Hair-follicle associated pluripotent (HAP)-cell-sheet implantation enhanced wound healing in diabetic db/db mice.

Diabetes often results in chronic ulcers that fail to heal. Effective treatment for diabetic wounds has not been achieved, although stem-cell-treatment has shown promise. Hair-follicle-associated-pluripotent (HAP)-stem-cells from bulge area of mouse hair follicle have been shown to differentiate into keratinocytes, vascular endothelial cells, smooth muscle cells, and some other types of cells. In the present study, we developed HAP-cell-sheets to determine their effects on wound healing in type-2 diabetes mellitus (db/db) C57BL/6 mouse model. Flow cytometry analysis showed cytokeratin 15 expression in 64% of cells and macrophage expression in 3.6% of cells in HAP-cell-sheets. A scratch cell migration assay in vitro showed the ability of fibroblasts to migrate and proliferate was enhanced when co-cultured with HAP-cell-sheets. To investigate in vivo effects of the HAP-cell-sheets, they were implanted into 10 mm circular full-thickness resection wounds made on the back of db/db mice. Wound closure was facilitated in the implanted group until day 16. The thickness of epithelium and granulation tissue volume at day 7 were significantly increased by the implantation. CD68 positive area and TGF-ß1 positive area were significantly increased; meanwhile, iNOS positive area was reduced at day 7 in the HAP-cell-sheets implanted group. After 21 days, CD68 positive areas in the implanted group were reduced to under the control group level, and TGF-ß1 positive area had no difference between the two groups. These observations strongly suggest that the HAP-cell-sheets implantation is efficient to facilitate early macrophage activity and to suppress inflammation level. Using immuno-double-staining against CD34 and α-SMA, we found more vigorous angiogenesis in the implanted wound tissue. The present results suggest autologous HAP-cell-sheets can be used to heal refractory diabetic ulcers and have clinical promise.

Rat hair-follicle-associated pluripotent (HAP) stem cells can differentiate into atrial or ventricular cardiomyocytes in culture controlled by specific supplementation.

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells.

Direct implantation of hair-follicle-associated pluripotent (HAP) stem cells repairs intracerebral hemorrhage and reduces neuroinflammation in mouse model.

Intracerebral hemorrhage (ICH) is a leading cause of mortality with ineffective treatment. Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into neurons, glial cells and many other types of cells. HAP stem cells have been shown to repair peripheral-nerve and spinal-cord injury in mouse models. In the present study, HAP stem cells from C57BL/6J mice were implanted into the injured brain of C57BL/6J or nude mice with induced ICH. After allo transplantation, HAP stem cells differentiated to neurons, astrocytes, oligodendrocytes, and microglia in the ICH site of nude mice. After autologous transplantation in C57BL/6J mice, HAP stem cells suppressed astrocyte and microglia infiltration in the injured brain. The mRNA expression levels of IL-10 and TGF-ß1, measured by quantitative Real-Time RT-PCR, in the brain of C57BL/6J mice with ICH was increased by HAP-stem-cell implantation compared to the non-implanted mice. Quantitative sensorimotor function analysis, with modified limb-placing test and the cylinder test, demonstrated a significant functional improvement in the HAP-stem-cell-implanted C57BL/6J mice, compared to non-implanted mice. HAP stem cells have critical advantages over induced pluripotent stem cells, embryonic stem cells as they do not develop tumors, are autologous, and do not require genetic manipulation. The present study demonstrates future clinical potential of HAP-stem-cell repair of ICH, currently a recalcitrant disease.

Hair-follicle-associated pluripotent (HAP) stem cells differentiate into mature beating cardiomyocyte sheets on flexible substrates in vitro.

