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Dive into the research topics where Shinichiro Kuno is active.

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Featured researches published by Shinichiro Kuno.


Plastic and Reconstructive Surgery | 2012

The fate of adipocytes after nonvascularized fat grafting: evidence of early death and replacement of adipocytes.

Hitomi Eto; Harunosuke Kato; Hirotaka Suga; Noriyuki Aoi; Kentaro Doi; Shinichiro Kuno; Kotaro Yoshimura

Background: Clinical outcomes following fat grafting are variable and technique dependent, and it is unknown how the graft is revascularized. The authors recently observed that living and dead adipocytes can be differentiated not with hematoxylin and eosin staining but with immunohistochemistry for perilipin. Methods: The viability of cellular components (adipocytes, adipose stem/stromal/progenitor cells, vascular endothelial cells, and hematopoietic cells) in human adipose tissue was evaluated using (1) stored lipoaspirates, (2) cultured cells, and (3) organ-cultured adipose tissue. In addition, the groin fat pad (150 to 200 mg) in mice was transplanted under the scalp, and the graft was stained at 0, 1, 2, 3, 5, 7, or 14 days. Results: In vitro studies revealed that adipocytes are most susceptible to death under ischemic conditions, although adipose-derived stromal cells can remain viable for 3 days. The in vivo study indicated that most adipocytes in the graft began to die on day 1, and only some of the adipocytes located within 300 &mgr;m of the tissue edge survived. The number of proliferating cells increased from day 3, and an increase in viable adipocyte area was detected from day 7, suggesting that repair/regeneration of the dead tissue had begun. Conclusions: The authors show convincing evidence of very dynamic remodeling of adipose tissue after nonvascularized grafting. The authors observed three zones from the periphery to the center of the graft: the surviving area (adipocytes survived), the regenerating area (adipocytes died, adipose-derived stromal cells survived, and dead adipocytes were replaced with new ones), and the necrotic area (both adipocytes and adipose-derived stromal cells died).


Journal of Tissue Engineering and Regenerative Medicine | 2013

Stromal vascular fraction isolated from lipo‐aspirates using an automated processing system: bench and bed analysis

Kentaro Doi; Shinsuke Tanaka; Hideo Iida; Hitomi Eto; Harunosuke Kato; Noriyuki Aoi; Shinichiro Kuno; Toshitsugu Hirohi; Kotaro Yoshimura

The heterogeneous stromal vascular fraction (SVF), containing adipose‐derived stem/progenitor cells (ASCs), can be easily isolated through enzymatic digestion of aspirated adipose tissue. In clinical settings, however, strict control of technical procedures according to standard operating procedures and validation of cell‐processing conditions are required. Therefore, we evaluated the efficiency and reliability of an automated system for SVF isolation from adipose tissue. SVF cells, freshly isolated using the automated procedure, showed comparable number and viability to those from manual isolation. Flow cytometric analysis confirmed an SVF cell composition profile similar to that after manual isolation. In addition, the ASC yield after 1 week in culture was also not significantly different between the two groups. Our clinical study, in which SVF cells isolated with the automated system were transplanted with aspirated fat tissue for soft tissue augmentation/reconstruction in 42 patients, showed satisfactory outcomes with no serious side‐effects. Taken together, our results suggested that the automated isolation system is as reliable a method as manual isolation and may also be useful in clinical settings. Automated isolation is expected to enable cell‐based clinical trials in small facilities with an aseptic room, without the necessity of a good manufacturing practice‐level cell processing area. Copyright


Laboratory Investigation | 2012

Therapeutic potential of fibroblast growth factor-2 for hypertrophic scars: upregulation of MMP-1 and HGF expression

Hitomi Eto; Hirotaka Suga; Noriyuki Aoi; Harunosuke Kato; Kentaro Doi; Shinichiro Kuno; Yasuhiko Tabata; Kotaro Yoshimura

Although hypertrophic scars (HTSs) and keloids are challenging problems, their pathogenesis is not well understood, making therapy difficult. We showed that matrix metalloproteinase (MMP)-1 expression was downregulated in HTS compared with normal skin from the same patients, whereas type 1 and 3 collagen and transforming growth factor-β (TGF-β) were upregulated. These differences, however, were not seen in cultured fibroblasts, suggesting the involvement of microenvironmental factors in the pathogenesis of HTS. Fibroblast growth factor-2 (FGF-2) highly upregulated the expression of MMP-1 and hepatocyte growth factor (HGF) in both HTS-derived and control fibroblasts; the upregulation was reversed by extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors. An animal study using human HTS tissue implanted into nude mice indicated that controlled-release FGF-2 resulted in significantly less weight and decreased hydroxyproline content in HTS. Degradation of collagen fibers in FGF-2-treated HTS was also confirmed histologically. Western blotting showed that FGF-2-treated HTS expressed significantly higher MMP-1 protein than control. Decreased MMP-1 expression may be an important transcriptional change in HTS, and its reversal as well as upregulation of HGF by FGF-2 could be a new therapeutic approach for HTS.


