Yuko Hamada

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.

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.

From hair to heart: nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells differentiate to beating 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 CD34-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. In the present study, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. We separated the mouse vibrissa hair follicle into 3 parts (upper, middle, and lower), and suspended each part separately in DMEM containing 10% FBS. All three parts of hair follicle differentiated to beating cardiac muscle cells as well as neurons, glial cells, keratinocytes and smooth muscle cells. The differentiation potential to cardiac muscle is greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol and inhibited by propanolol. HAP stem cells have potential for regenerative medicine for heart disease as well as nerve and spinal cord repair.

Observations on angiopoietin 2 in patients with angiosarcoma

SIR, Vascular remodelling in host tissues surrounding growing tumours is implicated in the successful development of tumour neovasculature. Cooperation between vascular endothelial growth factor (VEGF) and angiopoietins (Angs) is considered to be critical in this context. VEGF is a prime regulator of endothelial cell proliferation, angiogenesis, vasculogenesis and vascular permeability. We have recently established a human angiosarcoma cell line, ISO-HAS. We have previously demonstrated that tumour cells of the angiosarcoma cell line ISO-HAS secrete VEGF-A protein. Tie2 is an endothelium-specific receptor tyrosine kinase known to play a role in tumour angiogenesis. Modulation of Tie2 receptor activity by its Ang ligands is crucial for angiogenesis, blood vessel maturation and integrity of the vascular endothelium. Ang1 and Ang2 are respectively proangiogenic and antiangiogenic owing to their respective agonist and antagonist signalling action through the Tie2 receptor. It has recently been reported that in the presence of endogenous VEGF-A, Ang2 promotes a rapid increase in capillary diameter, remodelling of the basal lamina and proliferation and migration of endothelial cells, and stimulates sprouting of new blood vessels in vivo. By contrast, Ang2 promotes endothelial cell death and vessel regression if the activity of endogenous VEGF is inhibited. These observations support a model for regulation of vascularity where VEGF can convert the consequence of Ang2 stimulation from antiangiogenic to proangiogenic. In the present study, we have demonstrated that tumour cells of the angiosarcoma cell line ISO-HAS express mRNA of Ang2 and its receptor, Tie2, and secrete the Ang2 protein, and that serum levels of Ang2 increase with advancing stages of the tumour in patients with angiosarcoma. A human angiosarcoma cell line (ISO-HAS) and a murine phenotypic angiosarcoma cell line (ISO-S1) maintained in our laboratory and normal human endothelial cells (HMvEC) (Morinaga, Yokohama, Japan) were used in this study. ISO-HAS cells were derived from the periauricular metastatic tissue of an 84-year-old Japanese man. ISO-S1 cells were cultured in a complete medium, and ISO-HAS cells were cultured in a complete medium mixed with 50% (v ⁄ v) of the conditioned medium of ISO-S1. Complete medium consisted of high-glucose Dulbecco’s modified Eagle’s medium (Gibco-BRL, Gaithersburg, MD, U.S.A.) supplemented with 15% (v ⁄ v) heat-inactivated fetal calf serum (JRH Biosciences, Lanexa, KS, U.S.A.). HMvEC were cultured in the medium provided by the manufacturer. Polymerase chain reaction (PCR) was performed with primers specific to Ang1, Ang2 and Tie2 (which have been reported by Zhang et al.), and to b-actin as a control. For PCR, 1 lL of cDNA was added to a 25-lL reaction mixture containing 10 mmol L Tris–HCl pH 9Æ0, 50 mmol L KCl, 1Æ5 mmol L MgCl2, 0Æ1% (w ⁄ v) gelatin, 0Æ2 mmol L deoxyribonucleoside triphosphates, 25 pmol L 5¢ and 3¢ oligonucleotide primers, and 2Æ5 U of Taq polymerase (Takara Shuzo Co., Kyoto, Japan). A DNA thermocycler 480 (PerkinElmer Cetus, Norwalk, CT, U.S.A.) was used for one cycle of 95 C for 9 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 58 C for 30 s, and a final cycle of 72 C for 7 min. The PCR product was subjected to electrophoresis in 1Æ5% agarose gel and was visualized by staining with ethidium bromide. We investigated 11 elderly patients (seven men and four women; mean age 75Æ3 years, range 67–84) with definite angiosarcoma of the face and scalp. The serum level of VEGF was measured using a human Ang2 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, U.S.A.) according to the manufacturer’s protocol. Normal control sera were obtained from 18 healthy volunteers (10 men and eight women; mean age 70Æ3 years, range 62–81). We also analysed Ang2 protein levels by ELISA in the conditioned media of ISO-HAS cells and HMvEC. ISO-HAS cells and HMvEC were treated with trypsin, plated out at a density of 5Æ0 · 10 cells per well in 24-multiwell plates, and allowed to attach overnight. After 24 h, cell-free culture supernatants were removed and assayed for VEGF protein levels by ELISA. We have previously demonstrated that tumour cells of the angiosarcoma cell line ISO-HAS secrete VEGF-A protein. In addition, we have observed serum VEGF protein levels to increase with advancing tumour stage in the patient from whom the ISO-HAS cells were derived. In the present

Nestin-positive hair follicle pluripotent stem cells can promote regeneration of impinged peripheral nerve injury

Nestin‐positive, keratin 15 (K15)‐negative multipotent hair follicle stem cells are located above the hair follicle bulge. We have termed this location the hair follicle pluripotent stem cell area. We have previously shown that transplantation of nestin‐expressing hair follicle stem cells can regenerate peripheral nerve and spinal cord injuries. In the present study, we regenerated the impinged sciatic nerve by transplanting hair follicle pluripotent stem cells. Human hair follicle stem cells were transplanted around the impinged sciatic nerve of ICR nude (nu/nu) mice. The hair follicle stem cells were transplanted between impinged sciatic nerve fragments of the mouse where they differentiated into glial fibrillary acidic protein‐positive Schwann cells and promoted the recovery of pre‐existing axons. The regenerated sciatic nerve functionally recovered. These multipotent hair follicle stem cells thereby provide a potential accessible, autologous source of stem cells for regeneration therapy of nerves degenerated by compression between bony or other hard surfaces.

