Kensei Katsuoka

Nestin-Linked Green Fluorescent Protein Transgenic Nude Mouse for Imaging Human Tumor Angiogenesis

We report here a novel transgenic nude mouse for the visualization of human tumor angiogenesis. We have recently shown that the neural stem cell marker nestin is expressed in hair follicle stem cells and blood vessel networks in the skin of C57/B6 transgenic mice with nestin regulatory element-driven green fluorescent protein (ND-GFP). Others have shown ND-GFP is expressed in the brain, pancreas, and testes in these mice. In the present study, the nestin ND-GFP gene was crossed into nude mice on the C57/B6 background to obtain ND-GFP nude mice. ND-GFP was expressed in the brain, spinal cord, pancreas, stomach, esophagus, heart, lung, blood vessels of glomeruli, blood vessels of skeletal muscle, testes, hair follicles, and blood vessel network in the skin of ND-GFP nude mice. Human lung cancer, pancreatic cancer, and colon cancer cell lines as well as a murine melanoma cell line and breast cancer tumor cell line expressing red fluorescent protein were implanted orthotopically, and a red fluorescent protein-expressing human fibrosarcoma was implanted s.c. in the ND-GFP nude mice. These tumors grew extensively in the ND-GFP mice. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumors, visualized by dual-color fluorescence imaging. Results of immunohistochemical staining showed that CD31 was expressed in the ND-GFP-expressing nascent blood vessels. The ND-GFP transgenic nude mouse model enables the visualization of nascent angiogenesis in human and mouse tumor progression. These results suggest that this model is useful for the imaging of the angiogenesis of human as well as rodent tumors and visualization of the efficacy of angiogenetic inhibitors.

Multipotent hair follicle stem cells promote repair of spinal cord injury and recovery of walking function

The mouse hair follicle is an easily accessible source of actively growing, pluripotent adult stem cells. C57BL transgenic mice, labeled with the fluorescent protein GFP, afforded follicle stem cells whose fate could be followed when transferred to recipient animals. These cells appear to be relatively undifferentiated since they are positive for the stem cell markers nestin and CD34 but negative for the keratinocyte marker keratin 15. These hair follicle stem cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Implanting hair follicle stem cells into the gap region of severed sciatic or tibial nerves greatly enhanced the rate of nerve regeneration and restoration of nerve function. The transplanted follicle cells transdifferentiated mostly into Schwann cells, which are known to support neuron regrowth. The treated mice regained the ability to walk essentially normally. In the present study, we severed the thoracic spinal chord of C57BL/6 immunocompetent mice and transplanted GFP-expressing hair follicle stem cells to the injury site. Most of the transplanted cells also differentiated into Schwann cells that apparently facilitated repair of the severed spinal cord. The rejoined spinal cord reestablished extensive hind-limb locomotor performance. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury. Thus, hair follicle stem cells provide an effective accessible, autologous source of stem cells for the promising treatment of peripheral nerve and spinal cord injury.

Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral‐nerve injury: An advantageous alternative to ES and iPS cells

The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral‐, plasmid‐ or transposon‐mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection. To address these fundamental problems, our group is developing hair follicle pluripotent stem (hfPS) cells. Our previous studies have shown that mouse hfPS cells can differentiate to neurons, glial cells in vitro, and other cell types, and can promote nerve and spinal cord regeneration in vivo. hfPS cells are located above the hair follicle bulge in what we have termed the hfPS cell area (hfPSA) and are nestin positive and keratin 15 (K‐15) negative. Human hfPS cells can also differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In the present study, human hfPS cells were transplanted in the severed sciatic nerve of the mouse where they differentiated into glial fibrillary‐acidic‐protein (GFAP)‐positive Schwann cells and promoted the recovery of pre‐existing axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. The hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regenerative medicine that have important advantages over ES or iPS cells. J. Cell. Biochem. 107: 1016–1020, 2009.

Hair Follicle–Derived Blood Vessels Vascularize Tumors in Skin and Are Inhibited by Doxorubicin

We have recently shown that the neural-stem cell marker nestin is expressed in hair follicle stem cells and the blood vessel network interconnecting hair follicles in the skin of transgenic mice with nestin regulatory element-driven green fluorescent protein (ND-GFP). The hair follicles were shown to give rise to the nestin-expressing blood vessels in the skin. In the present study, we visualized tumor angiogenesis by dual-color fluorescence imaging in ND-GFP transgenic mice after transplantation of the murine melanoma cell line B16F10 expressing red fluorescent protein. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumor. Results of immunohistochemical staining showed that the blood vessel-specific antigen CD31 was expressed in ND-GFP-expressing nascent blood vessels. ND-GFP expression was diminished in the vessels with increased blood flow. Progressive angiogenesis during tumor growth was readily visualized during tumor growth by GFP expression. Doxorubicin inhibited the nascent tumor angiogenesis as well as tumor growth in the ND-GFP mice transplanted with B16F10-RFP. This model is useful for direct visualization of tumor angiogenesis and evaluation of angiogenic inhibitors.

Expression of IL-18 in psoriasis.

