J. Peter Rubin

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.

Stromal vascular progenitors in adult human adipose tissue

The in vivo progenitor of culture‐expanded mesenchymal‐like adipose‐derived stem cells (ADSC) remains elusive, owing in part to the complex organization of stromal cells surrounding the small vessels, and the rapidity with which adipose stromal vascular cells adopt a mesenchymal phenotype in vitro. Immunohistostaining of intact adipose tissue was used to identify three markers (CD31, CD34, and CD146), which together unambiguously discriminate histologically distinct inner and outer rings of vessel‐associated stromal cells, as well as capillary and small vessel endothelial cells. These markers were used in multiparameter flow cytometry in conjunction with stem/progenitor markers (CD90 and CD117) to further characterize stromal vascular fraction (SVF) subpopulations. Two mesenchymal and two endothelial populations were isolated by high speed flow cytometric sorting, expanded in short term culture, and tested for adipogenesis. The inner layer of stromal cells in contact with small vessel endothelium (pericytes) was CD146+/α‐SMA+/CD90±/CD34−/CD31−; the outer adventitial stromal ring (designated supra adventitial‐adipose stromal cells, SA‐ASC) was CD146−/α‐SMA−/CD90+/CD34+/CD31−. Capillary endothelial cells were CD31+/CD34+/CD90+ (endothelial progenitor), whereas small vessel endothelium was CD31+/CD34−/CD90− (endothelial mature). Flow cytometry confirmed these expression patterns and revealed a CD146+/CD90+/CD34+/CD31− pericyte subset that may be transitional between pericytes and SA‐ASC. Pericytes had the most potent adipogenic potential, followed by the more numerous SA‐ASC. Endothelial populations had significantly reduced adipogenic potential compared with unsorted expanded SVF cells. In adipose tissue, perivascular stromal cells are organized in two discrete layers, the innermost consisting of CD146+/CD34− pericytes, and the outermost of CD146−/CD34+ SA‐ASC, both of which have adipogenic potential in culture. A CD146+/CD34+ subset detected by flow cytometry at low frequency suggests a population transitional between pericytes and SA‐ASC.

Regional Anatomic and Age Effects on Cell Function of Human Adipose-Derived Stem Cells

Adipose tissue has been shown to contain adult mesenchymal stem cells that have therapeutic applications in regenerative medicine. There is evidence that the ability of adipose precursor cells to grow and differentiate varies among fat depots and changes with age. Defining these variations in cell function and molecular mechanisms of adipogenesis will facilitate the development of cell-based therapies. We compared cells harvested from 5 different subcutaneous (SC) adipose depots in 12 female patients classified into 3 age ranges (25–30, 40–45, and 55–60 years old). Capacity for differentiation of isolated adipose-derived stem cells (ASCs) with and without ciglitazone, a strong peroxisome proliferatoractivated receptors (PPAR)-γ agonist, was assessed in vitro. ASCs were also characterized by lipolytic function, proliferation, and sensitivity to apoptosis. Additionally, PPAR-γ-2 protein expression was determined. We observed a difference in the apoptotic susceptibility of ASCs from various SC depots, with the superficial abdominal depot (above Scarpas layer) significantly more resistant to apoptosis when compared with the 4 other depots. We have also demonstrated that a PPAR-γ agonist aids in the induction of differentiation in cells from all depots and ages. Although sensitivity to apoptosis was linked to anatomic depot, differences in cell proliferation were related primarily to age. Stimulated free glycerol release has been shown to be highest in the arm depot. The arm depot has also consistently shown expression of PPAR-γ-2 with and without a PPAR-γ agonist. Younger patients have increased PPAR-γ-2 expression in all depots, whereas the older patients have consistent elevated expression only in the arm and thigh depots. We have shown there is variability in function of ASCs that have been harvested from different SC depots. Additionally, we have shown age-related changes in function. These data will help select patients and cell harvest sites most suitable for tissue engineering therapies.

The Tunica Adventitia of Human Arteries and Veins As a Source of Mesenchymal Stem Cells

We previously demonstrated that human pericytes, which encircle capillaries and microvessels, give rise in culture to genuine mesenchymal stem cells (MSCs). This raised the question as to whether all MSC are derived from pericytes. Pericytes and other cells defined on differential expression of CD34, CD31, and CD146 were sorted from the stromal vascular fraction of human white adipose tissue. Besides pericytes, CD34+ CD31- CD146- CD45- cells, which reside in the outmost layer of blood vessels, the tunica adventitia, natively expressed MSC markers and gave rise in culture to clonogenic multipotent progenitors identical to standard bone marrow-derived MSC. Despite common MSC features and developmental properties, adventitial cells and pericytes retain distinct phenotypes and genotypes through culture. However, in the presence of growth factors involved in vascular remodeling, adventitial cells acquire a pericytes-like phenotype. In conclusion, we demonstrate the co-existence of 2 separate perivascular MSC progenitors: pericytes in capillaries and microvessels and adventitial cells around larger vessels.