Plastic and Reconstructive Surgery | 2015

Degeneration, regeneration, and cicatrization after fat grafting: dynamic total tissue remodeling during the first 3 months.

Harunosuke Kato; Kazuhide Mineda; Hitomi Eto; Kentaro Doi; Shinichiro Kuno; Kahori Kinoshita; Koji Kanayama; Kotaro Yoshimura

Background: Fat grafting is promising, but clinical outcomes are not always predictable. The mechanisms of tissue revascularization/regeneration, and tissue necrosis and subsequent absorption/fibrosis of the graft, are poorly understood. Methods: An autologous inguinal fat pad was transplanted under the scalp of mice, and detailed cellular events during the first 3 months were investigated with immunohistochemistry. Results: Except for the most superficial surviving zone, death of all adipocytes was confirmed at 1 week. Perilipin-positive small new adipocytes appeared at 1 week and peaked in number at 4 weeks in the regenerating zone (the second zone). In the most central necrotizing zone, adipogenesis did not occur and many inflammatory cells were observed after 2 weeks. CD34+/Ki67+ proliferating adipose stem/progenitor cells were seen at 1 to 4 weeks, but the majority of proliferating cells were MAC2+ monocytes/macrophages. Although CD206− M1 macrophages surrounded oil droplets for phagocytosis, CD206+ M2 macrophages appeared in areas where adipocyte replacement failed and formed multiple layers for cicatrization of oil drop spaces. Adipogenesis was complete by 12 weeks, but stabilization of nonregenerated areas was still ongoing at that time. Lipid droplets derived from dead adipocytes were absorbed slowly and thus aided adipose remodeling by maintaining the space until adipocyte regeneration. Conclusions: Dynamic remodeling after fat grafting was confirmed. Adipocyte fate differed, depending on the microenvironment: intact survival, replacement with a new adipocyte, or replacement with cicatrization/oil cyst. This detailed understanding will help refine surgical grafting procedures and postoperative evaluation.


Stem Cells and Development | 2013

Characterization of Human Adipose Tissue-Resident Hematopoietic Cell Populations Reveals a Novel Macrophage Subpopulation with CD34 Expression and Mesenchymal Multipotency

Hitomi Eto; Hisako Ishimine; Kahori Kinoshita; Kanako Watanabe-Susaki; Harunosuke Kato; Kentaro Doi; Shinichiro Kuno; Akira Kurisaki; Kotaro Yoshimura

Adipose tissue (AT) is composed of mature adipocytes and stromal vascular fraction (SVF) cells, including adipose stem/stromal cells (ASCs). We characterized hematopoietic cells residing in human nonobese AT by analyzing the SVF isolated from human lipoaspirates and peripheral blood (PB). Flow cytometry revealed that AT-resident hematopoietic cells consisted of AT-resident macrophages (ATMs) or lymphocytes with a negligible number of granulocytes. AT-resident lymphocytes were composed of helper T cells and natural killer cells. Almost no B cells and few cytotoxic T cells were observed in nonobese AT. More than 90% of ATMs were M2 state CD206(+) macrophages (CD45(+)/CD14(+)) that were located in the periendothelium or interstitial spaces between adipocytes. We also discovered a novel subpopulation of CD34(+)/CD206(+) ATMs (11.1% of CD206(+)ATMs) that localized in the perivascular region. Microarray of noncultured CD34(+)/CD206(+) ATMs, CD34(-)/CD206(+) ATMs, CD45(-)/CD31(-)/CD34(+) ASCs, and PB-derived circulating monocytes revealed that CD34(+)/CD206(+) ATMs shared characteristics with ASCs and circulating monocytes. Unlike CD34(-)/CD206(+) ATMs, CD34(+)/CD206(+) ATMs could grow in adherent culture and were capable of differentiating into multiple mesenchymal (adipogenic, osteogenic, and chondrogenic) lineages, similar to ASCs. CD34(+)/CD206(+) ATMs grew rapidly and lost expression of CD45, CD14, and CD206 by passage 3, which resulted in a similar expression profile to ASCs. Thus, this novel ATM subpopulation (CD45(+)/CD14(+)/CD34(+)/CD206(+)) showed distinct biological properties from other ATMs and circulating monocytes/macrophages. The CD34(+)/CD206(+) ATMs possessed characteristics similar to ASCs, including adherence, localization, morphology, and mesenchymal multipotency. This AT-resident subpopulation may have migrated from the bone marrow and may be important to tissue maintenance and remolding.


