that of the liver surface). Similar findings were confirmed in four animals for each group. j, qPCR analysis of gross AAV genomic copies in ulcer tissues (left) and AAV genomic copies per mouse diploid genome (right) in animals 3 days after administration of GFPNLS-AAV with or without collagen gel (four animals for each group). The displayed values are the minimum (bottom range), mean (holizontal line), and maximum (top range). Overlaid dot plots indicate the distribution of the data. k, qPCR analysis of AAV genome copies per mouse diploid genome in liver in animals 3 days after administration of GFPNLS-AAV with or without collagen gel (four animals for each group). Three different lobes of liver tissues were analysed for each animal. The displayed values are the minimum (bottom range), mean (holizontal line), and maximum (top range) for each animal. Overlaid dot plots indicate the distribution of the data. j, k, Differences between groups were analysed with two-sided Student’s t-test. l, Schematic of the experimental design for epithelialization of ulcer in large chamber. m, Representative appearances of an ulcer treated by DGTM-AAVs and collagen gel administration (protocol I, n = 2, similar findings in both), an ulcer treated by DGTM-AAVs and collagen gel administration with application of Rock inhibitor (protocol II, n = 2, similar findings in both), an ulcer treated by DGTM-AAVs and collagen gel administration with application of FGF2 (protocol III, n = 3, similar findings in all), and an ulcer treated by DGTM-AAVs and collagen gel administration with application of Rock inhibitor and FGF2 (protocol IV, n = 3, similar findings in all). Yellow arrows indicate the initial emergence of visually identifiable epithelial tissues. External diameter of chamber, 14.3 mm. Source data

  8. Extended Data Fig. 8 in vivo iSEPs have high clonogenic ability and tumorigenic potential compatible with non-malignant epithelial cells.

    a, Schematic representation of clonogenicity assessment by expansion of single-cell-derived clones performed for neonatal primary keratinocytes (NPKs), primary keratinocytes from adult mice and in vivo iSEPs. b, Representative images of large (left), medium (middle), and small (right) single-cell-derived colonies of primary keratinocytes and iSEPs in 96-well plate. Similar findings were obtained from 122 (from three NPKs and five primary keratinocytes) and 169 (five 3M iSEPs and five 6M iSEPs) clones. c, Representative images of plates stained with Rhodanile blue staining. Similar differences between primary keratinocyte and 3M iSEPs were confirmed in five sets of primary keratinocytes and iSEPs isolated from the same animal. d, Occupied areas of single-cell-derived colonies in 12-well plates. iSEPs (n = 10) constantly show higher clonogenic ability than primary keratinocytes (n = 8). The displayed values are the minimum (bottom whisker), 25th percentile (bottom of box), median (line in box), 75th percentile (top of box), and maximum (top whisker). Overlaid dot plots indicate the distribution of the data (n = 12 for NPK1–3 and 6M iSEPs 1–5, n = 15 for PK1, n = 21 for PK2, n = 18 for PK3–4, n = 14 for PK5, n = 18 for 3M iSEPs 1, n = 16 for 3M iSEPs 2, n = 29 for 3M iSEPs 3, n = 24 for 3M iSEPs 4, and n = 22 for 3M iSEPs 5). e, Representative images of soft-agar assay. In vivo iSEPs showed no colony formation in DMEM (DMEM + 10% FBS), KFM and CKFM. Scale bar, 200 μm. Similar findings were confirmed in one HeLa, five primary keratinocytes, five 3M iSEPs, and five 6M iSEPs. f, Number of colonies (>30 μm) in one well (six-well plate) (n = 3, technical replicates). Averages of triplicates are shown. Overlaid dot plots indicate the distribution of the data. g, Bright field and fluorescence images showing the representative morphology of mES cells from C57BL/6-TfN(act-EGFP) mice, fixed HeLa cells, primary keratinocytes from Krt14cre;LSLtdTomato mice, and in vivo iSEPs from PdgfracreER;LSLtdTomato mice after in vivo reprogramming with DGTM-AAV. hMito, human mitochondria. Scale bar, 200 μm. Similar findings were confirmed once for mES and HeLa, five times for primary keratinocytes, and ten times for in vivo iSEPs. h, Schematic representation of the tumorigenic/teratogenic assay by subcutaneous injection of the cells to immunodeficient mice. i, Volume of the nodules 28 days after injection. Top, volume of mES-cell-derived nodules. Asterisks indicate the day on which nodules were collected (*, day 24; **, day 16) owing to animal-welfare concerns about the size or properties of the nodule. j, Appearances of the largest nodules for HeLa, primary keratinocytes, and iSEPs. Similar findings were confirmed in three animals for each. Scale bars, 5 mm. kn, H&E staining and fluorescence analysis of the largest nodule of 3M iSEPs 1 (k), primary keratinocytes (l), mES cells (m), and HeLa cells (n). For mES cells and HeLa cells teratogenicity and transfacial invasion were confirmed, respectively. Dotted outlines indicate the position of magnified panels (bottom). Similar findings were observed in other samples for each cells (n = 9 for iSEPs, n = 3 for primary keratinocytes, mES, and Hela cells). Red scale bar, 5 mm; blue scale bar, 2 mm; black scale bar, 500 μm; yellow scale bar, 200 μm. o, Schematic representation of biodistribution assessment of iSEPs. luciferase-expressing iSEPs were prepared using retroviral transduction. p, In vivo luminescent images 56 days after the subcutaneous injection of luciferase-expressing iSEPs. q, Luminescent images of dissected organs and tissues. Luciferase expression was confined to site of injection. M, muscle beneath the transplants; Sk + N, nodule and overlaid skin. p, q, Similar findings were confirmed in three animals for each iSEP group. Source data