Cardiomyocytes have been differentiated from various stem cells such as human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), but it is difficult to produce mature cardiomyocytes. We showed rat hair-follicle-associated pluripotent (HAP) stem cells have pluripotency and produced mature beating cardiomyocyte sheets differentiated from rat HAP stem cells. The upper parts of rat vibrissa hair follicles were cultured in 10% FBS DMEM and stained with antibodies of the ectoderm, mesoderm, endoderm system to show the differentiation of multiple cell types. Moreover, HAP stem cells were cultured under three different conditions to decide the most suitable culture conditions for making beating cardiomyocyte sheets. The beating cardiomyocyte sheets were shown to be mature by staining sarcomere structures. Isoproterenol alone and the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) effectively induced beating long-fiber cardiomyocytes, which formed beating sheets, only in the presence of all four agents. Flexible substrates were essential for the differentiation of sheets of mature beating cardiomyocytes for HAP stem cells. The features of the cardiomyocytes differentiated from HAP stem cells demonstrate they have clinical potential for heart regeneration.

Chronic spinal cord injury functionally repaired by direct implantation of encapsulated hair-follicle-associated pluripotent (HAP) stem cells in a mouse model: Potential for clinical regenerative medicine.

Chronic spinal cord injury (SCI) is a highly debilitating and recalcitrant disease with limited treatment options. Although various stem cell types have shown some clinical efficacy for injury repair they have not for SCI. Hair-follicle-associated pluripotent (HAP) stem cells have been shown to differentiate into neurons, Schwan cells, beating cardiomyocytes and many other type of cells, and have effectively regenerated acute spinal cord injury in mouse models. In the present report, HAP stem cells from C57BL/6J mice, encapsulated in polyvinylidene fluoride membranes (PFM), were implanted into the severed thoracic spinal cord of C57BL/6J or athymic nude mice in the early chronic phase. After implantation, HAP stem cells differentiated to neurons, astrocytes and oligodendrocytes in the regenerated thoracic spinal cord of C57BL/6J and nude mice. Quantitative motor function analysis, with the Basso Mouse Scale for Locomotion (BMS) score, demonstrated a significant functional improvement in the HAP-stem-cell-implanted mice, compared to non-implanted mice. HAP stem cells have critical advantages over other stem cells: they do not develop teratomas; do not loose differentiation ability when cryopreserved and thus are bankable; are autologous, readily obtained from anyone; and do not require genetic manipulation. HAP stem cells therefore have greater clinical potential for SCI repair than induced pluripotent stem cells (iPSCs), neuronal stem cells (NSCs)/neural progenitor cells (NPCs) or embryonic stem cells (ESCs). The present report demonstrates future clinical potential of HAP-stem-cell repair of chronic spinal cord injury, currently a recalcitrant disease.

Hair-Follicle-Associated Pluripotent (HAP) Stem Cells Can Extensively Differentiate to Tyrosine-Hydroxylase-Expressing Dopamine-Secreting Neurons.

Hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of hair follicles from mice and humans and have been shown to differentiate to neurons, glia, keratinocytes, smooth muscle cells, melanocytes and beating cardiac muscle cells in vitro. Subsequently, we demonstrated that HAP stem cells could effect nerve and spinal-cord regeneration in mouse models, differentiating to Schwann cells and neurons in this process. HAP stem cells can be banked by cryopreservation and preserve their ability to differentiate. In the present study, we demonstrated that mouse HAP stem cells cultured in neural-induction medium can extensively differentiate to dopaminergic neurons, which express tyrosine hydroxylase and secrete dopamine. These results indicate that the dopaminergic neurons differentiated from HAP stem cells may be useful in the future to improve the symptoms of Parkinson's disease in the clinic.

Gray-edged line sign of scabies burrow.

