Kotaro Yoshimura

CD8 + effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity

Inflammation is increasingly regarded as a key process underlying metabolic diseases in obese individuals. In particular, obese adipose tissue shows features characteristic of active local inflammation. At present, however, little is known about the sequence of events that comprises the inflammatory cascade or the mechanism by which inflammation develops. We found that large numbers of CD8+ effector T cells infiltrated obese epididymal adipose tissue in mice fed a high-fat diet, whereas the numbers of CD4+ helper and regulatory T cells were diminished. The infiltration by CD8+ T cells preceded the accumulation of macrophages, and immunological and genetic depletion of CD8+ T cells lowered macrophage infiltration and adipose tissue inflammation and ameliorated systemic insulin resistance. Conversely, adoptive transfer of CD8+ T cells to CD8-deficient mice aggravated adipose inflammation. Coculture and other in vitro experiments revealed a vicious cycle of interactions between CD8+ T cells, macrophages and adipose tissue. Our findings suggest that obese adipose tissue activates CD8+ T cells, which, in turn, promote the recruitment and activation of macrophages in this tissue. These results support the notion that CD8+ T cells have an essential role in the initiation and propagation of adipose inflammation.

Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT)

BACKGROUND AIMS Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population. METHODS Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature. RESULTS In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction. CONCLUSIONS The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters.

Cell-Assisted Lipotransfer for Cosmetic Breast Augmentation: Supportive Use of Adipose-Derived Stem/Stromal Cells

BackgroundLipoinjection is a promising treatment but has some problems, such as unpredictability and a low rate of graft survival due to partial necrosis.MethodsTo overcome the problems with lipoinjection, the authors developed a novel strategy known as cell-assisted lipotransfer (CAL). In CAL, autologous adipose-derived stem (stromal) cells (ASCs) are used in combination with lipoinjection. A stromal vascular fraction (SVF) containing ASCs is freshly isolated from half of the aspirated fat and recombined with the other half. This process converts relatively ASC-poor aspirated fat to ASC-rich fat. This report presents the findings for 40 patients who underwent CAL for cosmetic breast augmentation.ResultsFinal breast volume showed augmentation by 100 to 200 ml after a mean fat amount of 270 ml was injected. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Cyst formation or microcalcification was detected in four patients. Almost all the patients were satisfied with the soft and natural-appearing augmentation.ConclusionsThe preliminary results suggest that CAL is effective and safe for soft tissue augmentation and superior to conventional lipoinjection. Additional study is necessary to evaluate the efficacy of this technique further.

Characterization of Freshly Isolated and Cultured Cells Derived From the Fatty and Fluid Portions of Liposuction Aspirates

Liposuction aspirates (primarily saline solution, blood, and adipose tissue fragments) separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and contain adipose‐derived adherent stromal cells (ASCs). Here we define cells isolated from the fluid portion of liposuction aspirates as liposuction aspirate fluid (LAF) cells. Stromal vascular fractions (SVF) were isolated separately from both portions and characterized under cultured and non‐cultured conditions. A comparable number of LAF and PLA cells were freshly isolated, but fewer LAF cells were adherent. CD34+CD45− cells from fresh LAF isolates were expanded by adherent culture, suggesting that LAF cells contain ASCs. Although freshly isolated PLA and LAF cells have distinct cell surface marker profiles, adherent PLA and LAF cells have quite similar characteristics with regard to growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed significant increased expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD151 and decreased expression of CD31 and CD45. Multicolor FACS analysis revealed that SVF are composed of heterogeneous cell populations including blood‐derived cells (CD45+), ASCs (CD31−CD34+CD45−CD90+CD105−CD146−), endothelial (progenitor) cells (CD31+CD34+CD45−CD90+CD105lowCD146+), pericytes (CD31−CD34−CD45−CD90+CD105−CD146+), and other cells. After plating, ASCs showed a dramatic increase in CD105 expression. Although some adherent ASCs lost CD34 expression with increasing culture time, our culture method maintained CD34 expression in ASCs for at least 10–20 weeks. These results suggest that liposuction‐derived cells may be useful and valuable for cell‐based therapies. J. Cell. Physiol.

