Comparative epigenomic analysis of murine and human adipogenesis.

We report the generation and comparative analysis of genome-wide chromatin state maps, PPARγ and CTCF localization maps, and gene expression profiles from murine and human models of adipogenesis. The data provide high-resolution views of chromatin remodeling during cellular differentiation and allow identification of thousands of putative preadipocyte- and adipocyte-specific cis-regulatory elements based on dynamic chromatin signatures. We find that the specific locations of most such elements differ between the two models, including at orthologous loci with similar expression patterns. Based on sequence analysis and reporter assays, we show that these differences are determined, in part, by evolutionary turnover of transcription factor motifs in the genome sequences and that this turnover may be facilitated by the presence of multiple distal regulatory elements at adipogenesis-dependent loci. We also utilize the close relationship between open chromatin marks and transcription factor motifs to identify and validate PLZF and SRF as regulators of adipogenesis.

Circulating Osteoprogenitor Cells Have a Mixed Immune and Mesenchymal Progenitor Function in Humans.

BACKGROUND: Circulating osteoprogenitors (COP) are a population of cells in the peripheral circulation that possess functional and phenotypical characteristics of multipotent stromal cells (MSCs). This population has a solid potential to become an abundant, accessible, and replenishable source of MSCs with multiple potential clinical applications. However, a comprehensive functional characterization of COP cells is still required to test and fully develop their use in clinical settings. METHODS: This study characterized COP cells by comparing them to bone marrow-derived MSCs (BM-MSCs) and adipose-derived MSCs (ASCs) through detailed transcriptomic and proteomic analyses. RESULTS: We demonstrate that COP cells have a distinct gene and protein expression pattern with a significantly stronger immune footprint, likely owing to their hematopoietic lineage. In addition, regarding progenitor cell differentiation and proliferation pathways, COP cells have a similar expression pattern to BM-MSCs and ASCs. CONCLUSION: COP cells are a unique but functionally similar population to BM-MSCs and ASCs, sharing their proliferation and differentiation capacity, thus presenting an accessible source of MSCs with strong potential for translational regenerative medicine strategies.

Adipose Tissue in Breast Cancer Microphysiological Models to Capture Human Diversity in Preclinical Models.

Female breast cancer accounts for 15.2% of all new cancer cases in the United States, with a continuing increase in incidence despite efforts to discover new targeted therapies. With an approximate failure rate of 85% for therapies in the early phases of clinical trials, there is a need for more translatable, new preclinical in vitro models that include cellular heterogeneity, extracellular matrix, and human-derived biomaterials. Specifically, adipose tissue and its resident cell populations have been identified as necessary attributes for current preclinical models. Adipose-derived stromal/stem cells (ASCs) and mature adipocytes are a normal part of the breast tissue composition and not only contribute to normal breast physiology but also play a significant role in breast cancer pathophysiology. Given the recognized pro-tumorigenic role of adipocytes in tumor progression, there remains a need to enhance the complexity of current models and account for the contribution of the components that exist within the adipose stromal environment to breast tumorigenesis. This review article captures the current landscape of preclinical breast cancer models with a focus on breast cancer microphysiological system (MPS) models and their counterpart patient-derived xenograft (PDX) models to capture patient diversity as they relate to adipose tissue.

CD146 expression regulates osteochondrogenic differentiation of human adipose-derived stem cells.

