Kenichiro Tsuchiyama

Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts

The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single cell. They can be isolated as stage-specific embryonic antigen-3/CD105 double-positive cells. When human fibroblasts were separated into Muse and non-Muse cells and transduced with Oct3/4, Sox2, Klf4, and c-Myc, iPS cells were generated exclusively from Muse cells but not from non-Muse cells. Although some colonies were formed from non-Muse cells, they were unlike iPS cells. Furthermore, epigenetic alterations were not seen, and some of the major pluripotency markers were not expressed for the entire period during iPS cell generation. These findings were confirmed further using cells transduced with a single polycistronic virus vector encoding all four factors. The results demonstrate that in adult human fibroblasts a subset of preexisting adult stem cells whose properties are similar in some respects to those of iPS cells selectively become iPS cells, but the remaining cells make no contribution to the generation of iPS cells. Therefore this system seems to fit the elite model rather than the stochastic model.

Functional Melanocytes Are Readily Reprogrammable from Multilineage-Differentiating Stress-Enduring (Muse) Cells, Distinct Stem Cells in Human Fibroblasts

The induction of melanocytes from easily accessible stem cells has attracted attention for the treatment of melanocyte dysfunctions. We found that multilineage-differentiating stress-enduring (Muse) cells, a distinct stem cell type among human dermal fibroblasts, can be readily reprogrammed into functional melanocytes, whereas the remainder of the fibroblasts do not contribute to melanocyte differentiation. Muse cells can be isolated as cells positive for stage-specific embryonic antigen-3, a marker for undifferentiated human embryonic stem cells, and differentiate into cells representative of all three germ layers from a single cell, while also being nontumorigenic. The use of certain combinations of factors induces Muse cells to express melanocyte markers such as tyrosinase and microphthalmia-associated transcription factor and to show positivity for the 3,4-dihydroxy-L-phenylalanine reaction. When Muse cell-derived melanocytes were incorporated into three-dimensional (3D) cultured skin models, they localized themselves in the basal layer of the epidermis and produced melanin in the same manner as authentic melanocytes. They also maintained their melanin production even after the 3D cultured skin was transplanted to immunodeficient mice. This technique may be applicable to the efficient production of melanocytes from accessible human fibroblasts by using Muse cells, thereby contributing to autologous transplantation for melanocyte dysfunctions, such as vitiligo.

Neuroselective transcutaneous electrical stimulation reveals neuronal sensitization in atopic dermatitis

BACKGROUND The neuroselective transcutaneous electrical stimulator (NTES) can provoke itch and/or pain by the application of a 5-Hz alternating current. OBJECTIVE We sought to examine whether there is any difference in the perception of the stimulus evoked by the NTES between patients with atopic dermatitis (AD) and healthy control subjects. METHODS In all, 24 healthy control subjects and 24 patients with AD (nonlesional skin) were stimulated on 7 body sites using the NTES. Qualitative differences in the evoked perceptions and quantitative differences in the current intensity required to evoke perception were statistically analyzed. RESULTS The NTES preferentially evoked itch in patients with AD. The current perception threshold was statistically lower in AD than in healthy control subjects on 3 body sites. LIMITATIONS Tests were performed on limited body areas. CONCLUSION We demonstrated that the NTES can reveal neuronal sensitization to itch in nonlesional atopic skin.

Successful adalimumab treatment of a psoriasis vulgaris patient with hemodialysis for renal failure: A case report and a review of the previous reports on biologic treatments for psoriasis patients with hemodialysis for renal failure

The efficacy and safety of biologic treatments have been established in patients with moderate to severe psoriasis, but there are few reports on biologic therapy for patients with psoriasis complicated by end‐stage renal failure on hemodialysis (HD). In this report, we demonstrated the efficacy and safety of adalimumab for patients with severe psoriasis on HD. A 46‐year‐old Japanese man with a 14‐year history of psoriasis was referred to our clinic in September 2009. He had developed hypertension and renal failure during a 7‐year history of cyclosporin treatment. With the infliximab treatment, he achieved 75% improvement of the Psoriasis Area and Severity Index (PASI) score within 3 months from the PASI of 42.3 before the treatment. However, his renal failure gradually deteriorated, and HD was initiated at 1 year after the introduction of infliximab. Because of hydration during the i.v. injection of infliximab, he developed pulmonary edema with every infliximab treatment after starting HD. We switched to ustekinumab treatment, but his psoriasis was not improved. Then, we switched to adalimumab and achieved a PASI‐100 response within 2 months. The patient received adalimumab treatment for more than a year without any adverse effects. In addition to our case, five articles reported cases of psoriasis patients with renal failure on HD who were treated with biologics. The psoriatic lesions were improved by biologics in these cases, and no severe adverse effects on the renal function were reported. Thus, biologics are a reasonable treatment option for patients with severe psoriasis with renal failure on HD.