In situ Expression of CD40 and CD40 Ligand in Psoriasis

Background: The interaction between CD40 and CD40 ligand (CD40L) provides a signal that contributes to the initiation of cellular immune responses. However, little information on the in vivo expression of CD40 and CD40L in cutaneous inflammation has been reported. Objective: To investigate the potential role of CD40-mediated signals in the pathogenesis of psoriasis. Materials and Methods: In situ CD40 and CD40L expression was examined immunohistochemically in different stages of psoriatic lesions: fully developed and initial pinpoint. Results: In normal skin, faintly positive immunoreactivity for CD40 was seen in the basal keratinocytes and dermal endothelial cells. These showed almost the same intensity as that seen in psoriatic lesional skin. In the dermal infiltrates of psoriatic lesions, CD40 was intensely expressed and some of these positive cells appeared to be dendritic in shape. Whereas CD40 expression was observed almost in all specimens of psoriatic lesions, the expression of CD40L was predominantly detected in the initial pinpoint lesions of psoriasis. These seemed to be distributed close to CD40-positive cells. Conclusion: These results suggested that CD40L-triggered signals could be involved in the early stage of psoriatic lesion formation.

Multipotent nestin-expressing stem cells capable of forming neurons are located in the upper, middle and lower part of the vibrissa hair follicle

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells. Nestin-expressing cells were initially identified in the hair follicle bulge area (BA) using a transgenic mouse model in which the nestin promoter drives the green fluorescent protein (ND-GFP). The hair-follicle ND-GFP-expressing cells are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. Subsequently, we showed that the nestin-expressing stem cells could affect nerve and spinal cord regeneration after injection in mouse models. In the present study, we separated the mouse vibrissa hair follicle into three parts (upper, middle and lower). Each part of the follicle was cultured separately in DMEM-F12 containing B-27 and 1% methylcellulose supplemented with basic FGF. After 2 mo, the nestin-expressing cells from each of the separated parts of the hair follicle proliferated and formed spheres. Upon transfer of the spheres to RPMI 1640 medium containing 10% FBS, the nestin-expressing cells in the spheres differentiated to neurons, as well as glia, keratinocytes, smooth muscle cells and melanocytes. The differentiated cells were produced by spheres which formed from nestin-expressing cells from all segments of the hair follicle. However, the differentiation potential is greatest in the upper part of the follicle. This result is consistent with trafficking of nestin-expressing cells throughout the hair follicle from the bulge area to the dermal papilla that we previously observed. The nestin-expressing cells from the upper part of the follicle produced spheres in very large amounts, which in turn differentiated to neurons and other cell types. The results of the present study demonstrate that multipotent, nestin-expressing stem cells are present throughout the hair follicle and that the upper part of the follicle can produce the stem cells in large amounts that could be used for nerve and spinal cord repair.

Direct transplantation of uncultured hair-follicle pluripotent stem (hfPS) cells promotes the recovery of peripheral nerve injury

We previously showed that the stem cell marker nestin is expressed in hair follicle stem cells which suggested their pluripotency. We subsequently showed that the nestin‐expressing hair‐follicle pluripotent stem (hfPS) cells can differentiate in culture to neurons, glial cells, keratinocytes, and other cell types and can promote regeneration of peripheral nerve and spinal cord injuries upon injection to the injured nerve or spinal cord. The location of the hfPS cells has been termed the hfPS cell area (hfPSCA). Previously, hfPS cells were cultured for 1–2 months before transplantation to the injured nerve or spinal cord which would not be optimal for clinical application of these cells for nerve or spinal cord repair, since the patient should be treated soon after injury. In the present study, we addressed this issue by directly using the upper part of the hair follicle containing the hfPSCA, without culture, for injection into the severed sciatic nerve in mice. After injection of hfPSCA, the implanted hfPS cells grew and promoted joining of the severed nerve. The transplanted hfPS cells differentiated mostly to glial cells forming myelin sheaths, which promoted axonal growth and functional recovery of the severed nerve. These results suggest that the direct transplantation of the uncultured upper part of the hair follicle containing the hfPSA is an important method to promote the recovery of peripheral nerve injuries and has significant clinical potential. J. Cell. Biochem. 110: 272–277, 2010.

Isoproterenol directs hair follicle-associated pluripotent (HAP) stem cells to differentiate in vitro to cardiac muscle cells which can be induced to form beating heart-muscle tissue sheets.

ABSTRACT Nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of the follicle. Previous studies have shown that HAP stem cells can differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. HAP stem cells effected nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. The differentiation potential to cardiac muscle cells was greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol. In the present study, we 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. These results demonstrate that HAP stem cells have great potential to form beating cardiac muscle cells in tissue sheets.

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.