Hirotaka Suga

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).

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.

Functional Implications of CD34 Expression in Human Adipose-Derived Stem/Progenitor Cells

CD34 is frequently used as a marker of adipose-derived stem/stromal/progenitor cells (ASCs). However, CD34 expression in human ASCs (hASCs) decreases over time in culture, and the implications of this remain unclear. In this study, we sorted shortly-cultured hASCs into CD34+ and CD34- fractions and compared their biological functions. Results indicated that CD34+ hASCs were more proliferative and had a greater ability to form colonies. In contrast, CD34- cells showed a greater ability for differentiation into adipogenic and osteogenic lineages. Both CD34+ and CD34- cells showed similar abilities in migration and capillary formation in response to growth factors. Expression levels of endothelial progenitor markers (Flk-1, FLT1, and Tie-2) were higher in CD34+ cells, whereas those of pericyte markers (CD146, NG2, and alpha-smooth muscle actin) were higher in CD34- cells. Both CD34+ and CD34- cells showed similar expression levels of vascular endothelial growth factor and hepatocyte growth factor, although hypoxia affected the expression levels. Together these results suggest that CD34 expression in hASCs may correlate with replicative capacity, differentiation potentials, expression profiles of angiogenesis-related genes, and immaturity or stemness of the cells. Loss of CD34 expression may be related to the physiological process of commitment and/or differentiation from immature status into specific lineages such as adipose, bone, or smooth muscle.

Characterization of Structure and Cellular Components of Aspirated and Excised Adipose Tissue

Background: Adipose tissue is an easily accessible tissue for use as a soft-tissue filler and source of adult multipotent cells, called adipose-derived stem/stromal/progenitor cells. However, many aspects of its anatomy remain unclear because of the fragile structure of adipocytes and adipose tissue. Methods: Aspirated (n = 15) or intact (n = 9) subcutaneous adipose tissue samples were evaluated by (1) whole-mount histology with triple-fluorescence staining for three-dimensional visualization of living adipose tissue, (2) glycerol-3-phosphate dehydrogenase assay, (3) multicolor flow cytometry (CD34, CD31, and CD45), and (4) adherent cell culture of stromal vascular fraction cells for viable adipose-derived stromal cell yield. Results: Whole-mount histology revealed the presence of a capillary network running alongside adipocytes, which was partly disrupted in aspirated adipose tissues. Aspirated adipose tissue also lacked large vasculature structures and showed significantly larger numbers of small lipid droplets (ruptured adipocytes) (p = 0.00016) and dead cells (p = 0.0038) compared with excised adipose tissue. Adipocyte number was less than 20 percent of total cellularity, and vasculature-associated cells, including endothelial cells and adipose-derived stromal cells, constituted over one-half of total cells in both intact and aspirated adipose tissue. Glycerol-3-phosphate dehydrogenase assay suggested that greater than 30 percent and 5 percent of adipocytes were ruptured in aspirated and excised adipose tissue, respectively (p = 0.032). Multicolor flow cytometric analysis indicated a much higher percentage of blood-derived cells (CD45+) contaminated in aspirated adipose tissue (p = 0.0038), and adipose-derived stromal cell yield in aspirated adipose tissue was approximately one-half that in excised tissue (p = 0.011). Conclusion: The authors’ results indicate the differential structure and cellular composition of the two tissues, and significant tissue damage and progenitor yield deficiency in aspirated adipose tissue.

Adipose tissue remodeling under ischemia: death of adipocytes and activation of stem/progenitor cells.

Background: Following various types of plastic surgery, such as adipose grafting and flap elevation, adipose tissue undergoes ischemia, leading to hypoxia and nutrient depletion. However, few studies have examined ischemic and/or hypoxic changes in adipose tissue. Methods: The authors established surgically induced ischemia models by severing blood vessels supplying the inguinal fat pads in mice. The partial pressure of oxygen in adipose tissue was measured with an oxygen monitor, and ischemic changes were analyzed by whole-mount staining, immunohistochemistry, flow cytometry, and Western blotting. The authors also examined cell survival under a hypoxic condition in vitro. Results: Models for three degrees (mild, intermediate, and severe) of ischemia showed approximately 75, 55, and 20 percent of the partial pressure of oxygen level in normal adipose tissue (50.5 ± 1.3 mm Hg), respectively. Adipose tissue atrophy with substantial fibrosis on day 28 was seen, depending on the severity of ischemia. Intermediate and severe ischemia induced elevated expression of hypoxia-inducible factor 1&agr; and fibroblast growth factor 2 on day 1 and degenerative changes (i.e., apoptosis, necrosis, and macrophage infiltration and phagocytosis) in adipose tissue. Dead cells included adipocytes, vascular endothelial cells, and blood-derived cells, but not adipose-derived stem/progenitor cells. Subsequent to degenerative changes, regenerative changes were seen, including angiogenesis, adipogenesis, and proliferation of cells (adipose-derived stem/progenitor cells, vascular endothelial cells, and blood cells). The authors found that, in vitro, the experimentally differentiated adipocytes underwent apoptosis and/or necrosis under severe hypoxia, but adipose-derived stem/progenitor cells remained viable. Conclusions: Severe ischemia/hypoxia induces degenerative changes in adipose tissue and subsequent adaptive tissue remodeling. Adipocytes die easily under ischemic conditions, whereas adipose-derived stem/progenitor cells are activated and contribute to adipose tissue repair.

