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1.  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 (IFATS) and Science and the International Society for Cellular Therapy (ISCT) 
Cytotherapy  2013;15(6):641-648.
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.
doi:10.1016/j.jcyt.2013.02.006
PMCID: PMC3979435  PMID: 23570660
adipose-derived stromal/stem cells; adipose tissue; characterization; function; phenotype; stromal vascular fraction
2.  Correction: Pericytes Derived from Adipose-Derived Stem Cells Protect against Retinal Vasculopathy 
PLoS ONE  2013;8(9):10.1371/annotation/679017bf-abd5-44ce-9e20-5e7af1cd3468.
doi:10.1371/annotation/679017bf-abd5-44ce-9e20-5e7af1cd3468
PMCID: PMC3776879  PMID: 24069115
3.  Long-term In-Vivo Tumorigenic Assessment of Human Culture-expanded Adipose Stromal/Stem Cells 
Experimental Cell Research  2011;318(4):416-423.
After more than a decade of extensive experimentation, the promise of stem cells to revolutionize the field of medicine has negotiated their entry into clinical trial. Adipose tissue specifically holds potential as an attainable and abundant source of stem cells. Currently undergoing investigation are adipose stem cell (ASC) therapies for diabetes and critical limb ischemia, among others. In the enthusiastic pursuit of regenerative therapies, however, questions remain regarding ASC persistence and migration, and, importantly, their safety and potential for neoplasia. To date, assays of in vivo ASC activity have been limited by early end points. We hypothesized that with time, ASCs injected subcutaneously undergo removal by normal tissue turnover and homeostasis, and by the host’s immune system. In this study, a high dose of culture expanded ASCs were formulated and implanted as multicellular aggregates into immunocompromised mice, which were maintained for over one year. Animals were monitored for toxicity, and surviving cells quantified at study endpoint. No difference in growth/weight or lifespan was found between cell-treated and vehicle treated animals, and no malignancies were detected in treated animals. Moreover, real-time PCR for a human specific sequence, ERV-3, detected no persistent ASCs. With the advent of clinical application, clarification of currently enigmatic stem cell properties has become imperative. Our study represents the longest duration determination of stem cell activity in vivo, and contributes strong evidence in support of the safety of adipose derived stem cell applications.
doi:10.1016/j.yexcr.2011.12.002
PMCID: PMC3264753  PMID: 22185824
Adipose derived stem cell; Mesenchymal Stem Cell Transplantation/toxicity
4.  Collagen nanofibres are a biomimetic substrate for the serum-free osteogenic differentiation of human adipose stem cells 
Electrospinning has recently gained widespread attention as a process capable of producing nanoscale fibres that mimic native extracellular matrix. In this study, we compared the osteogenic differentiation behaviour of human adipose stem cells (ASCs) on a 3D nanofibre matrix of type I rat tail collagen (RTC) and a 2D RTC collagen-coated substrate, using a novel serum-free osteogenic medium. The serum-free medium significantly enhanced the numbers of proliferating cells in culture, compared to ASCs in traditional basal medium containing 10% animal serum, highlighting a potential clinical role for in vitro stem cell expansion. Osteogenic differentiation behaviour was assessed at days 7, 14 and 21 using quantitative real-time RT–PCR analysis of the osteogenic genes collagen I (Coll I), alkaline phosphatase (ALP), osteopontin (OP), osteonectin (ON), osteocalcin (OC) and core-binding factor-α (cbfa1). All genes were upregulated (>one-fold) in ASCs cultured on nanofibre scaffolds over 2D collagen coatings by day 21. Synthesis of mineralized extracellular matrix on the scaffolds was assessed on day 21 with Alizarin red staining. These studies demonstrate that 3D nanoscale morphology plays a critical role in regulating cell fate processes and in vitro osteogenic differentiation of ASCs under serum-free conditions.
doi:10.1002/term.85
PMCID: PMC3122962  PMID: 18493910
adipose stem cells; serum-free medium; nanofibres; electrospinning; bone tissue engineering
5.  Functional Binding of Human Adipose-Derived Stromal Cells: Effects of Extraction Method & Hypoxia Pretreatment 
Annals of plastic surgery  2008;60(4):437-444.