Tissue engineering aims to develop innovative approaches to repair tissue defects. The use of adipose-derived stem cells (ASCs) in tissue regeneration was extensively investigated for osteochondrogenesis. Among the ASC population, ASCs expressing the CD146 were demonstrated to be multipotent and considered as perivascular stem cells, although the functional role of CD146 expression in these cells remains unclear. Herein, we investigated the influence of CD146 expression on osteochondrogenic differentiation of ASCs. Our results showed that, in two-dimensional culture systems, sorted CD146+ ASCs proliferated less and displayed higher adipogenic and chondrogenic potential than CD146- ASCs. The latter demonstrated a higher osteogenic capacity. Besides this, CD146+ ASCs in three-dimensional Matrigel/endothelial growth medium (EGM) cultures showed the highest angiogenic capability. When cultured in three-dimensional collagen scaffolds, CD146+ ASCs showed a spontaneous chondrogenic differentiation, further enhanced by the EGM medium's addition. Finally, CD146- ASCs seeded on hexafluoroisopropanol silk scaffolds displayed a greater spontaneous osteogenetic capacity. Altogether, these findings demonstrated a functional and relevant influence of CD146 expression in ASC properties and osteochondrogenic commitment. Exploiting the combination of specific differentiation properties of ASC subpopulations and appropriate culture systems could represent a promising strategy to improve the efficacy of new regenerative therapies.

Adenosine triphosphate enhances osteoblast differentiation of rat dental pulp stem cells via the PLC-IP3 pathway and intracellular Ca 2+ signaling.

Intracellular Ca2+ signals are essential for stem cell function and play a significant role in the differentiation process. Dental pulp stem cells (DPSCs) are a potential source of stem cells; however, the mechanisms controlling cell differentiation remain largely unknown. Utilizing rat DPSCs, we examined the effect of adenosine triphosphate (ATP) on osteoblast differentiation and characterized its mechanism of action using real-time Ca 2+ imaging analysis. Our results revealed that ATP enhanced osteogenesis as indicated by Ca 2+ deposition in the extracellular matrix via Alizarin Red S staining. This was consistent with upregulation of osteoblast genes BMP2, Mmp13, Col3a1, Ctsk, Flt1, and Bgn. Stimulation of DPSCs with ATP (1-300 µM) increased intracellular Ca 2+ signals in a concentration-dependent manner, whereas histamine, acetylcholine, arginine vasopressin, carbachol, and stromal-cell-derived factor-1α failed to do so. Depletion of intracellular Ca 2+ stores in the endoplasmic reticulum by thapsigargin abolished the ATP responses which, nevertheless, remained detectable under extracellular Ca 2+ free condition. Furthermore, the phospholipase C (PLC) inhibitor U73122 and the inositol triphosphate (IP 3 ) receptor inhibitor 2-aminoethoxydiphenyl borate inhibited the Ca 2+ signals. Our findings provide a better understanding of how ATP controls osteogenesis in DPSCs, which involves a Ca 2+ -dependent mechanism via the PLC-IP 3 pathway. This knowledge could help improve osteogenic differentiation protocols for tissue regeneration of bone structures.

Non-toxic freezing media to retain the stem cell reserves in adipose tissues.

Subcutaneous adipose tissue is a rich source of stromal vascular fraction (SVF) and adipose-derived stromal/stem cells (ASCs) that are inherently multipotent and exhibit regenerative properties. In current practice, lipoaspirate specimens harvested from liposuction surgeries are routinely discarded as a biohazard waste due to a lack of simple, cost effective, and validated cryopreservation protocols. The aim of this study is to develop a xenoprotein-free cryoprotective agent cocktail that will allow for short-term (up to 6 months) preservation of lipoaspirate tissues suitable for fat grafting and/or stromal/stem cell isolation when stored at achievable temperatures (-20 °C or -80 °C). Lipoaspirates donated by three consenting healthy donors undergoing elective cosmetic liposuction surgeries were suspended in five freezing media (FM1: 10% DMSO and 35% BSA; FM2: 2% DMSO and 43% BSA; FM3: 10% DMSO and 35% lipoaspirate saline; FM4: 2% DMSO and 6% HSA; and FM5: 40% lipoaspirate saline and 10% PVP) all suspended in 1X DMEM/F12 and frozen using commercially available freezers (-20 °C or -80 °C) and stored at least for a 1 month. After 1 month of freezing storage, SVF cells and ASCs were isolated from the frozen-thawed lipoaspirates by digestion with collagenase type I. Cell viability was evaluated by fluorescence microscopy after staining with acridine orange and ethidium bromide. The SVF isolated from lipoaspirates frozen at -80 °C retained comparable cell viability with the tested freezing media (FM2, FM3, FM4) comparable with the conventional DMSO and animal serum media (FM1), whereas the FM5 media resulted in lower viability. In contrast, tissues frozen and stored at -20 °C did not yield live SVF cells after thawing and collagenase digestion. The surface marker expression (CD90, CD29, CD34, CD146, CD31, and CD45) of ASCs from frozen lipoaspirates at -80 °C in different cryoprotectant media were also evaluated and no significant differences were found between the groups. The adipogenic and osteogenic differentiation potential were studied by histochemical staining and gene expression by qRT-PCR. Oil Red O staining for adipogenesis revealed that the CPA media FM1, FM4 and FM5 displayed robust differentiation. Alizarin Red S staining for osteogenesis revealed that FM1 and FM4 media displayed superior differentiation in comparison to other tested media. Measurement of adipogenic and osteogenic gene expression by qRT-PCR provided similar outcomes and indicated that FM4 CPA media comparable with FM1 for adipogenesis and osteogenesis.