An Acellular Biologic Scaffold Promotes Skeletal Muscle Formation in Mice and Humans with Volumetric Muscle Loss

Scaffolds composed of cell-free extracellular matrix promote de novo formation of functional skeletal muscle tissue in sites of volumetric muscle loss. Cell-Free Matrix Refills Muscle In traumatic accidents, or even in surgery, large amounts of skeletal muscle can be lost, resulting in pain and loss of function. Although muscle has the ability to regenerate naturally, it cannot refill massive defects, such as those seen in volumetric muscle loss (VML). In response, Sicari and colleagues devised a biomaterial scaffold that can be surgically implanted at the site of VML, encouraging local muscle regeneration and improving function in both mice and humans. The biomaterial used in this study was made up of bladder tissue that had been stripped of cells, leaving behind only the protein scaffold called the extracellular matrix (ECM). Sicari et al. first tested it in a mouse model of VML. In mice treated with ECM, they saw signs of new skeletal muscle formation, characterized by muscle markers desmin and myosin heavy chain, as well as striated (striped) tissue organization. The new muscle also appeared to be innervated, which is necessary for function. The authors translated this preclinical work into a clinical study of five patients with VML and saw outcomes similar to the mice. Six months after ECM implantation at the site of muscle loss, all patients showed signs of new muscle and blood vessels. Three of the five patients showed 20% or greater improvement in limb strength during physical therapy. The two patients without functional changes did report improvements in nonfunctional tasks, such as balance, as well as an improvement in quality of life. Because of the widespread availability and known safety of cell-free ECM-based materials, the approach described by Sicari et al. may translate to regeneration of other human tissues in addition to muscle. Biologic scaffolds composed of naturally occurring extracellular matrix (ECM) can provide a microenvironmental niche that alters the default healing response toward a constructive and functional outcome. The present study showed similarities in the remodeling characteristics of xenogeneic ECM scaffolds when used as a surgical treatment for volumetric muscle loss in both a preclinical rodent model and five male patients. Porcine urinary bladder ECM scaffold implantation was associated with perivascular stem cell mobilization and accumulation within the site of injury, and de novo formation of skeletal muscle cells. The ECM-mediated constructive remodeling was associated with stimulus-responsive skeletal muscle in rodents and functional improvement in three of the five human patients.

Validation of the Caprini Risk Assessment Model in Plastic and Reconstructive Surgery Patients

BACKGROUND The Venous Thromboembolism Prevention Study (VTEPS) Network is a consortium of 5 tertiary referral centers established to examine venous thromboembolism (VTE) in plastic surgery patients. We report our midterm analyses of the studys control group to evaluate the incidence of VTE in patients who receive no chemoprophylaxis, and validate the Caprini Risk Assessment Model (RAM) in plastic surgery patients. STUDY DESIGN Medical record review was performed at VTEPS centers for all eligible plastic surgery patients between March 2006 and June 2009. Inclusion criteria were Caprini score ≥3, surgery under general anesthesia, and postoperative hospital admission. Patients who received chemoprophylaxis were excluded. Dependent variables included symptomatic deep vein thrombosis (DVT) or pulmonary embolism (PE) within the first 60 postoperative days and time to DVT or PE. RESULTS We identified 1,126 historic control patients. The overall VTE incidence was 1.69%. Approximately 1 in 9 (11.3%) patients with Caprini score >8 had a VTE event. Patients with Caprini score >8 were significantly more likely to develop VTE when compared with patients with Caprini score of 3 to 4 (odds ratio [OR] 20.9, p < 0.001), 5 to 6 (OR 9.9, p < 0.001), or 7 to 8 (OR 4.6, p = 0.015). Among patients with Caprini score 7 to 8 or Caprini score >8, VTE risk was not limited to the immediate postoperative period (postoperative days 1-14). In these high-risk patients, more than 50% of VTE events were diagnosed in the late (days 15-60) postoperative period. CONCLUSIONS The Caprini RAM effectively risk-stratifies plastic and reconstructive surgery patients for VTE risk. Among patients with Caprini score >8, 11.3% have a postoperative VTE when chemoprophylaxis is not provided. In higher risk patients, there was no evidence that VTE risk is limited to the immediate postoperative period.

Body image and quality of life in post massive weight loss body contouring patients.

Objective: Because post‐bariatric surgery patients undergo massive weight loss, the resulting skin excess can lead to both functional problems and profound dissatisfaction with appearance. Correcting skin excess could improve all these corollaries, including body image. Presently, few data are available documenting body image and weight‐related quality of life in this population.