Human adipose-derived stromal cells (hASCs) were evaluated in vitro for their ability to bind vascular adhesion and extracellular matrix proteins in order to arrest (firmly adhere) under physiological flow conditions. hASCs were flowed through a parallel plate flow chamber containing substrates presenting immobilized Type I Collagen, fibronectin, E-selectin, L-selectin, P-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1) under static and laminar flow conditions (wall shear stress = 1 dyn/cm2). hASCs were able to firmly adhere to Type I Collagen, fibronectin, VCAM-1, and ICAM-1 substrates, but not to any of the selectins. Pretreatment with hypoxia increased the ability of hASCs isolated by liposuction to adhere to VCAM-1 and ICAM-1, but this effect was not seen in cells isolated by tissue excision. These results indicate that hASCs possess the ability to adhere key adhesion proteins, illustrate the importance of hASC harvest procedure, and suggest mechanisms for homing in a setting where interaction with inflamed or injured tissue is necessary.
doi:10.1097/SAP.0b013e318095a771
PMCID: PMC2829884  PMID: 18362576
Adipose-derived stromal cells; hypoxia; liposuction; parallel plate flow chamber; adhesion cascade
6.  IFATS Series: The Role of Human Adipose-Derived Stromal Cells in Inflammatory Microvascular Remodeling and Evidence of a Perivascular Phenotype 
Stem cells (Dayton, Ohio)  2008;26(10):2682-2690.
A growing body of literature suggests that human adipose-derived stromal cells (hASCs) possess developmental plasticity both in vitro and in vivo, and might represent a viable cell source for therapeutic angiogenesis and tissue engineering. We investigate their phenotypic similarity to perivascular cell types, ability to contribute to in vivo microvascular remodeling, and ability to modulate vascular stability. We evaluated hASC surface expression of vascular and stem/progenitor cell markers in vitro, as well as any effects of PDGF-BB and VEGF165 on in vitro hASC migration. To ascertain in vivo behavior of hASCs in an angiogenic environment, hASCs were isolated, expanded in culture, labeled with a fluorescent marker, and injected into adult nude rat mesenteries that were stimulated to undergo microvascular remodeling. 10, 30, and 60 days after injection, tissues from anesthetized animals were harvested and processed with immunohistochemical techniques to determine hASC quantity, positional fate in relation to microvessels, and expression of endothelial and perivascular cell markers. After 60 days, 29% of hASCs exhibited perivascular morphologies compared to 11% of injected human lung fibroblasts. hASCs exhibiting perivascular morphologies also expressed markers characteristic of vascular pericytes: smooth muscle α-actin (SMA) (10%) and NG2 (8%). In tissues treated with hASCs, vascular density was significantly increased over age-matched controls lacking hASCs. This study demonstrates that hASCs express pericyte lineage markers in vivo and in vitro, exhibit increased migration in response to PDGF-BB in vitro, exhibit perivascular morphology when injected in vivo, and contribute to increases in microvascular density during angiogenesis by migrating toward vessels.
doi:10.1634/stemcells.2008-0030
PMCID: PMC2672107  PMID: 18436860
adipose-derived stromal cells; microcirculation; pericyte; angiogenesis
7.  Agent-Based Model of Therapeutic Adipose-Derived Stromal Cell Trafficking during Ischemia Predicts Ability To Roll on P-Selectin 
PLoS Computational Biology  2009;5(2):e1000294.
Intravenous delivery of human adipose-derived stromal cells (hASCs) is a promising option for the treatment of ischemia. After delivery, hASCs that reside and persist in the injured extravascular space have been shown to aid recovery of tissue perfusion and function, although low rates of incorporation currently limit the safety and efficacy of these therapies. We submit that a better understanding of the trafficking of therapeutic hASCs through the microcirculation is needed to address this and that selective control over their homing (organ- and injury-specific) may be possible by targeting bottlenecks in the homing process. This process, however, is incredibly complex, which merited the use of computational techniques to speed the rate of discovery. We developed a multicell agent-based model (ABM) of hASC trafficking during acute skeletal muscle ischemia, based on over 150 literature-based rules instituted in Netlogo and MatLab software programs. In silico, trafficking phenomena within cell populations emerged as a result of the dynamic interactions between adhesion molecule expression, chemokine secretion, integrin affinity states, hemodynamics and microvascular network architectures. As verification, the model reasonably reproduced key aspects of ischemia and trafficking behavior including increases in wall shear stress, upregulation of key cellular adhesion molecules expressed on injured endothelium, increased secretion of inflammatory chemokines and cytokines, quantified levels of monocyte extravasation in selectin knockouts, and circulating monocyte rolling distances. Successful ABM verification prompted us to conduct a series of systematic knockouts in silico aimed at identifying the most critical parameters mediating hASC trafficking. Simulations predicted the necessity of an unknown selectin-binding molecule to achieve hASC extravasation, in addition to any rolling behavior mediated by hASC surface expression of CD15s, CD34, CD62e, CD62p, or CD65. In vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 µm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications.