Decellularized Adipose Tissue: Biochemical Composition, in vivo Analysis and Potential Clinical Applications.

Decellularized tissues are gaining popularity as scaffolds for tissue engineering; they allow cell attachment, proliferation, differentiation, and are non-immunogenic. Adipose tissue is an abundant resource that can be decellularized and converted in to a bio-scaffold. Several methods have been developed for adipose tissue decellularization, typically starting with freeze thaw cycles, followed by washes with hypotonic/hypertonic sodium chloride solution, isopropanol, detergent (SDS, SDC and Triton X-100) and trypsin digestion. After decellularization, decellularized adipose tissue (DAT) can be converted into a powder, solution, foam, or sheet to allow for convenient subcutaneous implantation or to repair external injuries. Additionally, DAT bio-ink can be used to 3D print structures that closely resemble physiological tissues and organs. Proteomic analysis of DAT reveals that it is composed of collagens (I, III, IV, VI and VII), glycosaminoglycans, laminin, elastin, and fibronectin. It has also been found to retain growth factors like VEGF and bFGF after decellularization. DAT inherently promotes adipogenesis when seeded with adipose stem cells in vitro, and when DAT is implanted subcutaneously it is capable of recruiting host stem cells and forming adipose tissue in rodents. Furthermore, DAT has promoted healing in rat models of full-thickness skin wounds and peripheral nerve injury. These findings suggest that DAT is a promising candidate for repair of soft tissue defects, and is suitable for breast reconstruction post-mastectomy, wound healing, and adipose tissue regeneration. Moreover, since DAT's form and stiffness can be altered by physicochemical manipulation, it may prove suitable for engineering of additional soft and hard tissues.

Age, Diet and Epidermal Signaling Modulate Dermal Fibroblasts' Adipogenic Potential.

The recognition of a distinct fat depot, the dermal white adipose tissue (dWAT), points out the complexity of the interaction among skin resident cells: keratinocytes, dermal fibroblasts (DFs) and adipocytes in response to physiological (diet, age) and pathological (injury) stimulations. dWAT has been recognized as a significant contributor to thermoregulation, hair cycle, immune response, wound healing and scarring. In this study, we examined age- and diet-related changes in dWAT modulation and DFs' adipogenic potential. The data showed that diet modulates dWAT expansion predominantly by hypertrophy, whereas age affects the pool of adipocyte progenitor cells in the skin indicating its role in dWAT hyperplasia. Analysis of DFs' migratory abilities in the model of skin explants isolated from the skin of young, old, low (LFD)- or high (HFD)-fat diet C56BL/6 mice revealed that HFD, regardless of animal age has the most profound stimulatory impact of DF migration. We determined that the adipogenic potential of DFs is comparable to stromal vascular fraction (SVF) of inguinal fat depot and ear mesenchymal stem cells (EMSC). We also showed the stimulatory role of epidermally expressed transcription factor Foxn1 on adipogenic signaling: bone morphogenetic protein 2 (Bmp2) and insulin-like growth factor 2 (Igf2) in keratinocytes.