Plastic and Reconstructive Surgery | 2014

Chronic Inflammation and Progressive Calcification as a Result of Fat Necrosis: The Worst Outcome in Fat Grafting

Kazuhide Mineda; Shinichiro Kuno; Harunosuke Kato; Kahori Kinoshita; Kentaro Doi; Ichiro Hashimoto; Hideki Nakanishi; Kotaro Yoshimura

Background: Autologous fat injection into the breast has been performed widely for breast augmentation and reconstruction because of recent technical and scientific advancements. However, it is important to learn what occurs and how problematic it can be if fat grafting is not conducted appropriately. Methods: Oil cysts were explanted from three subjects who underwent cosmetic fat grafting to the breast 2, 4, and 6 years previously. The oil cyst samples were examined histopathologically. Computed tomographic, magnetic resonance imaging, and mammographic images obtained sequentially after fat grafting were also analyzed. Results: The cyst wall consisted of innermost and outermost fibrous layers and intermediate tissue that contained the regular adipose portion, a degenerated adipose portion, and a fibrous area. Eggshell-like macrocalcifications were seen in the inner surface. Numerous inflammatory cells, mainly MAC2+/CD206+ anti-inflammatory M2 macrophages, were observed in the degenerated adipose portion. Oil cysts with a longer history showed more calcifications in the innermost layer and a larger fibrous area adjacent to the degenerated fat portion than those with a shorter history. These histopathologic findings and clinical computed tomographic images revealed that oil cysts continued to be inflammatory and calcifications continued to develop over several years. Conclusions: After fat necrosis, long-term chronic inflammation persists and calcification seems to progress without limits. Oil cysts are the worst outcome of fat grafting and must be avoided by standardizing meticulous injection techniques. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.


Stem Cells Translational Medicine | 2015

Therapeutic Potential of Adipose-Derived SSEA-3-Positive Muse Cells for Treating Diabetic Skin Ulcers

Kahori Kinoshita; Shinichiro Kuno; Hisako Ishimine; Noriyuki Aoi; Kazuhide Mineda; Harunosuke Kato; Kentaro Doi; Koji Kanayama; Jingwei Feng; Takanobu Mashiko; Akira Kurisaki; Kotaro Yoshimura

Stage‐specific embryonic antigen‐3 (SSEA‐3)‐positive multipotent mesenchymal cells (multilineage differentiating stress‐enduring [Muse] cells) were isolated from cultured human adipose tissue‐derived stem/stromal cells (hASCs) and characterized, and their therapeutic potential for treating diabetic skin ulcers was evaluated. Cultured hASCs were separated using magnetic‐activated cell sorting into positive and negative fractions, a SSEA‐3+ cell‐enriched fraction (Muse‐rich) and the remaining fraction (Muse‐poor). Muse‐rich hASCs showed upregulated and downregulated pluripotency and cell proliferation genes, respectively, compared with Muse‐poor hASCs. These cells also released higher amounts of certain growth factors, particularly under hypoxic conditions, compared with Muse‐poor cells. Skin ulcers were generated in severe combined immunodeficiency (SCID) mice with type 1 diabetes, which showed delayed wound healing compared with nondiabetic SCID mice. Treatment with Muse‐rich cells significantly accelerated wound healing compared with treatment with Muse‐poor cells. Transplanted cells were integrated into the regenerated dermis as vascular endothelial cells and other cells. However, they were not detected in the surrounding intact regions. Thus, the selected population of ASCs has greater therapeutic effects to accelerate impaired wound healing associated with type 1 diabetes. These cells can be achieved in large amounts with minimal morbidity and could be a practical tool for a variety of stem cell‐depleted or ischemic conditions of various organs and tissues.


Dermatologic Surgery | 2011

A Prospective Randomized Controlled Study of Oral Tranexamic Acid for Preventing Postinflammatory Hyperpigmentation After Q-Switched Ruby Laser

Harunosuke Kato; Jun Araki; Hitomi Eto; Kentaro Doi; Rintaro Hirai; Shinichiro Kuno; Takuya Higashino; Kotaro Yoshimura

BACKGROUND Postinflammatory hyperpigmentation (PIH) is the most common skin complication in Asians after invasive cosmetic treatments. OBJECTIVE To determine whether oral tranexamic acid (TA) reduces the incidence of PIH after Q‐switched ruby laser (QSRL) treatment. METHODS AND MATERIALS Thirty‐two Japanese women underwent QSRL treatment for senile lentigines on the face. They were randomly divided into two groups that did (n=15) and did not (n=17) receive oral TA treatment (750 mg/d) for the first 4 weeks after QSRL treatment. Nineteen participants had melasma‐like maculae at baseline. Clinical and colorimetric assessments were performed at baseline and 2 and 4 weeks later. RESULTS Pigmentation was effectively treated using QSRL at 2 weeks, but PIH was frequently seen at 4 weeks. There was no significant difference in the incidence of PIH between participants who received oral TA and those who did not. The presence of melasma did not influence the effectiveness of the treatment. CONCLUSION Although oral TA has been reported to have depigmentation effects, it may not be effective for preventing PIH after QSRL. Considering the dosage and duration of treatment, an optimal protocol may be needed to induce the efficacy of this treatment to achieve the PIH‐preventing effect of oral TA. The authors have indicated no significant interest with commercial supporters.