The osteogenic potential of adipose-derived stem cells for the repair of rabbit calvarial defects

Introduction: Bone replacement is often necessary during reconstruction of craniofacial anomalies or trauma. Adipose-derived stem cells (ASCs) possess osteogenic potential and are a promising cell source for bone tissue engineering. The present study was designed to assess the osteogenic potential and utility of using ASCs to regenerate bone in a rabbit calvarial defect model. Methods: Rabbit ASCs were seeded on gelatin foam (GF) scaffolds and induced in osteogenic medium containing bone morphogenetic protein (BMP)-2. Thirty-four 8-mm calvarial defects were randomly treated with autograft, no treatment, GF scaffold, GF + ASCs, or GF + osteoinduced ASCs. After 6 weeks, calvaria were harvested and underwent histologic and radiologic analyses to compare healing between the treatment groups. Results: Defects treated with autograft underwent complete healing. Radiologically, there were no significant (P > 0.05) differences in healing among empty defects, and those treated with GF alone or GF plus osteoinduced ASCs. Osteoinduced ASCs exhibited significantly (P < 0.05) greater healing than noninduced ASCs. Conclusion: Preimplantation osteoinduction of ASCs enhances their osteogenic capacity. Lack of a significant osteogenic effect of ASCs on calvarial healing at 6 weeks may be secondary to use of noncritical-sized defects. Larger defects would likely demonstrate the osteogenic potential of ASCs more definitively.

Mesenchymal markers on human adipose stem/progenitor cells

The stromal‐vascular fraction (SVF) of adipose tissue is a rich source of multipotent stem cells. We and others have described three major populations of stem/progenitor cells in this fraction, all closely associated with small blood vessels: endothelial progenitor cells (EPC, CD45−/CD31+/CD34+), pericytes (CD45−/CD31−/CD146+), and supra‐adventitial adipose stromal cells (SA‐ASC, CD45−/CD31−/CD146−/CD34+). EPC are luminal, pericytes are adventitial, and SA‐ASC surround the vessel like a sheath. The multipotency of the pericytes and SA‐ASC compartments is strikingly similar to that of CD45−/CD34−/CD73+/CD105+/CD90+ bone marrow‐derived mesenchymal stem cells (BM‐MSC). Here, we determine the extent to which this mesenchymal pattern is expressed on the three adipose stem/progenitor populations. Eight independent adipose tissue samples were analyzed in a single tube (CD105‐FITC/CD73‐PE/CD146‐PETXR/CD14‐PECY5/CD33‐PECY5/CD235A‐PECY5/CD31‐PECY7/CD90‐APC/CD34‐A700/CD45‐APCCY7/DAPI). Adipose EPC were highly proliferative with (14.3 ± 2.8)% (mean ± SEM) having >2N DNA. About half (53.1 ± 7.6)% coexpressed CD73 and CD105, and (71.9 ± 7.4)% expressed CD90. Pericytes were less proliferative [(8.2 ± 3.4)% >2N DNA)] with a smaller proportion [(29.6 ± 6.9)% CD73+/CD105+, (60.5 ± 10.2)% CD90+] expressing mesenchymal associated markers. However, the CD34+ subset of CD146+ pericytes were both highly proliferative [(15.1 ± 3.6)% with >2N DNA] and of uniform mesenchymal phenotype [(93.3 ± 3.7)% CD73+/CD105+, (97.8 ± 0.7)% CD90+], suggesting transit amplifying progenitor cells. SA‐ASC were the least proliferative [(3.7 ± 0.8)%>2N DNA] but were also highly mesenchymal in phenotype [(94.4 ± 3.2)% CD73+/CD105+, (95.5 ± 1.2)% CD90+]. These data imply a progenitor/progeny relationship between pericytes and SA‐ASC, the most mesenchymal of SVF cells. Despite phenotypic and functional similarities to BM‐MSC, SA‐ASC are distinguished by CD34 expression.

Role of Gender and Anatomical Region on Induction of Osteogenic Differentiation of Human Adipose-derived Stem Cells

Adipose-derived stem cells (ASCs) display multilineage plasticity and, under appropriate conditions, can mineralize their extracellular matrix and undergo osteogenesis. The aims of this study are to examine in vitro osteogenic differentiation properties of ASCs to assess the role of gender, fat depot, and optimal duration as variables for differentiation. Human ASCs were isolated from superficial and deep adipose layers of the abdominoplasty specimens obtained from patients undergoing elective surgeries. ASCs were cultured in osteogenic media (OM). After 1, 2, and 4 weeks of differentiation, cultures were assessed for markers of osteogenesis. Alkaline phosphatase (AP), alizarin red (AR) and Masson trichrome (MT) stainings for osteoblastic transformation, matrix mineralization, and collagen production; enzyme-linked immunosorbent assay (ELISA) for Gla-osteocalcin; and Western blot analysis for osteonectin protein expression were performed. Osteogenic differentiation began as early as 1 week. Cells exhibited a vertical growth pattern, lacunae formed in the cultures, matrix volume increased, and mineralization was observed. Differences in AP staining were most evident during the first week. AR activity progressively increased over 4 weeks, and collagen was secreted only by differentiated ASCs. There was no significant difference in the degree of osteogenic differentiation between the ASCs from both depots in the female. In the male, the superficial depot ASCs differentiated faster and more efficiently than those of the deep depot. Male ASCs from both depots differentiated more effectively than female ASCs from both depots. We describe a hierarchy of osteogenic differentiation potential based on gender and anatomic harvest site by layering adipose tissues of the abdominal wall. ASCs derived from male superficial layer were most efficient in achieving osteogenesis. In future clinical applications using stem cells for osseous healing, these gender and depot differences will guide our clinical methods.