Adipose tissue mitochondrial dysfunction in human obesity is linked to a specific DNA methylation signature in adipose-derived stem cells.

BACKGROUND: A functional population of adipocyte precursors, termed adipose-derived stromal/stem cells (ASCs), is crucial for proper adipose tissue (AT) expansion, lipid handling, and prevention of lipotoxicity in response to chronic positive energy balance. We previously showed that obese human subjects contain a dysfunctional pool of ASCs. Elucidation of the mechanisms underlying abnormal ASC function might lead to therapeutic interventions for prevention of lipotoxicity by improving the adipogenic capacity of ASCs. METHODS: Using epigenome-wide association studies, we explored the impact of obesity on the methylation signature of human ASCs and their differentiated counterparts. Mitochondrial phenotyping of lean and obese ASCs was performed. TBX15 loss- and gain-of-function experiments were carried out and western blotting and electron microscopy studies of mitochondria were performed in white AT biopsies from lean and obese individuals. RESULTS: We found that DNA methylation in adipocyte precursors is significantly modified by the obese environment, and adipogenesis, inflammation, and immunosuppression were the most affected pathways. Also, we identified TBX15 as one of the most differentially hypomethylated genes in obese ASCs, and genetic experiments revealed that TBX15 is a regulator of mitochondrial mass in obese adipocytes. Accordingly, morphological analysis of AT from obese subjects showed an alteration of the mitochondrial network, with changes in mitochondrial shape and number. CONCLUSIONS: We identified a DNA methylation signature in adipocyte precursors associated with obesity, which has a significant impact on the metabolic phenotype of mature adipocytes.

Concise Review: Using Fat to Fight Disease: A Systematic Review of Nonhomologous Adipose-Derived Stromal/Stem Cell Therapies.

The objective of this Review is to describe the safety and efficacy of adipose stem/stromal cells (ASC) and stromal vascular fraction (SVF) in treating common diseases and the next steps in research that must occur prior to clinical use. Pubmed, Ovid Medline, Embase, Web of Science, and the Cochrane Library were searched for articles about use of SVF or ASC for disease therapy published between 2012 and 2017. One meta-analysis, 2 randomized controlled trials, and 16 case series were included, representing 844 human patients. Sixty-nine studies were performed in preclinical models of disease. ASCs improved symptoms, fistula healing, remission, and recurrence rates in severe cases of inflammatory bowel disease. In osteoarthritis, ASC and SVF improved symptom-related, functional, radiographic, and histological scores. ASC and SVF were also shown to improve clinical outcomes in ischemic stroke, multiple sclerosis, myocardial ischemia, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, chronic liver failure, glioblastoma, acute kidney injury, and chronic skin wounds. These effects were primarily paracrine in nature and mediated through reduction of inflammation and promotion of tissue repair. In the majority of human studies, autologous ASC and SVF from liposuction procedures were used, minimizing the risk to recipients. Very few serious, treatment-related adverse events were reported. The main adverse event was postprocedural pain. SVF and ASC are promising therapies for a variety of human diseases, particularly for patients with severe cases refractory to current medical treatments. Further randomized controlled trials must be performed to elaborate potential safety and efficacy prior to clinical use. Stem Cells 2018;36:1311-1328.

Co-Transplantation of Adipose Tissue-Derived Stromal Cells and Olfactory Ensheathing Cells for Spinal Cord Injury Repair.