A quantitative analysis of multilineage-differentiating stress-enduring (Muse) cells in human adipose tissue and efficacy of melanocytes induction

We have shown previously that multilineage-differentiating stress-enduring (Muse) cells in neonatal fibroblasts can differentiate into functional melanocytes. In this study, we quantitate Muse cells in adipose-mesenchymal stem cells (adipose-MSCs) of human subcutaneous tissue obtained from 11 subjects of various ages, and measured efficacy of melanocytes induction from Adipose-MSC-derived Muse cells (hASC-Muse cells). There was a statistically significant negative correlation between the age of donors and the numbers of adipose-MSCs recovered per g fat as well as the percentage of SSEA3+ cells in the adipose-MSC populations, but isolated hASC-Muse cells showed pluripotency and growth curves equally regardless the age of donors. Adipose-Muse cells sequentially expressed melanocyte-related genes including KIT, MITF, TYRP1 PMEL, DCT, melanocortin 1 receptor (MC1R), and TYR at a comparable level to melanocytes during 6-week culture. Parallel with MC1R expression, adipose-Muse cells increased melanin content by α-MSH stimulation. By quantitating the cell numbers recovered at each step, we found that 10g of adipose tissue could produce at least 2.5×106 melanocytes after 6 weeks of culture. These studies suggest that induction of melanocytes from adipose-Muse is a novel approach to obtain sufficient numbers of melanocytes for clinical application and in vitro study of melanocyte differentiation.

Successful Treatment of Segmental Vitiligo in Children with the Combination of 1-mm Minigrafts and Phototherapy

Background: Minigrafts using a 1-mm biopsy punch (1-mm minigrafts) are being increasingly used to treat vitiligo. However, there have been few reports of the use of 1-mm minigrafts in pediatric patients. Objective: To examine the effectiveness of combination therapy with 1-mm minigrafts and phototherapy in children with segmental vitiligo. Methods: Minigrafts were placed in 13 patients aged ≤16 years with segmental vitiligo. Following surgery, 11 patients underwent irradiation with excimer laser light and 2 with narrow-band ultraviolet B light. Results: A mean repigmentation of 81.6% was obtained. A particularly high mean repigmentation of 87.9% was seen in patients aged ≤12 years, indicating greater efficacy in these patients than in patients aged ≥13 years (mean, 67.5%). Although a transient cobblestone appearance occurred as an adverse effect, it improved over time. Conclusions: Combined treatment of segmental vitiligo with 1-mm minigrafts and phototherapy can be performed safely and is highly effective in young patients.

Therapeutic drug monitoring of patients with psoriasis during tumour necrosis factor (TNF)-α antagonist treatment using a novel interleukin-8 reporter cell line

Tumour necrosis factor (TNF)‐α antagonist therapy is currently used for moderate and severe psoriasis. However, this treatment has several drawbacks, including interindividual variability in clinical response and secondary loss of effectiveness.

Adult-Onset Acquired Partial Lipodystrophy Accompanied by Rheumatoid Arthritis

Lipodystrophy is a group of metabolic disorders, possibly caused by autoimmune disease. In this report, we describe a case of adult-onset acquired partial lipodystrophy accompanied by rheumatoid arthritis without a family history. Interestingly, immunohistochemical staining revealed dense infiltration of IL-27-producing cells as well as MMP-7- and MMP-28-expressing cells, both of which have been reported to facilitate the development of autoimmune disease. Our present case might suggest possible mechanisms for acquired partial lipodystrophy.

Toll-like receptors 2 and 3 enhance melanogenesis and melanosome transport in human melanocytes

Because little is known about how the innate immune response influences skin pigmentation, we examined whether Toll‐like receptor (TLR) agonists participate in melanogenesis and melanosome transportation. We observed that TLR2/2 agonist HKLM and TLR3 agonist Poly(I:C) increased the amount of extracellular melanin from primary human epidermal melanocytes. HKLM, but not Poly(I:C), increased the melanogenic genes such as tyrosinase and dopachrome tautomerase. Poly(I:C) increased the expression of Rab27A, a molecule that facilitates melanosome transport to perimembranous actin filament. UVB irradiation induced Rab27A and melanosome transportation in a similar manner of Poly(I:C). SiRNA for TLR3 or Rab27A suppressed the perimembranous accumulation of Gp100‐positive vesicles in melanocytes and decreased melanin transfer to neighboring keratinocytes induced by both Poly(I:C) and UVB. These results suggest that the microenvironment in the epidermis and innate immune stimuli, such as microbiome and ultraviolet represented here by TLR2 and TLR3 agonists, could affect the melanogenesis in human melanocytes.

Efficacy of oral cholecalciferol on rhododendrol-induced vitiligo: A blinded randomized clinical trial

Rhododendrol (RD), 4‐(4‐hydroxyphenyl)‐2‐butanol, inhibits melanin synthesis and has been used for skin‐whitening cosmetic products. RD has been very effective in lightening skin pigmentation, but some persons have developed so‐called RD vitiligo, in which vitiligo starts on the face, neck and hands where topical RD has been applied and even extended over skin areas where RD has not been applied. RD vitiligo lesions in some patients have lasted for years and have been resistant to conventional vitiligo treatments. We examined the effects of cholecalciferol on RD vitiligo in a blinded randomized clinical trial. Forty‐eight female RD vitiligo patients were recruited for the trial and were randomized into two groups: the vitamin D (VD)‐intervention group that received daily 5000 IU cholecalciferol for 5 months and the control group. Three blinded investigators scored vitiligo improvement by comparing photographic images of baseline and at 5‐month observation. Serum 25(OH)D3 of RD vitiligo patients was not significantly different from age‐matched healthy volunteers. Twenty‐two in the VD‐intervention group and 23 in the control group completed the 5‐month observation. Serum 25(OH)D3 levels were significantly increased after the 5‐month VD intervention, while the control group did not change. The improvement scores were significantly higher in the VD‐intervention group than the control group. The improvement scores were positively correlated with the serum 25(OH)D3 levels after the 5‐month intervention period but not before the treatment. This blinded randomized clinical trial showed favor in administrating 5000 IU cholecalciferol daily to RD vitiligo patients.