A scabies burrow is created by a mature female mite laying eggs through the stratum corneum, representing a kind of scabies eruption. We have noticed that the edges of the scabies burrow sometime appear as blackish-gray lines. We named these lines the "gray-edged line" sign, as a new feature of scabies burrows. The gray-edged line sign has the following two tendencies: (i) it is rarely seen on the palm or sole; and (ii) when the burrow follows a curved course, the gray-edged line often forms on the outer wall. Explaining the formation of this sign from clinical findings was difficult, so the aim of the present study was to elucidate the mechanisms underlying the gray-edged line sign. This retrospective study involved collection of data from electronic medical records of patients treated for scabies in our department between April 2015 and February 2020. We treated 32 scabies patients, including 4 patients with the gray-edged line sign. We analyzed clinical features, dermoscopy, histopathology and special stains. Fontana-Masson staining showed melanin staining in three parts: feces; some keratinocytes around the scabies burrows; and the mouth and legs of the scabies mite. The gray-edged line sign appears to represent mite feces containing melanin.

Expression of anti-aging type-XVII collagen (COL17A1/BP180) in hair follicle-associated pluripotent (HAP) stem cells during differentiation.

Hair-follicle-associated pluripotent (HAP) stem cells reside in the upper part of the bulge area of the the hair follicle. HAP stem cells are nestin-positive and keratin 15-negative and have the capacity to differentiate into various types of cells in vitro. HAP stem cells are also involved in nerve and spinal cord regeneration in mouse models. Recently, it was shown that the DNA-damage response in non-HAP hair follicle stem cells induces proteolysis of type-XVII collagen (COL17A1/BP180), which is involved in hair-follicle stem-cell maintenance. COL17A1 proteolysis stimulated hair-follicle stem-cell aging, characterized by the loss of stemness signatures and hair-follicle miniaturization associated with androgenic alopecia. In the present study, we demonstrate that HAP stem cells co-express nestin and COL17A1 in vitro and in vivo. The expression of HAP stem cell markers (nestin and SSEA1) increased after HAP stem-cell colonies were formed, then decreased after differentiation to epidermal keratinocytes. In contrast COL17A1 increased after differentiation to epidermal keratinocytes. These results suggest that COL17A1 is important in differentiation of HAP stem cells.

Hair-Follicle-Associated Pluripotent (HAP) Stem Cells Encapsulated on Polyvinylidene Fluoride Membranes (PFM) Promote Functional Recovery from Spinal Cord Injury.

Our previous studies showed that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells, which reside in the bulge area of the hair follicle, could restore injured nerve and spinal cord and differentiate into cardiac muscle cells. Here we transplanted mouse green fluorescent protein (GFP)-expressing HAP stem-cell colonies enclosed on polyvinylidene fluoride membranes (PFM) into the severed thoracic spinal cord of nude mice. After seven weeks of implantation, we found the differentiation of HAP stem cells into neurons and glial cells. Our results also showed that PFM-captured GFP-expressing HAP stem-cell colonies assisted complete reattachment of the thoracic spinal cord. Furthermore, our quantitative motor function analysis with the Basso Mouse Scale for Locomotion (BMS) score demonstrated a significant improvement in the implanted mice compared to non-implanted mice with a severed spinal cord. Our study also showed that it is easy to obtain HAP stem cells, they do not develop teratomas, and do not loose differentiation ability when cryopreserved. Collectively our results suggest that HAP stem cells could be a better source compared to induced pluripotent stem cells (iPS) or embryonic stem (ES) cells for regenerative medicine, specifically for spinal cord repair.

Implanted hair-follicle-associated pluripotent (HAP) stem cells encapsulated in polyvinylidene fluoride membrane cylinders promote effective recovery of peripheral nerve injury.