  9. Extended Data Fig. 9 Exogenous gene expression and transcriptional profiles of iSEPs.

    a, Integration analysis of TP63, GRHL2, TFAP2A and MYC. RNA-seq reads were mapped simultaneously to human and mouse transcriptomes and the sum of the normalized transcript counts for all variants of TP63, GRHL2, TFAP2A, and MYC is shown for Pdgfra+ ADSCs, in vitro iSEPs, 1M in vivo iSEPs, 3M in vivo iSEPs, 6M in vivo iSEPs and primary keratinocytes. Human transcripts are derived from the AAV genome. Data from three Pdgfra+ ADSCs, three in vitro iSEPs, three 1M in vivo iSEPs, five 3M in vivo iSEPs, six 6M in vivo iSEPs and three primary keratinocytes. b, Clustered heat map showing the normalized expression of the top expressed genes across all conditions. c, Clustered keratinocyte marker gene expression represented as a heat map. d, Differentially expressed genes were found between primary keratinocytes and Pdgfra+ ADSCs (red), in vivo iSEPs (blue) and in vitro iSEPs (green). Overlap of genes that are significantly up- (top) or downregulated (bottom) in primary keratinocytes are shown. e, f, Top five Gene Ontology (GO) enrichment terms overrepresented for genes upregulated in primary keratinocytes (e) or genes upregulated in in vivo iSEPs (f). g, h, Top five enriched transcription factors known to bind to promoters of genes upregulated in primary keratinocytes (g) or genes upregulated in in vivo iSEPs (h). eh, Bar plots show −log(adjusted P value) significance of overrepresentation. Enrichment testing was carried out using HOMER for Gene Ontology enrichment testing (hypergeometric test with Benjamini and Yekutieli general multiple testing correction), and WebGestalt for transcription-factor enrichment testing (hypergeometic test with Benjamini–Hochberg multiple testing correction) on genes upregulated in primary keratinocytes (n = 399), or upregulated in in vivo iSEPs (n = 632). bh, Data for Pdgfra+ mADSCs, in vitro iSEPs, 6M and 3M in vivo iSEPs were from three experiments. i, Normalized expression values were k-means clustered to reveal the top five patterns of gene expression. Cluster A, Mesench (mesenchymal); cluster B, late; cluster C, sustained; cluster D, early; cluster E, transient. j, Heat map showing relative normalized expression of keratinocyte marker genes. k, Analysis of the enrichment of the top Gene Ontology terms are shown for each cluster of genes. General terms with >100 genes were filtered out to remove overly general terms. l, Top five transcription factors enriched in promoters of clustered genes. k, l, Bar plots show –log(adjusted P value) significance of overrepresentation. Enrichment testing was carried out using HOMER for Gene Ontology enrichment testing (hypergeometric test with Benjamini and Yekutieli general multiple testing correction), and WebGestalt for transcription factor enrichment testing (hypergeometic test with Benjamini–Hochberg multiple testing correction) on genes in clusters A (n = 1,966), B (n = 396), C (n = 2,501), D (n = 400), and E (n = 1,386). il, Data are from three Pdgfra+ mADSCs, three 1M in vivo iSEPs, two 3M in vivo iSEPs, three 6M in vivo iSEPs. Source data

  10. Extended Data Fig. 10 Description of wound healing with in vivo reprogramming.

    During physiological wound healing, epidermal defects are repaired from the other epidermis. On the other hand, in vivo reprogramming allows de novo epithelialization and greatly enhances the capacity for the regeneration of cutaneous defects.

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Figures 1-2 and Supplementary Methods

  2. Reporting Summary

  3. Supplementary Tables

    This file contains Supplementary Tables 1-13

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https://doi.org/10.1038/s41586-018-0477-4

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