Patients suffering from spinal cord injury (SCI) still have a dismal prognosis. Despite all the efforts developed in this area, currently there are no effective treatments. Therefore, cell therapies have been proposed as a viable alternative to the current treatments used. Adipose tissue-derived stromal cells (ASCs) and olfactory ensheathing cells (OECs) have been used with promising results in different models of SCI, namely due to the regenerative properties of the secretome of the first, and the guidance capability of the second. Using an in vitro model of axonal growth, the dorsal root ganglia explants, we demonstrated that OECs induce neurite outgrowth mainly through cell-cell interactions, while ASCs' effects are strongly mediated by the release of paracrine factors. A proteomic analysis of ASCs' secretome revealed the presence of proteins involved in VEGF, PI3K, and Cadherin signaling pathways, which may be responsible for the effects observed. Then, the cotransplantation of ASCs and OECs showed to improve motor deficits of SCI-rats. Particular parameters of movement such as stepping, coordination, and toe clearance were improved in rats that received the transplant of cells, in comparison to nontreated rats. A histological analysis of the spinal cord tissues revealed that transplantation of ASCs and OECs had a major effect on the reduction of inflammatory cells close the lesion site. A slight reduction of astrogliosis was also evident. Overall, the results obtained with the present work indicate that the cotransplantation of ASCs and OECs brings important functional benefits to the injured spinal cord. Stem Cells 2018;36:696-708.

Adipose Stromal Vascular Fraction-Mediated Improvements at Late-Stage Disease in a Murine Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a common neurodegenerative disease and remains an unmet clinical challenge. In MS, an autoimmune response leads to immune cell infiltration, inflammation, demyelination, and lesions in central nervous system (CNS) tissues resulting in tremors, fatigue, and progressive loss of motor function. These pathologic hallmarks are effectively reproduced in the murine experimental autoimmune encephalomyelitis (EAE) model. The stromal vascular fraction (SVF) of adipose tissue is composed of adipose-derived stromal/stem cells (ASC), adipocytes, and various leukocytes. The SVF can be culture expanded to generate ASC lines. Clinical trials continue to demonstrate the safety and efficacy of ASC therapies for treating several diseases. However, little is known about the effectiveness of the SVF for neurodegenerative diseases, such as MS. At late-stage disease, EAE mice show severe motor impairment. The goal for these studies was to test the effectiveness of SVF cells and ASC in EAE mice after the onset of neuropathology. The clinical scoring, behavior, motor function, and histopathologic analyses revealed significant improvements in EAE mice treated with the SVF or ASC. Moreover, SVF treatment mediated more robust improvements to CNS pathology than ASC treatment based on significant modulations of inflammatory factors. The most pronounced changes following SVF treatment were the high levels of interleukin-10 in the peripheral blood, lymphoid and CNS tissues along with the induction of regulatory T cells in the lymph nodes which indicate potent immunomodulatory effects. The data indicate SVF cells effectively ameliorated the EAE immunopathogenesis and supports the potential use of SVF for treating MS. Stem Cells 2017;35:532-544.

Bone Marrow Adipocyte Developmental Origin and Biology.

PURPOSE OF REVIEW: This review explores how the relationships between bone marrow adipose tissue (BMAT) adipogenesis with advancing age, obesity, and/or bone diseases (osteopenia or osteoporosis) contribute to mechanisms underlying musculoskeletal pathophysiology. RECENT FINDINGS: Recent studies have re-defined adipose tissue as a dynamic, vital organ with functions extending beyond its historic identity restricted solely to that of an energy reservoir or sink. "State of the art" methodologies provide novel insights into the developmental origin, physiology, and function of different adipose tissue depots. These include genetic tracking of adipose progenitors, viral vectors application, and sophisticated non-invasive imaging modalities. While constricted within the rigid bone cavity, BMAT vigorously contributes to local and systemic metabolic processes including hematopoiesis, osteogenesis, and energy metabolism and undergoes dynamic changes as a function of age, diet, bone topography, or sex. These insights will impact future research and therapies relating to osteoporosis.