Cell‐Assisted Lipotransfer for Facial Lipoatrophy: Efficacy of Clinical Use of Adipose‐Derived Stem Cells

BACKGROUND Lipoinjection is a promising treatment, but its efficacy in recontouring facial lipoatrophy remains to be established. OBJECTIVE The objective was to evaluate the efficacy and adverse effects of lipoinjection and supplementation of adipose-derived stem/stromal cells (ASCs) to adipose grafts. METHODS To overcome drawbacks of autologous lipoinjection, we have developed a novel strategy called cell-assisted lipotransfer (CAL). In CAL, stromal vascular fraction containing ASCs was freshly isolated from half of an aspirated fat sample and attached to the other half of aspirated fat sample with the fat acting as a scaffold. This process converts relatively ASC-poor aspirated fat into ASC-rich fat. We performed conventional lipoinjection (non-CAL; n=3) or CAL (n=3) on six patients with facial lipoatrophy due to lupus profundus or Parry-Romberg syndrome. RESULTS All patients obtained improvement in facial contour, but the CAL group had a better clinical improvement score than did the non-CAL patients, although the difference did not reach statistical significance (p=.11). Adipose necrosis was found in one non-CAL case who took perioperative oral corticosteroids. CONCLUSION Our results suggest that CAL is both effective and safe and potentially superior to conventional lipoinjection for facial recontouring.

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

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).

One-stage transfer of the latissimus dorsi muscle for reanimation of a paralyzed face: a new alternative.

&NA; The two‐stage method combining neurovascular freemuscle transfer with cross‐face nerve grafting is now a widely accepted procedure for dynamic smile reconstruction in cases with long established unilateral facial paralysis. Although the results are promising, the two operations, about 1 year apart, exert an economic burden on the patients and require a lengthy period before obtaining results. Sequelae such as hypoesthesia, paresthesia, and conspicuous scar on the donor leg for harvesting a sural nerve graft also cannot be disregarded. To overcome such drawbacks of the two‐stage method, we report a refined technique utilizing one‐stage microvascular free transfer of the latissimus dorsi muscle. Its thoracodorsal nerve is crossed through the upper lip and sutured to the contralateral intact facial nerve branches. Reinnervation of the transferred muscle is established at a mean of 7 months postoperatively, which is faster than that of the two‐stage method. In our present series with 24 patients, 21 patients (more than 87 percent) believed that their results were excellent or satisfactory, which also compares well with the results of the two‐stage method combining free‐muscle transfer with cross‐face nerve graft. (Plast. Reconstr. Surg. 102: 941, 1998.)

Progenitor-Enriched Adipose Tissue Transplantation as Rescue for Breast Implant Complications

Abstract:  Breast enhancement with artificial implants is one of the most frequently performed cosmetic surgeries but is associated with various complications, such as capsular contracture, that lead to implant removal or replacement at a relatively high rate. For replacement, we used transplantation of progenitor‐supplemented adipose tissue (cell‐assisted lipotransfer; CAL) in 15 patients. The stromal vascular fraction containing adipose tissue progenitor cells obtained from liposuction aspirates was used to enrich for progenitor cells in the graft. Overall, clinical results were very satisfactory, and no major abnormalities were seen on magnetic resonance imaging or mammogram after 12 months. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Surviving fat volume at 12 months was 155 ± 50 mL (Right; mean ± SD) and 143 ± 80 mL (Left) following lipoinjection from an initial mean of 264 mL. These preliminary results suggest that CAL is a suitable methodology for the replacement of breast implants.

Adipose-derived stem/progenitor cells: roles in adipose tissue remodeling and potential use for soft tissue augmentation.

Many features of adipose tissue-specific stem/progenitor cells, such as physiological function and localization, have recently been examined. Adipose-tissue turnover is very slow and its perivascular progenitor cells differentiate into adipocytes in the next generation. The progenitor cells play important roles in physiological turnover, hyperplasia and atrophy of adipose tissue, as well as in incidental remodeling, such as postinjury repair. Adipose tissue has been used as an autologous filler for soft tissue defects, despite unpredictable clinical results and a low rate of graft survival, which may be due to the relative deficiency of progenitor cells in graft materials. A novel transplantation strategy, termed cell-assisted lipotransfer, involves the enrichment of adipose progenitor cells in grafts; preliminary results suggest this approach to be safe and effective.

Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: Reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue

Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB‐MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper‐exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB‐MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donors birth. This resulted in 90% success in isolation of CB‐MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM‐MSC) and adipose tissue (AT‐MSC) as reference controls, we observed that CB‐MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 109 cells required for cell therapies. CB‐MSC showed karyotype stability after prolonged expansion. Functionally, CB‐MSC could be more readily induced to differentiate into chondrocytes than could BM‐MSC and AT‐MSC. CB‐MSC showed immunosuppressive activity equal to that of BM‐MSC and AT‐MSC. Collectively, our data indicate that viable CB‐MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system. J. Cell. Biochem. 112: 1206–1218, 2011.