Recessive dystrophic epidermolysis bullosa (RDEB) is a cutaneous genetic disease, which is characterized by the loss of functional type VII collagen. The epidermis becomes extremely fragile without functional type VII collagen, and the patients suffer from repetitive blistering and scar formation. RDEB is caused by mutations in the COL7A1 gene (coding type VII collagen), and most of the patients have two different mutations on each COL7A1 gene. Interestingly, it is known that a part of the skin in the RDEB patients can be reverted to a normal appearance; this phenomenon is called revertant mosaicism. One possible mechanism inducing revertant mosaicism is a chromosome crossover by which the two mutations on COL7A1 are gathered on one chromosome, resulting in the recovery of normal COL7A1 gene on the other chromosome. Revertant mosaicism, also referred as natural gene therapy, has attracted much attention for elucidating the pathogenesis of RDEB and developing a treatment for the disease. However, it has been technically challenging to identify whether the two pathogenic mutations are on the same chromosome, and this difficulty has been a barrier for the field. In this study, we report an efficient method using long-read sequencing to analyze revertant mosaicism. We improved an existing method, nCATS, which uses CRISPR/Cas9 technology to enrich the target region of the genome, by using barcoded adapters and introducing a pre-amplification step. We believe that this method will be useful not only for RDEB but also for the analysis of revertant mosaicism in many other genetic diseases.

Our Research Group for Rare and Intractable Skin Diseases operates within the Project for Research on Intractable Diseases of the Ministry of Health, Labour, and Welfare of Japan and is conducting research on eight rare intractable skin diseases. Five of these are monogenic disorders (epidermolysis bullosa, congenital ichthyoses, oculocutaneous albinism, pseudoxanthoma elasticum, and hereditary angioedema), and for a sixth [generalized pustular psoriasis (GPP)], genetic predisposing factors are important. This review introduces our activities for raising public awareness of these six intractable hereditary skin diseases and summarizes our recent achievements in clarifying the situation of medical treatments for these diseases in Japan. We note our current progress in elucidating the pathogeneses of these diseases and in developing new treatment methods, and we discuss our progress in establishing clinical practice guidelines. A nationwide survey on epidermolysis bullosa and a clinical survey on congenital ichthyoses are progressing. The Angioedema Activity Score and the Angioedema Quality-of-Life Questionnaire, the latter of which is a quality-of-life evaluation tool, have been established for hereditary angioedema. Registries of patients with oculocutaneous albinism and pseudoxanthoma elasticum have been created, and the registry for the latter has achieved its target of 170 cases. For GPP, the results of our survey on clinical practice were published in 2021. Information regarding all six of these hereditary skin diseases has been disseminated to academic societies, medical professionals, patients, and the general public.

Cutaneous development is initiated by cross-talk between ectodermal epithelia and underlying mesenchymal cells, resulting in stratification of the epidermis and buddings of the follicular appendages. The ectoderm-derived mesenchymal cells, so-called ectomesenchyme, are known to emerge from neural fold in mouse embryo. The ectomesenchyme is characterized by both ectodermal and mesenchymal phenotypes, expressing protein zero (P0) and platelet-derived growth factor receptor alpha (PDGFRa), respectively. However, precise rolls of them in the cutaneous development are largely unknown. In this study, we traced the ectomesenchymal lineages using P0-Cre;tdTomato mice and PDGFRa-Cre;tdTomato mice. Confocal microscopy observations of the fetal skin identified a robust emergence of the ectomesenchymal keratinocytes(KCs) at the cranio-facial region, which eventually extended from the rostral to the caudal, and from the dorsal to the abdominal regions. Single cell RNA/ATAC-seq and FACS analyses of the neonate skin revealed that the ectomesenchymal KCs dominantly occupied an integrin a6-positive and Thy1-positive keratinocyte population, which is known as an epidermal stem cell-enriched fraction, resulting in about 80% occupation of them in the cranio-facio-dorsal skin of the adult mice. The robust emergence of the ectomesenchymal KCs was confirmed by substantial induction of keratinocytes from human iPS cell-derived ectomesenchyme in vitro. Altogether, this is the first demonstration of ectomesenchyme as a major source of epidermal stem cells in the skin.