Serially Transplanted Nonpericytic CD146(-) Adipose Stromal/Stem Cells in Silk Bioscaffolds Regenerate Adipose Tissue In Vivo.

Progenitors derived from the stromal vascular fraction (SVF) of white adipose tissue (WAT) possess the ability to form clonal populations and differentiate along multiple lineage pathways. However, the literature continues to vacillate between defining adipocyte progenitors as "stromal" or "stem" cells. Recent studies have demonstrated that a nonpericytic subpopulation of adipose stromal cells, which possess the phenotype, CD45(-) /CD31(-) /CD146(-) /CD34(+) , are mesenchymal, and suggest this may be an endogenous progenitor subpopulation within adipose tissue. We hypothesized that an adipose progenitor could be sorted based on the expression of CD146, CD34, and/or CD29 and when implanted in vivo these cells can persist, proliferate, and regenerate a functional fat pad over serial transplants. SVF cells and culture expanded adipose stromal/stem cells (ASC) ubiquitously expressing the green fluorescent protein transgene (GFP-Tg) were fractionated by flow cytometry. Both freshly isolated SVF and culture expanded ASC were seeded in three-dimensional silk scaffolds, implanted subcutaneously in wild-type hosts, and serially transplanted. Six-week WAT constructs were removed and evaluated for the presence of GFP-Tg adipocytes and stem cells. Flow cytometry, quantitative polymerase chain reaction, and confocal microscopy demonstrated GFP-Tg cell persistence, proliferation, and expansion, respectively. Glycerol secretion and glucose uptake assays revealed GFP-Tg adipose was metabolically functional. Constructs seeded with GFP-Tg SVF cells or GFP-Tg ASC exhibited higher SVF yields from digested tissue, and higher construct weights, compared to nonseeded controls. Constructs derived from CD146(-) CD34(+) -enriched GFP-Tg ASC populations exhibited higher hemoglobin saturation, and higher frequency of GFP-Tg cells than unsorted or CD29(+) GFP-Tg ASC counterparts. These data demonstrated successful serial transplantation of nonpericytic adipose-derived progenitors that can reconstitute adipose tissue as a solid organ. These findings have the potential to provide new insights regarding the stem cell identity of adipose progenitor cells.

Human Adipose Stromal/Stem Cells from Obese Donors Show Reduced Efficacy in Halting Disease Progression in the Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis.

Multiple sclerosis is an autoimmune disease that affects the white matter of the central nervous system and involves inflammation and demyelination. The recent advances in our understanding of adipose-derived stromal/stem cells (ASCs) and the utilization of these cells in clinical settings to treat diseases have made it essential to identify the most effective ASCs for therapy. Studies have not yet investigated the impact of obesity on the therapeutic efficacy of ASCs. Obesity is characterized by adipocyte hyperplasia and hypertrophy and can extend to metabolic and endocrine dysfunction. Investigating the impact obesity has on ASC biology will determine whether these cells are suitable for use in regenerative medicine. The therapeutic efficacy of ASCs isolated from lean subjects (body mass index [BMI] < 25; lnASCs) and obese subjects (BMI > 30; obASCs) were determined in murine experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Compared with the EAE disease-modifying effects of lnASCs, obASCs consistently failed to alleviate clinical symptoms or inhibit inflammation in the central nervous system. When activated, obASCs expressed higher mRNA levels of several pro-inflammatory cytokines compared with lnASCs. Additionally, conditioned media (CM) collected from the obASCs markedly enhanced the proliferation and differentiation of T cells; whereas, CM from lnASC did not. These results indicate that obesity reduces, or eliminates, the anti-inflammatory effects of human ASCs such that they may not be a suitable cell source for the treatment of autoimmune diseases. The data suggest that donor demographics may be particularly important when identifying suitable stem cells for treatment.

Characterization of an Acellular Scaffold for a Tissue Engineering Approach to the Nipple-Areolar Complex Reconstruction.