Stem Cells Translational Medicine | 2015

Therapeutic Potential of Human Adipose-Derived Stem/Stromal Cell Microspheroids Prepared by Three-Dimensional Culture in Non-Cross-Linked Hyaluronic Acid Gel

Kazuhide Mineda; Jingwei Feng; Hisako Ishimine; Hitomi Takada; Kentaro Doi; Shinichiro Kuno; Kahori Kinoshita; Koji Kanayama; Harunosuke Kato; Takanobu Mashiko; Ichiro Hashimoto; Hideki Nakanishi; Akira Kurisaki; Kotaro Yoshimura

Three‐dimensional culture of mesenchymal stem/stromal cells for spheroid formation is known to enhance their therapeutic potential for regenerative medicine. Spheroids were prepared by culturing human adipose‐derived stem/stromal cells (hASCs) in a non‐cross‐linked hyaluronic acid (HA) gel and compared with dissociated hASCs and hASC spheroids prepared using a nonadherent dish. Preliminary experiments indicated that a 4% HA gel was the most appropriate for forming hASC spheroids with a relatively consistent size (20–50 µm) within 48 hours. Prepared spheroids were positive for pluripotency markers (NANOG, OCT3/4, and SOX‐2), and 40% of the cells were SSEA‐3‐positive, a marker of the multilineage differentiating stress enduring or Muse cell. In contrast with dissociated ASCs, increased secretion of cytokines such as hepatocyte growth factor was detected in ASC spheroids cultured under hypoxia. On microarray ASC spheroids showed upregulation of some pluripotency markers and downregulation of genes related to the mitotic cell cycle. After ischemia‐reperfusion injury to the fat pad in SCID mice, local injection of hASC spheroids promoted tissue repair and reduced the final atrophy (1.6%) compared with that of dissociated hASCs (14.3%) or phosphate‐buffered saline (20.3%). Part of the administered hASCs differentiated into vascular endothelial cells. ASC spheroids prepared in a HA gel contain undifferentiated cells with therapeutic potential to promote angiogenesis and tissue regeneration after damage.


Plastic and Reconstructive Surgery | 2015

Differential contributions of graft-derived and host-derived cells in tissue regeneration/remodeling after fat grafting.

Kentaro Doi; Fusa Ogata; Hitomi Eto; Harunosuke Kato; Shinichiro Kuno; Kahori Kinoshita; Koji Kanayama; Jingwei Feng; Ichiro Manabe; Kotaro Yoshimura

Background: Recent research indicates that the adipose tissue of nonvascularized grafts is completely remodeled within 3 months, although origins of next-generation cells are unclear. Methods: Inguinal fat pads of green fluorescent protein mice and wild-type mice were cross-transplanted beneath the scalp. At 1, 2, 4, and 12 weeks after transplantation, grafted fat was harvested, weighed, and analyzed through immunohistochemistry, whole-mount staining, and flow cytometry of cell isolates. Bone marrow of green fluorescent protein mice was transplanted to wild-type mice (after irradiation). Eight weeks later, these mice also received fat grafts, which were analyzed as well. Results: The majority of host-derived cells detected during remodeling of grafted fat were macrophages (>90 percent at the early stage; 60 percent at 12 weeks). Cell origins were analyzed at 12 weeks (i.e., when completely regenerated). At this point, mature adipocytes were largely derived from adipose-derived stem/stromal cells of grafts. Although vascular wall constituents were chiefly graft derived, vascular endothelial cells originated equally from graft and host bone marrow. Adipose-derived stem/stromal cells of regenerated fat were an admixture of grafted, host nonbone marrow, and host bone marrow cells. Conclusions: The above findings underscore the importance of adipose stem/stromal cells in the grafted fat for adipocyte regeneration. Host bone marrow and local tissues contributed substantially to capillary networks and provided new adipose-derived stem/stromal cells. An appreciation of mechanisms that are operant in this setting stands to improve clinical outcomes of fat grafting and cell-based therapies.

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Akira Kurisaki

National Institute of Advanced Industrial Science and Technology

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