In the process of the cutaneous wound healing, fibroblasts play important roles for regenerating the injured tissues. We previously reported that the necrotic cells release abundant nuclear proteins including high mobility group box 1 (HMGB1), which induces accumulation of the circulating platelet-derived growth factor receptor alpha (PDGFRa)-positive mesenchymal stem/progenitor cells to promote regeneration of the necrotic injury. However, the characters of the necrotic injury/HMGB1-induced mesenchymal stem/progenitor cells in the skin are still largely unknown. In this study, we tried to characterize them by single cell RNA-seq analyses of the skin engrafted on the back of mice. Cell clustering analysis identified a unique epithelioid fibroblast sub-cluster among the fibroblast cluster, characterized by expression of keratin 5 and interferon regulatory factor 6 (IRF6), a transcriptional factor regulating mesenchymal-epithelial transition. Immunohistochemical analyses showed the epithelioid fibroblasts mainly distributed in the subcutaneous fat tissues of the skin graft, indicating that the epithelioid fibroblasts were not originated from the necrotic epidermis. Systemic HMGB1 peptide administration gave rise to a similar but distinct sub-cluster among the epithelioid fibroblast cluster, indicating divergence of the epithelioid fibroblasts, and also induced an emergence of PDGFRa-positive mesenchymal keratinocytes as a unique sub-cluster among the keratinocyte cluster. Relations between the epithelioid fibroblasts and the mesenchymal keratinocytes are currently unknown. Nevertheless, we believe that those necrotic injury/HMGB1-induced cells play pivotal roles for regenerating the necrotic injury of the skin.

Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable skin genetic disease characterized by the skin fragility leading to the recurrent blistering. The mutations in COL7A1 gene, encoding type VII collagen (C7) that forms anchoring fibrils, cause RDEB. RDEB patients are known to show variable prognosis, likely influenced by the position of pathogenic mutations. However, as the mutations in COL7A1 gene distribute across the gene without any apparent hotspots and the majority of RDEB patients harbor the mutations in a compound heterozygous manner, the genotype-phenotype correlation is not fully elucidated. Here, we propose a fast and robust framework to understand how each pathogenic mutation impact of the function of type VII collagen using i-GONAD, a CRISPR based method for rapid genome editing. i-GONAD removes time- and cost-consuming steps in generating genetically modified mice and enables to establish multiple genetically modified mice in a single laboratory. As a proof-of-concept study, we chose three COL7A1 mutations relatively common in Japanese RDEB patients (E2857X, c.5818delC, and 6573+1G>C). Using i-GONAD, we introduced these three mutations to the corresponding locations in the mouse genome. Mice with the 5818delC type mutation in a homozygous manner died soon after birth with blistering. The mice with E2857X and 6573+1G>C type homozygous mutations showed milder RDEB related phenotype. Mice with the c.5818delC/E2857X mutations, most of the RDEB patients have mutations in a compound heterozygous manner, presented an intermediate phenotype between the c.5818delC and E2857X homozygous mice. These phenotypes in each mouse well recapitulated the phenotypes seen in human patients. Together, we have shown that our method is a powerful tool to analyze how the COL7A1 mutations detected in RDEB patients impact on the skin integrity. We believe the proposed strategy will facilitate studies to develop a better RDEB therapy.