A significant number of patients undergo mastectomies and breast reconstructions every year using many surgical-based techniques to reconstruct the nipple-areolar complex (NAC). Described herein is a tissue engineering approach that may permit a human NAC onlay graft during breast reconstruction procedures. By applying decellularization, which is the removal of cellular components from tissue, to an intact whole donor NAC, the extracellular matrix (ECM) structure of the NAC is preserved. This creates a biologically derived scaffold for cells to repopulate and regenerate the NAC. A detergent-based decellularization method was used to derive whole NAC scaffolds from nonhuman primate rhesus macaque NAC tissue. Using both histological and quantitative analyses for the native and decellularized tissues, the derived ECM graft was assessed. The bioactivity of the scaffold was evaluated following cell culture with bone marrow-derived mesenchymal stem cells (BMSCs). The data presented here demonstrate that scaffolds are devoid of cells and retain ECM integrity and a high degree of bioactivity. The content of collagen and glycosaminoglycans were not significantly altered by the decellularization process, whereas the elastin content was significantly decreased. The proliferation and apoptosis of seeded BMSCs were found to be approximately 65 and <1.5%, respectively. This study characterizes the successful decellularization of NAC tissue as compared to native NACs based on structural protein composition, lubricating protein retention, the maintenance of adhesion molecules, and bioactivity when reseeded with cells. These histological and quantitative analyses provide the foundation for a novel approach to NAC reconstruction.

Foxn1 and Mmp-9 expression in intact skin and during excisional wound repair in young, adult, and old C57Bl/6 mice.

The transcription factor Foxn1 is essential for skin development. Our previous studies performed on young C57BL/6J mice model showed that Foxn1 acts as regulator of the skin wound healing process. The present study extended our initial research regarding the expression and potential role of Foxn1 in the intact and wounded skin as a function of animal age and stage of the wound healing process. We analyzed Foxn1 and Mmp-9 expression in the intact and postinjured skin of young, adult, and old C57BL/6J and transgenic Foxn1::Egfp mice. The similar levels of epidermal Foxn1 mRNA expression were detected in young and adult C57BL/6J mice and higher levels in old animals. Postinjured skin tissues displayed a gradual decrease of Foxn1 mRNA expression at Days 1, 5, and 7 after injury. Foxn1-eGFP positive cells were abundant at wound margin and in re-epithelialized epidermis at postwounded Days 1, 5, and 7 and colocalized with E-cadherin and Mmp-9. Postwounded skin at Days 14-36 displayed Foxn1-eGFP cells in the epidermis and in the dermal part of the skin (papillary dermis). A subset of Foxn1-eGFP positive cells in the papillary dermis expressed the myofibroblast marker αSMA. Flow cytometric analysis of cells isolated from postwounded (Day 7) skin tissues showed a significant increase in the percentage of Foxn1-eGFP positive cells with phenotype of double positivity for E-cadherin/N-cadherin (epithelial/mesenchymal markers). Collectively, these data identify the transcription factor Foxn1 as a potential key epidermal regulator modifying both epidermal and dermal healing processes after cutaneous wounding.

Obesity inhibits the osteogenic differentiation of human adipose-derived stem cells.

BACKGROUND: Craniomaxillofacial defects secondary to trauma, tumor resection, or congenital malformations are frequent unmet challenges, due to suboptimal alloplastic options and limited autologous tissues such as bone. Significant advances have been made in the application of adipose-derived stem/stromal cells (ASCs) in the pre-clinical and clinical settings as a cell source for tissue engineering approaches. To fully realize the translational potential of ASCs, the identification of optimal donors for ASCs will ensure the successful implementation of these cells for tissue engineering approaches. In the current study, the impact of obesity on the osteogenic differentiation of ASCs was investigated. METHODS: ASCs isolated from lean donors (body mass index <25; lnASCs) and obese donors (body mass index >30; obASCs) were induced with osteogenic differentiation medium as monolayers in an estrogen-depleted culture system and on three-dimensional scaffolds. Critical size calvarial defects were generated in male nude mice and treated with scaffolds implanted with lnASCs or obASCs. RESULTS: lnASCs demonstrated enhanced osteogenic differentiation in monolayer culture system, on three-dimensional scaffolds, and for the treatment of calvarial defects, whereas obASCs were unable to induce similar levels of osteogenic differentiation in vitro and in vivo. Gene expression analysis of lnASCs and obASCs during osteogenic differentiation demonstrated higher levels of osteogenic genes in lnASCs compared to obASCs. CONCLUSION: Collectively, these results indicate that obesity reduces the osteogenic differentiation capacity of ASCs such that they may have a limited suitability as a cell source for tissue engineering.