Hair follicle-associated-pluripotent (HAP) stem cells are located in the bulge area of the hair follicle, express the stem-cell marker, nestin, and have been shown to differentiate to nerve cells, glial cells, keratinocytes, smooth muscle cells, cardiac muscle cells, and melanocytes. Transplanted HAP stem cells promote the recovery of peripheral nerve and spinal cord injuries and have the potential for heart regeneration as well. In the present study, we implanted mouse green fluorescent protein (GFP)-expressing HAP stem-cell spheres encapsulated in polyvinylidene fluoride (PVDF)-membrane cylinders into the severed sciatic nerve of immunocompetent and immunocompromised (nude) mice. Eight weeks after implantation, immunofluorescence staining showed that the HAP stem cells differentiated into neurons and glial cells. Fluorescence microscopy showed that the HAP stem cell hair spheres promoted rejoining of the sciatic nerve of both immunocompetent and immunodeficient mice. Hematoxylin and eosin (H&E) staining showed that the severed scatic nerves had regenerated. Quantitative walking analysis showed that the transplanted mice recovered the ability to walk normally. HAP stem cells are readily accessible from everyone, do not form tumors, and can be cryopreserved without loss of differentiation potential. These results suggest that HAP stem cells may have greater potential than iPS or ES cells for regenerative medicine.

Hypoxia Enhances Differentiation of Hair Follicle-Associated-Pluripotent (HAP) Stem Cells to Cardiac-Muscle Cells.

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair-follicle stem cells located in the bulge area which are termed hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells from mouse and human could form spheres in culture, termed hair spheres, which are keratin 15-negative and nestin-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Subsequently, we demonstrated that nestin-expressing stem cells could effect nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. We recently observed that isoproterenol directs HAP stem cells to differentiate to cardiac-muscle cells in large numbers in culture compared to HAP stem cells not supplemented with isoproterenol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. In the present study, we report that, under hypoxic conditions, HAP stem cells differentiated to troponin-positive cardiac-muscle cells at a higher rate that under normoxic conditions. Hypoxia did not influence the differentiation to other cell types. For future use of HAP stem cells for cardiac muscle regeneration, hypoxia should enhance the rate of differentiation thereby providing patients more opportunities to use their own HAP stem cells which are easily accessible, for this purpose. J. Cell. Biochem. 118: 554-558, 2017. © 2016 Wiley Periodicals, Inc.

Antibodies against cyclic citrullinated peptide in Japanese psoriatic arthritis patients.

Anti-cyclic citrullinated peptide antibodies (anti-CCP) are highly considered to indicate disease severity and be predictive markers in rheumatoid arthritis (RA). RA patients who are positive for anti-CCP tend to progress more frequently to joint deformity and functionally deteriorate more than negative patients. A study concerning the presence of anti-CCP in Japanese patients with psoriatic arthritis (PsA) has been published. Our aim was to clarify that anti-CCP could be a potentially useful marker in PsA patients. We herein describe a PsA patient with presence of anti-CCP. We examined anti-CCP in 15 patients with PsA, and compared with 18 controls who had other types of psoriasis. Three PsA patients were positive for anti-CCP, but no controls showed positive. The anti-CCP-positive patients had higher counts of radiographic erosion, higher prevalence rates of polyarticular disease, use of disease-modifying anti-rheumatic drugs, and the human leukocyte antigen DRB1*04 shared epitope than negative patients. Our study demonstrated that anti-CCP was potentially both predictive and a severity marker of joint involvement in PsA, the same as in RA.

Microscopic polyangiitis presenting urticarial erythema and Henoch-Schonlein purpura: two case reports.

Microscopic polyangiitis (MPA) is well known as a life-threatening member of a group of systemic vasculitis diseases. We report two cases of MPA. Case 1 was a 79-year-old-man who had been diagnosed with anti-neutrophil-cytoplasmic-antibody associated vasculitis (ANCA associated vasculitis) with alveolar hemorrhage and crescentric glomerulonephritis (CrGN). He presented with urticarial erythema in the abdomen, legs and back. The skin biopsy specimens showed leukocytoclastic vasculitis on the upper dermis. Case 2 was a 74-year-old-man, who presented with purpura on the abdomen, buttocks and legs that were similar to Henoch-Schonlein purpura (HSP). He also suffered from interstinal pneumonia. His renal biopsy specimens showed glomerulosclerosis and the peripheral pattern anti-neutrophil cytoplasmic antibody (P-ANCA) was positive. We reviewed the skin eruptions that had been reported with MPA, including our cases.