Plasma cell-free DNA (cfDNA) is frequently analyzed using liquid biopsy to investigate cancer markers. Accordingly, we hypothesized this concept could be applied to the field of exercise physiology. Here, we aimed to identify specific cfDNA (spcfDNA) sequences in the plasma of non-treated human participants using next generation sequencing (NGS) and to clearly define the dynamics regarding the amounts of spcfDNA-fragments upon extreme exercise, such as running a full marathon. NGS analysis was performed using cfDNA of pooled plasma collected from non-treated participants. We confirmed the TaqMan-qPCR assay had a high sensitivity and found the spcfDNA sequence abundance was 16,600-fold higher than a normal genomic region. We then used the TaqMan-qPCR assay to investigate the dynamics of the levels of spcfDNA-fragments upon running a full marathon. Quantities of the spcfDNA fragments were significantly increased post marathon. Furthermore, the amounts of spcfDNA fragments strongly correlated with the numbers of white blood cells and plasma myoglobin concentrations. These results suggest the spcfDNA fragments identified in this study were highly sensitive response markers to extreme physical stress. The findings of this study may provide new insights into exercise physiology and genome biology on the human.

Abstract Background High-mobility group box 1 protein (HMGB1) reportedly enhances CXCR4-positive bone marrow-derived mesenchymal stem cell (BM-MSC) recruitment to damaged tissue to promote tissue regeneration. Purpose Our aim of this study is to evaluate whether systemic administration of HMGB1 might promote tissue repair in a rat myocardial infarction (MI) model. Methods We prepared 26 MI model rats with high ligation of the left coronary artery. Two weeks later, HMGB1 (3 mg/kg/day) or phosphate-buffered saline (control: 3 mL/kg/day) was administered for 4 days via femoral vein. Cardiac performance was evaluated by ultrasonography, left ventricular (LV) remodeling via immunostaining. We then used immunostaining to examine MSC recruitment to damaged tissue in green fluorescent protein bone marrow transplantation (GFP-BMT) model rats, and also performed intravital imaging using two-photon microscopy to visualize BM-cells recruitment in real time. Results Compared with control rats, there was a significant improvement in the left ventricular ejection fraction of the HMGB1 group (HMGB1 vs. control: 48.6% ± 5.5% vs. 33.6% ± 5.4%; p<0.01) at 4 weeks after each administration. LV remodeling, characterized by interstitial fibrosis, cardiomyocyte hypertrophy, and a decrease of capillary density, was significantly attenuated in the HMGB1 group compared with control rats. On QT-PCR analysis, VEGF mRNA expression was significantly higher in the HMGB1 group than in the control (border zone; 1.6±0.6 vs. 1.1±0.2; p=0.02, septal zone; 1.1±0.1 vs. 0.9±0.1; p<0.01). In GFP-BMT rats, GFP+/PDGFR+ cells were significantly mobilized to the border zone in the HMGB1 group compared with the control (1331±197 vs. 615±45 /mm2; p<0.01), leading to formation of newly developed vasculature (Figure 1). In intravital imaging, more GFP+ cells were mobilized to the infarction area in the HMGB1 group than in the control, which was further enhanced at 12h later. Additionally, SDF-1 expression in the peri-infarction area increased significantly in MI rats compared with normal rats (MI vs. normal; 2.1±0.4 vs. 0.9±0.1; p<0.01), in where some cell-adhesions of vascular endothelial cells were destroyed. Conclusions Systemic administration of HMGB1 mobilized BM-MSCs to the damaged myocardium via the SDF-1/CXCR4 signaling complex. Those BM-MSCs might migrate to extracellular matrix in the border zone via the gap of each endothelial cell, leading to induction of angiogenesis and reduced fibrosis. Acknowledgement/Funding None

Recessive Dystrophic epidermolysis bullosa (RDEB) is a skin genetic disease caused by mutations in COL7A1 (coding type VII collagen). Type VII collagen is essential to maintain the fully functional dermal-epidermal junction, and the loss of type VII collagen results in detachment of epidermis. RDEB patients, without functional type VII collagen, suffer from the repetitive blistering and have high risk of early-onset aggressive squamous cell carcinoma. Currently, no fundamental treatment is available for RDEB. In this study, we aimed to develop therapeutics for RDEB using HMGB1 peptide, which we have previously identified as an endogenous SOS signal that can activates bone marrow stem/progenitor cells. Systemic injection of HMGB1 peptide in DEB model mice ameliorated mitten deformity and stenosis, and significantly extended the survival. This HMGB1 peptide treatment model provides a unique opportunity to investigate on how the DEB skin can be reconstructed. Thus, we comprehensively characterized the transcriptome (by RNA-seq) and epigenome (by ATAC-seq) of the skin from the HMGB1 peptide treated DEB model mice with single-cell resolution. Decrease in the number of immune cells upon the HMGB1 treatment was identified by both single cell RNA- and ATAC-seq analyses, indicating the treatment suppressed the inflammation. Furthermore, single cell ATAC-seq analysis depicted a sign of massive reprogramming in the transcription factor usage of the basal layer keratinocytes (contain stem/progenitor cells) in the HMGB1 peptide treated DEB model mice. These singe-cell level analyses suggested that the HMGB1 peptide can induce skin reconstruction by reprogramming the transcriptional program of skin stem/progenitor cells, and assured that the HMGB1 peptide serves as a promising option for the RDEB treatment.