Concise review: The obesity cancer paradigm: exploration of the interactions and crosstalk with adipose stem cells.

With the recognition of obesity as a global health crisis, researchers have devoted greater effort to defining and understanding the pathophysiological molecular pathways regulating the biology of adipose tissue and obesity. Obesity, the excessive accumulation of adipose tissue due to hyperplasia and hypertrophy, has been linked to an increased incidence and aggressiveness of colon, hematological, prostate, and postmenopausal breast cancers. The increased morbidity and mortality of obesity-associated cancers have been attributed to higher levels of hormones, adipokines, and cytokines secreted by the adipose tissue. The increased amount of adipose tissue also results in higher numbers of adipose stromal/stem cells (ASCs). These ASCs have been shown to impact cancer progression directly through several mechanisms, including the increased recruitment of ASCs to the tumor site and increased production of cytokines and growth factors by ASCs and other cells within the tumor stroma. Emerging evidence indicates that obesity induces alterations in the biologic properties of ASCs, subsequently leading to enhanced tumorigenesis and metastasis of cancer cells. This review will discuss the links between obesity and cancer tumor progression, including obesity-associated changes in adipose tissue, inflammation, adipokines, and chemokines. Novel topics will include a discussion of the contribution of ASCs to this complex system with an emphasis on their role in the tumor stroma. The reciprocal and circular feedback loop between obesity and ASCs as well as the mechanisms by which ASCs from obese patients alter the biology of cancer cells and enhance tumorigenesis will be discussed.

Platelet-Derived Growth Factor BB Enhances Osteogenesis of Adipose-Derived But Not Bone Marrow-Derived Mesenchymal Stromal/Stem Cells.

Tissue engineering using mesenchymal stem cells (MSCs) holds great promise for regenerating critically sized bone defects. While the bone marrow-derived MSC is the most widely studied stromal/stem cell type for this application, its rarity within bone marrow and painful isolation procedure have motivated investigation of alternative cell sources. Adipose-derived stromal/stem cells (ASCs) are more abundant and more easily procured; furthermore, they also possess robust osteogenic potency. While these two cell types are widely considered very similar, there is a growing appreciation of possible innate differences in their biology and response to growth factors. In particular, reports indicate that their osteogenic response to platelet-derived growth factor BB (PDGF-BB) is markedly different: MSCs responded negatively or not at all to PDGF-BB while ASCs exhibited enhanced mineralization in response to physiological concentrations of PDGF-BB. In this study, we directly tested whether a fundamental difference existed between the osteogenic responses of MSCs and ASCs to PDGF-BB. MSCs and ASCs cultured under identical osteogenic conditions responded disparately to 20 ng/ml of PDGF-BB: MSCs exhibited no difference in mineralization while ASCs produced more calcium per cell. siRNA-mediated knockdown of PDGFRß within ASCs abolished their ability to respond to PDGF-BB. Gene expression was also different; MSCs generally downregulated and ASCs generally upregulated osteogenic genes in response to PDGF-BB. ASCs transduced to produce PDGF-BB resulted in more regenerated bone within a critically sized murine calvarial defect compared to control ASCs, indicating PDGF-BB used specifically in conjunction with ASCs might enhance tissue engineering approaches for bone regeneration.