Preserved Proliferative Capacity and Multipotency of Human Adipose-Derived Stem Cells after Long-Term Cryopreservation

Background: Human adipose-derived stem (stromal) cells are promising as a regenerative therapy tool for defective tissues of mesenchymal lineage, including fat, bone, and cartilage, and blood vessels. In potential future clinical applications, adipose-derived stem cell cryopreservation could be an indispensable fundamental technology, as has occurred in other fields involving cell-based therapies using hematopoietic stem cells and umbilical cord blood cells. Methods: The authors examined the proliferative capacity and multipotency of human adipose-derived stem cells isolated from lipoaspirates of 18 patients in total before and after a 6-month cryopreservation following their defined protocol. Proliferative capacity was quantified by measuring doubling time in cell culture, and multipotency was examined with differentiation assays for chondrogenic, osteogenic, and adipogenic lineages. In addition, expression profiles of cell surface markers were determined by flow cytometry and compared between fresh and cryopreserved adipose-derived stem cells. Results: Cryopreserved adipose-derived stem cells fully retained the potential for differentiation into adipocytes, osteoblasts, and chondrocytes and for proliferative capacity. Flow cytometric analyses revealed that surface marker expression profiles remained constant before and after storage. Conclusions: Adipose-derived stem cells can be cryopreserved at least for up to 6 months under the present protocol without any loss of proliferative or differentiation potential. These results ensure the availability of autologous banked adipose-derived stem cells for clinical applications in the future.

IFATS collection: Fibroblast growth factor-2-induced hepatocyte growth factor secretion by adipose-derived stromal cells inhibits postinjury fibrogenesis through a c-Jun N-terminal kinase-dependent mechanism.

Adipose‐derived stem/stromal cells (ASCs) not only function as tissue‐specific progenitor cells but also are multipotent and secrete angiogenic growth factors, such as hepatocyte growth factor (HGF), under certain circumstances. However, the biological role and regulatory mechanism of this secretion have not been well studied. We focused on the role of ASCs in the process of adipose tissue injury and repair and found that among injury‐associated growth factors, fibroblast growth factor‐2 (FGF‐2) strongly promoted ASC proliferation and HGF secretion through a c‐Jun N‐terminal kinase (JNK) signaling pathway. In a mouse model of ischemia‐reperfusion injury of adipose tissue, regenerative changes following necrotic and apoptotic changes were seen for 2 weeks. Acute release of FGF‐2 by injured adipose tissue was followed by upregulation of HGF. During the adipose tissue remodeling process, adipose‐derived 5‐bromo‐2‐deoxyuridine‐positive cells were shown to be ASCs (CD31−CD34+). Inhibition of JNK signaling inhibited the activation of ASCs and delayed the remodeling process. In addition, inhibition of FGF‐2 or JNK signaling prevented postinjury upregulation of HGF and led to increased fibrogenesis in the injured adipose tissue. Increased fibrogenesis also followed the administration of a neutralizing antibody against HGF. FGF‐2 released from injured tissue acts through a JNK signaling pathway to stimulate ASCs to proliferate and secrete HGF, contributing to the regeneration of adipose tissue and suppression of fibrogenesis after injury. This study revealed a functional role for ASCs in the response to injury and provides new insight into the therapeutic potential of ASCs. STEM CELLS 2009;27:238–249

Differential expression of stem-cell-associated markers in human hair follicle epithelial cells

Several putative biomarkers have been suggested for identifying murine follicular stem cells; however, human hair follicles have a different pattern of biomarker expression, and follicular stem cell isolation methods have not been established. To isolate a stem cell population applicable to clinical settings, we conducted a comprehensive survey of the expression of stem-cell-associated (K15, CD200, CD34, and CD271) and other biomarkers (K1, K14, CD29, and CD49f) in immunohistological sections of the human epidermis and follicular outer root sheath (ORS). We also examined freshly isolated and cultured epidermal or follicular cells with single- and multicolor flow cytometry or immunocytochemistry. After sorting cells by CD200, CD34, and forward scatter (FSC) values (cell size), colony-forming assays were performed. We found that biomarkers were differentially expressed in the epidermis and ORS. Basal bulge cells were mainly K15+CD200+CD34−CD271−, and suprabasal cells were K15−CD200+CD34−CD271−. We categorized follicular cells into nine subpopulations according to biomarker expression profiles. The CD200+CD34− bulge cells had much higher colony-forming abilities than the CD34+ population, and were divided into two subpopulations: a CD200+CD34−FSChigh (K15-rich, basal) and a CD200+CD34–FSClow (K15-poor, suprabasal) population. The former formed fewer but larger-sized colonies than the latter. Follicular epithelial cell cultivation resulted in loss of K15, CD200, CD34, and CD271 expression, but maintenance of K14, CD29, and CD49f expression. We found that the bulge contained two populations with different localizations, cell sizes, and colony-forming abilities. We showed that K15, CD200, CD34, and CD271 were useful biomarkers for characterizing freshly isolated human follicular epithelial cells in diverse stages of differentiation.