Generalised severe recessive dystrophic epidermolysis bullosa (RDEB-GS) is a rare, incurable genetic disorder in which an essential skin component, type 7 collagen (C7), is defective or missing. As a result, skin and mucous membranes detach from the body, causing blisters and wounds. Bone marrow transplantation (BMT) can restore C7 in the skin, probably by supplying mesenchymal stem cells (MSCs) which turn into healthy new skin cells. However, BMT is a risky and unpleasant procedure because the drugs needed to stop the patient's immune system rejecting the donated bone marrow can themselves cause illness and sometimes death. Immune cells in the transplanted tissue may even attack the patient, causing graft versus host disease (GvHD). These doctors at the University of Minnesota tried a different BMT protocol in 10 children with RDEB-GS, using less intense immune-suppression pre-transplant. Three days post-transplant they gave an immunosuppressive drug called cyclophosphamide (PTCy). Two months later, when the patient's body had accepted the donor tissue, they were able to give more MSCs from the same donor. The graft failed in three patients, two of whom underwent repeat BMT followed in one by severe complications and death a year later. Successfully transplanted patients tended to show more C7 in their skin biopsies and improved symptoms with reductions in itch, pain and areas of blistered skin. None suffered GvHD in the short term. The authors conclude that PTCy BMT is feasible in people with RDEB-GS, even with “less-than-perfectly-matched” donors, and allows repeat “top-up” MSC grafts from the same donor.

Background Recessive dystrophic epidermolysis bullosa (RDEB) is a severe systemic genodermatosis lacking therapies beyond supportive care for its extensive, life-limiting manifestations. Objectives To report the safety and preliminary responses of 10 patients with RDEB to bone marrow transplant (BMT) with post-transplant cyclophosphamide (PTCy BMT) after reduced-intensity conditioning with infusions of immunomodulatory donor-derived mesenchymal stromal cells (median follow-up 16 months). Methods BMT toxicities, donor blood and skin engraftment, skin biopsies, photographic and dynamic assessments of RDEB disease activity were obtained at intervals from pre-BMT to 1 year post-BMT. Results Related donors varied from haploidentical (n = 6) to human leucocyte antigen (HLA)-matched (n = 3), with one HLA-matched unrelated donor. Transplant complications included graft failure (n = 3; two pursued a second PTCy BMT), veno-occlusive disease (n = 2), posterior reversible encephalopathy (n = 1) and chronic graft-versus-host disease (n = 1; this patient died). In the nine ultimately engrafted patients, median donor chimerism at 180 days after transplant was 100% in peripheral blood and 27% in skin. Skin biopsies showed stable (n = 7) to improved (n = 2) type VII collagen protein expression by immunofluorescence and gain of anchoring fibril components (n = 3) by transmission electron microscopy. Early signs of clinical response include trends toward reduced body surface area of blisters/erosions from a median of 49 center dot 5% to 27 center dot 5% at 100 days after BMT (P = 0 center dot 05), with parental measures indicating stable quality of life. Conclusions PTCy BMT in RDEB provides a means of attaining immunotolerance for future donor-derived cellular grafts (ClinicalTrials.gov identifier NCT02582775). What's already known about this topic? Severe, generalized recessive dystrophic epidermolysis bullosa (RDEB) is marked by great morbidity and early death. No cure currently exists for RDEB. Bone marrow transplant (BMT) is the only described systemic therapy for RDEB. What does this study add? The first description of post-transplant cyclophosphamide (PTCy) BMT for RDEB. PTCy was well tolerated and provided excellent graft-versus-host disease prophylaxis, replacing long courses of calcineurin inhibitors in patients receiving human leucocyte antigen-matched sibling BMT. What is the translational message? The PTCy BMT platform permits identification of a suitable related donor for most patients and for subsequent adoptive transfer of donor nonhaematopoietic cells after establishment of immunological tolerance.