Abstract Interleukin-18 (IL-18) is a novel cytokine that plays an important role in the T-helper 1 (Th1) response, primarily via its ability to induce IFN-γ production in T cells and NK cells. Human keratinocytes produce IL-18, as do monocytes and macrophages, which are the two major sources of this molecule. It is thought that IL-18 derived from keratinocytes might be involved in the cutaneous Th1-type immune response. In the present study, we investigated the expression of IL-18 in psoriatic lesional skin and attempted to determine whether immunoreactive IL-18 in crude extracts of psoriatic scales is processed to the mature, active form. Immunohistochemical and RT-PCR analysis showed that the expression of IL-18 was increased in psoriatic lesional skin relative to that in normal skin. Western blotting and an ELISA for IL-18 in combination demonstrated that the immunoreactive IL-18 in extracts of psoriatic scales contained the mature form of IL-18, but most of the IL-18 was pro-IL-18. No bioactivity of IL-18 or IFN-γ inducibility in human PBMC could be detected in psoriatic scales. Taken together, these findings indicate that keratinocyte-derived IL-18 participates in the development of the Th1 response in psoriatic lesions, and that its bioactivity appears to be tightly regulated in cutaneous inflammation.

The bulge area is the major hair follicle source of nestin-expressing pluripotent stem cells which can repair the spinal cord compared to the dermal papilla

Nestin has been shown to be expressed in the hair follicle, both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be pluripotent and be able to form neurons and other non-follicle cell types. The nestin-expressing pluripotent stem cells from the DP have been termed skin precursor or SKP cells. The objective of the present study was to determine the major source of nestin-expressing pluripotent stem cells in the hair follicle and to compare the ability of the nestin-expressing pluripotent stem cells from the BA and DP to repair spinal cord injury. Transgenic mice in which the nestin promoter drives GFP (ND-GFP) were used in order to observe nestin expression in the BA and DP. Nestin-expressing DP cells were found in early and middle anagen. The BA had nestin expression throughout the hair cycle and to a greater extent than the DP. The cells from both regions had very long processes extending from them as shown by two-photon confocal microscopy. Nestin-expressing stem cells from both areas differentiated into neuronal cells at high frequency in vitro. Both nestin-expressing DP and BA cells differentiated into neuronal and glial cells after transplantation to the injured spinal cord and enhanced injury repair and locomotor recovery within four weeks. Nestin-expressing pluripotent stem cells from both the BA and DP have potential for spinal cord regeneration, with the BA being the greater and more constant source.

Case Report. A case of chromoblastomycosis effectively treated with terbinafine. Characteristics of chromoblastomycosis in the Kitasato region, Japan

A 38‐year‐old male with history of trauma in the left gluteal region 20 years ago presented with a dark red skin eruption at the traumatized area. It gradually grew to form an erythematous plaque with a well‐defined border. Clinical findings and mycological cultures resulted in the diagnosis of chromoblastomycosis due to Fonsecaea pedrosoi. After initial administration of 5‐fluorocytosine and local heat an almost complete cure was achieved with terbinafine combined with local heat therapy. A review is given on the chromoblastomycosis cases observed in the Kitasato region in Japan.

Establishment of a human hemangiosarcoma cell line (ISO-HAS)

A cell line (ISO‐HAS) has been established from tumor tissue of a human hemangiosarcoma arising on the scalp by the use of conditioned medium from a murine‐phenotypic angiosarcoma cell line (ISOS‐1). Cells have been cultured for more than 2 years with up to 100 passages. The cells retained endothelial‐cell properties, such as a characteristic cobblestone appearance at confluency, contact‐inhibited growth, active uptake of acetylated low‐density lipoprotein labeled with 1,1‐dioctadecyl 1,3,3,3,3‐tetramethyl‐indocarbocyanine perchlorate (Dil‐Ac‐LDL) and CD31 expression. However, they were weakly positive for von‐Willebrand‐factor (vWf) antigen and for binding of Ulex europaeus agglutinin‐I (UEA‐1) lectin, and lacked tube‐formation activity. These findings indicate that ISO‐HAS is a poorly differentiated endothelial cell line. ISO‐HAS cells showed accumulation of p53 protein in the nuclei, and a new‐typed p53‐gene point mutation was found in exon 7 at codon 240. When inoculated s.c. into severe‐combined‐immunodeficiency (SCID) mice, the cells showed solid‐tumor growth that caused death. These properties suggest that ISO‐HAS is a malignant endothelial cell line with high tumorigenicity. Int. J. Cancer 81: 305–308, 1999.

Human and mouse hair follicles contain both multipotent and monopotent stem cells

No abstract.

Clinical significance of enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid

The NC16A domain of the 180-kDa bullous pemphigoid antigen (BP180) is the most immunogenic and, probably, pathogenic region in bullous pemphigoid (BP). In the present study, in order to determine whether serum level of circulating anti-BP180 autoantibodies is a valuable serum marker in BP, the immunoreactivity of sera against the NC16A domain of BP180 was measured using enzyme-linked immunosorbent assay (ELISA) in ten patients with BP. Serum levels of anti-BP180 autoantibodies correlated with the clinical course in BP patients, who received various therapeutic agents. The result suggests that this NC16A-ELISA is a useful method for evaluating the clinical course and efficacy of the therapy in patients with BP.