泛发型重度隐性营养不良性大疱性表皮松解症 (RDEB‐GS) 是一种罕见的不可治愈的遗传疾病,此疾病患者皮肤的主要成分 7 型胶原 (C7) 出现缺陷或缺失。 由此导致皮肤和粘膜与身体脱离,引发水疱和伤口。骨髓移植 (BMT) 可恢复皮肤中的 C7,其机理很可能是提供可转变为健康新皮肤细胞的间充质干细胞 (MSC)。但是,BMT 是一种存在风险的不适操作,因为需要用药来阻止患者免疫系统排斥捐献的骨髓,而这些药物本身可能导致疾病,有时甚至致死。移植组织中的免疫细胞甚至可能攻击患者,导致移植物抗宿主疾病 (GvHD)。 这些明尼苏达州立大学的医生们在 10 名 RDEB‐GS 儿童中尝试了一种不同的 BMT 方案,该方案使用较低强度的移植前免疫抑制。移植后 3 天,向他们给用称为环磷酰胺 (PTCy) 的免疫抑制药物。2 个月后,当患者身体已经接受供体组织时,他们可以从相同供体接受更多 MSC。 此移植在 3 名患者中失败,其中 2 名经历了重复的 BMT,1 名发生了严重并发症,并在 1 年后死亡。移植成功患者在皮肤活检中显示了 C7 增多和症状改善以及瘙痒、疼痛和水疱皮肤面积减少的趋势。短期无人发生 GvHD。 作者们得出结论,PTCy BMT 在 RDEB‐GS 患者中是可行的,甚至使用“不很完美匹配”的供体时也是如此,且允许通过相同供体重复“额外”MSC 移植。

Dystrophic epidermolysis bullosa (DEB) is a one of the most severe form of EB caused by mutations in COL7A1 (coding type VII collagen). DEB patients, without functional type VII collagen, suffer from the repetitive blistering and have high risk of early-onset aggressive squamous cell carcinoma. Recently, we found that a fragment of HMGB1 activates an endogenous tissue regeneration mechanism and ameliorates the DEB related manifestations in a DEB model mouse. This HMGB1 treatment model in the DEB mouse serves as a unique opportunity to investigate on how the skin can lose the integrity by loss of a single protein and how the damaged skin can be reconstructed. Here, we comprehensively characterized the transcriptome and epigenome of the HMGB1 treated DEB model mouse at a single cell level. We performed single cell RNA-seq and single cell ATAC-seq analyses on the skin. Both single cell RNA-seq and ATAC-seq analyses showed a decrease of immune cells by the HMGB1 treatment, indicating the treatment suppressed the inflammation. In addition, single cell RNA-seq analysis detected an increase of keratin 1/keratin 10 positive keratinocytes in the treated mice, suggesting a functional reconstruction of the DEB skin. Furthermore, single cell ATAC-seq analysis detected an increase of p63 active keratinocytes, providing another evidence for the skin reconstruction. These singe cell level data suggested that the HMGB1 treatment induced a skin reconstruction by reactivating skin stem/progenitor cells, and also serve as a foundation to design more effective DEB treatments.

Dystrophic epidermolysis bullosa (DEB) is an intractable skin genetic disease caused by mutations in the type VII collagen gene (COL7A1), which is a component of the dermal-epidermal junction. Previously, we have found that the damaged EB skin releases high mobility group box 1 (HMGB1), a transcriptional regulator known to localize in the nucleus, to mobilize bone marrow platelet-derived growth factor receptor alpha (Pdgfrα) positive cells into the damaged tissues. Here, we investigated whether the HMGB1 treatment could serve as a promising therapy for DEB. Systemic injection of HMGB1 significantly improved survival in the DEB model mice, while the untreated DEB model mice died at about 20 weeks after birth due to severe malnutrition. To understand the mechanisms of how HMGB1 ameliorates the clinical manifestations of DEB model mouse, we performed whole transcriptome analysis on the Pdgfrα positive cells at bulk and single-cell levels, and found that HMGB1 induced the anti-cell death pathway. Consistent with this anti-cell death gene profile, the number of the bone marrow Pdgfrα positive cells was maintained at physiological level in the HMGB1 treated DEB model mice, while the number of the bone marrow Pdgfrα positive cells significantly decreased without the HMGB1 treatment. Thus, these results indicated that the activation of tissue regeneration, possibly by maintaining the number of the bone marrow Pdgfrα positive cells during the course of disease, is a promising therapy for diseases of the cutaneous, and possibly non-cutaneous tissues with severe intractable damages, such as DEB.

Psoriasis is an autoimmune skin disorder characterized by severe inflammation and hyperproliferation of epidermal cells in the skin. Th17, IL-17 producing helper T, cells are known to play pivotal roles in the psoriasis pathogenesis. Although therapies targeting Th17 cells are being developed, effective treatment for psoriasis has not established. Previously, we have demonstrated that damaged tissues release high mobility group box protein 1(HMGB1) to promote tissue regeneration by mobilizing bone marrow mesenchymal stem cells (BM-MSCs). The BM-MSCs mobilized into the damaged tissues support efficient tissue regeneration by suppressing inflammation and by differentiating into multiple cell types. We have further identified the domain of HMGB1 which is essential and sufficient for activating BM-MSCs and showed that the HMGB1 domain peptide served as a promising drug for dystrophic epidermolysis bullosa (DEB), a skin genetic disorder characterized by severe blistering at skin. Because the HMGB1 therapy promotes tissue regeneration without targeting any specific molecules, this strategy can be potentially effective for a broad range of diseases with massive inflammation. Here, we tested whether the HMGB1 peptide treatment is effective for psoriasis using imiquimod (IMQ)-induced psoriasis-like dermatitis as a model. The systemic injection of HMGB1 peptide ameliorated the inflammatory manifestations of psoriasis. Consistent with these anti-inflammation activities, HMGB1 peptide decreased the gene expression of the inflammatory cytokines (IL-17A/F, IL-6, and IL-1β), suggesting that the HMGB1 peptide treatment could suppress activated Th17 cells. These data provide a foundation for the development a novel psoriasis therapy using a single defined endogenous molecule, HMGB1.

High mobility group box 1 (HMGB1) mainly localizes in the nucleus and regulates transcription by modulating chromatin structure. In a necrotic condition, HMGB1 is released into the extracellular milieu and plays pleiotropic roles in tissue regeneration and immunological processes. Previously, we have reported that the released HMGB1 mobilizes platelet-derived growth factor receptor alpha (PDGFRα) positive bone marrow cells into the damaged tissues using SDF-1α/CXCR4 axis. We have also shown that the HMGB1-induced PDGFRα positive cells can differentiate into both fibroblasts and keratinocytes, and provide type VII collagen (Col7) at the cutaneous basement membrane zone. Based on these results, we hypothesized that HMGB1 can serve as a potential drug for moderate form of dystrophic epidermolysis bullosa (DEB), which possess amino acid substitution mutations at least in one allele of Col7a1 gene to produce less-functional anchoring fibrils and induce dermal-epidermal separation. To test this, we systemically administered HMGB1 to Col7-hypomorphic mice. The treatment with HMGB1 significantly extended the survival with restoring the expression of Col7 at the basement membrane of the skin and the mucosa, while most of the untreated mice died within 20 weeks due to severe malnutrition. Correlated with the recovery of Col7 expression, the DEB symptoms were ameliorated. Collectively, these data position HMGB1 as a promising drug at least for moderate forms of DEB. In addition, our study provides a proof-of-concept for a drug which mobilizes multipotent cells from bone marrow, as a promising therapy for diseases of the cutaneous, and possibly non-cutaneous tissues with severe intractable damages, such as DEB.

Tissue regeneration requires efficient and coordinated replenishment of lost cells during tissue damage. In case of the limited sized tissue damage, cells can be replenished locally from neighboring healthy tissues. However, when the damage is massive, an additional mechanism to replenish cells are required for efficient tissue regeneration. Previously, we have shown that bone marrow cells function as the additional source to replenish cells to massively damaged tissues in mouse epidermolysis bullosa model, in which the epidermal-dermal junction at skin is massively disrupted due to a mutation in type VII collagen gene (Col7a1). Furthermore, we have clarified that the bone marrow cells are recruited by extracellular high mobility group box 1 (HMGB1), express platelet-derived growth factor receptor alpha (Pdgfrα) as a marker and can differentiate into multiple cell types, including epithelial cells expressing keratin 5. These data suggest that, when the damage is massive, cells are likely supplied via the circulation system for efficient tissue regeneration. However, these bone marrow cells involved in tissue regeneration have not successfully detected while the cells are circulating and analyzed its cell identity. Here, we used HMGB1 as a tool to mobilize bone marrow cells into the circulation and analyzed their characters. Systemic injection of HMGB1 significantly increased colony forming cells in the circulation and those HMGB1 mobilized cells showed higher proliferation capacity. In addition, we found that mesenchymal stem/stromal cells (MSCs), defined by its multipotency, are enriched in the circulation upon the HMGB1 injection. Our data clearly indicate that MSCs can be mobilized into the circulation by HMGB1 and suggest that tissue regeneration can be enhanced by a single defined factor. We will test the possibility of the use of HMGB1 as therapeutics for diseases with massive tissue damages, including epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is one of the most severe heritable blistering skin disease caused by genetic abnormality of type VII collagen (Col7). We previously reported that systemic administration of nuclear chromatin protein HMGB1 (high mobility group box 1) ameliorated phenotypes of the Col7-null mouse skin by inducing accumulation of bone marrow-derived PDGFR alpha-positive mesenchymal stem/progenitor cells (Pa cells). However, HMGB1 released from necrotic cells also induces inflammation via activating Toll-like receptors (TLRs) in the necrotic lesions. In this study, we identified Pa cell-activating domain (designated as KOI2 domain) locating at the different region from the TLR activating domain in the HMGB1 primary structure. Systemic administration of the synthetic KOI2 peptide significantly increased circulating Pa cells in wild-type mice without any evidence of inflammatory activation. Culture of the peripheral blood of the KOI2-administered mice generated a number of colonies which consisted of the Pa cells with both mesodermal and ectodermal differentiation capability. Intravenous administration of the KOI2 peptide in the Col7-hypomorphic mice significantly ameliorated digit fusion and intestinal stenosis, and drastically prolonged survival period of the RDEB model mice for more than a year. We further confirmed that intravenous administration of GMP-grade KOI2 peptide was safe and significantly increased mesenchymal cells in circulation of the healthy adult male volunteers in investigator-initiated phase I clinical trial. These data suggest that HMGB1-KOI2 peptide may function as an in vivo regeneration-inducing medicine for patients with severe tissue injury, such as RDEB.

RDEB is an incurable, often fatal, blistering genodermatosis. Mutations in COL7A1 result in a lack of anchoring fibrils (AFs) between the epidermal basement membrane and the dermal matrix. In 2010, we reported that BMCBT from healthy donors after myeloablative conditioning in 6 patients with RDEB increases C7 expression with parallel improvement in wound healing. As of January 2017, 32 RDEB patients age 5 months to 20 years have been treated by allogeneic BMCBT with follow up ranging from 0.3 to 9 years. The impact of BMCBT on C7 expression was assessed at days 100, 180, and 365 after treatment, and yearly thereafter. Mucocutaneous chimerism was >5% in 27 with increase in C7 expression documented in 12 subjects. Rudimentary AFs were detected in 16. In 13 patients we demonstrated immune-ultrastructural localization of C7 at the sites of AF-like structures at the dermal-epidermal junction, providing additional evidence for generation of new AFs. Based on direct observations and parental reports, 19 of 21 alive with chimerism had mucocutaneous improvement. The probability of survival at 2 years for BMCBT with HLA-matched donors was 69% for high-intensity conditioning (N=13); 81% for reduced-intensity conditioning (N=16). For BMCBT with HLA haploidentical donors and reduced-intensity conditioning, the survival is 100% (N=3). This suggests that patients with severe generalized RDEB can have sustained improvement in mucocutaneous integrity following BMCBT.

Recessive dystrophic epidermolysis bullosa (RDEB) is one of the most severe genetic blistering skin diseases caused by genetic dysfunction of type VII collagen (Col7), which anchor cutaneous basement membrane to the underlying dermis. Previously, we reported that necrotic epithelia of RDEB mouse skin released high mobility group box 1 (HMGB1), which then mobilized bone marrow mesenchymal stromal cells (MSCs) into the circulation, resulting in accumulation of MSCs in the RDEB mouse skin and promoting skin regeneration by anti-inflammatory and multi-differentiating activities of MSCs. We also demonstrated that transplanted bone marrow-derived MSCs could provide Col7 at the cutaneous basement membrane zone in the RDEB mouse skin. Under these backgrounds, we performed clinical trial of allogeneic MSC transplantation in three adult RDEB patients with chronic intractable ulcers continuing more than 5 years. Practically, 0.5 million of MSCs cultured from sex-mismatched healthy family donor bone marrow were subcutaneously inoculated at every 2cm distance around the chronic ulcer, and evaluated the ulcer area, expression of Col7 mRNA/ protein, formation of anchoring fibrils, and engraftment of the transplanted MSCs before and one year after the transplantation. All three cases showed almost complete healing of the intractable ulcers after one year. Among the three patients, one patient clearly demonstrated increase of Col7 protein and anchoring fibrils at the basement membrane, and two patients showed less but some engraftment of the allogeneic MSCs even after 1 year. These data clearly demonstrated that subcutaneous transplantation of allogeneic MSCs would be a therapeutic option for years-lasting intractable ulcers in severe generalized RDEB patients.

Cutaneous and hematopoietic cells synergize in wound healing. Skin fibroblasts and keratinocytes engage in epithelialization and scar formation. Bone marrow-derived inflammatory cells, such as neutrophils and macrophages, contribute to wound repair by phagocytosis and production of cytokines enabling skin repair. While non-hematopoietic bone marrow cells (eg, mesenchymal stromal cells, fibroblasts) also contribute to injury repair, it is unclear whether bone marrow or cord blood cells can functionally replace defective keratinocytes. Here we report early results using allogeneic BMCBT to ameliorate symptoms in patients with severe generalized JEB, a prototypical keratinocyte disorder. Skin engraftment was observed in 5 of the 10 patients (23% and 50% in those with α3 chain of L332-, 3% and 9% in β3 chain of L332-, and 12% in α6β4 integrin-deficient JEB) and associated with increased expression of α3 L332 or β3 chains of L332 but not α6β4 integrin, suggesting different disease mechanisms in these respective forms of JEB. For all JEB forms the overall 2-year survival after BMCBT was 40% (95% CI, 12-67%). Amelioration of symptoms was observed only in patients with mutations in the α3 chain of L332 (L332; N=2). No lasting benefit was observed in individuals with mutations in β3 chain of L332 (N=7) or α6β4 integrin (N=1). We anticipate our data to be a starting point for development of alternative therapies for β3 L332- or α6β4 integrin-deficient JEB, while BMCBT remains an option for those with α3 chain of L332-deficient severe generalized JEB.