Author Summary
Ischemic pathologies, such as acute myocardial infarction and peripheral vascular disease, continue to be associated with high morbidities and mortalities. Recently, therapies wherein adult stem cells are injected into the circulation have been shown to increase blood flow and help to restore tissue function following injury. Pre-clinical animal models and human trials have shown successes utilizing this approach, but variable trafficking efficiencies and low incorporation of cells into the injured tissue severely limit effectiveness and may preclude clinical adoption. To address this, we sought to study the complex process of how injected stem cells traffic through the microcirculation and home to sites of injury, in an effort to identify bottlenecks in this process that could be manipulated for therapeutic gain. We developed an agent-based computer model to speed the rate of discovery, and we identified a key cell–cell adhesion interaction that could be targeted to enhance stem cell homing efficiencies during injectable stem cell therapies.
doi:10.1371/journal.pcbi.1000294
PMCID: PMC2636895  PMID: 19247427
8.  NF-κB Regulates Mesenchymal Transition for the Induction of Non-Small Cell Lung Cancer Initiating Cells 
PLoS ONE  2013;8(7):e68597.
The epithelial-to-mesenchymal transition (EMT) is a de-differentiation process that has been implicated in metastasis and the generation of cancer initiating cells (CICs) in solid tumors. To examine EMT in non-small cell lung cancer (NSCLC), we utilized a three dimensional (3D) cell culture system in which cells were co-stimulated with tumor necrosis factor alpha (TNF) and transforming growth factor beta (TGFβ). NSCLC spheroid cultures display elevated expression of EMT master-switch transcription factors, TWIST1, SNAI1/Snail1, SNAI2/Slug and ZEB2/Sip1, and are highly invasive. Mesenchymal NSCLC cultures show CIC characteristics, displaying elevated expression of transcription factors KLF4, SOX2, POU5F1/Oct4, MYCN, and KIT. As a result, these putative CIC display a cancer “stem-like” phenotype by forming lung metastases under limiting cell dilution. The pleiotropic transcription factor, NF-κB, has been implicated in EMT and metastasis. Thus, we set out to develop a NSCLC model to further characterize the role of NF-κB activation in the development of CICs. Here, we demonstrate that induction of EMT in 3D cultures results in constitutive NF-κB activity. Furthermore, inhibition of NF-κB resulted in the loss of TWIST1, SNAI2, and ZEB2 induction, and a failure of cells to invade and metastasize. Our work indicates that NF-κB is required for NSCLC metastasis, in part, by transcriptionally upregulating master-switch transcription factors required for EMT.
doi:10.1371/journal.pone.0068597
PMCID: PMC3728367  PMID: 23935876
9.  Pericytes Derived from Adipose-Derived Stem Cells Protect against Retinal Vasculopathy 
PLoS ONE  2013;8(5):e65691.
Background
Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy.
Methodology/Principal Findings
We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection).
Conclusions/Significance
ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-β1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.
doi:10.1371/journal.pone.0065691
PMCID: PMC3669216  PMID: 23741506
10.  Human Adipose-Derived Stromal Cells Accelerate Diabetic Wound Healing: Impact of Cell Formulation and Delivery 
Tissue Engineering. Part A  2010;16(5):1595-1606.
Human adipose-derived stromal cells (ASCs) have been shown to possess therapeutic potential in a variety of settings, including cutaneous wound healing; however, it is unknown whether the regenerative properties of this cell type can be applied to diabetic ulcers. ASCs collected from elective surgical procedures were used to treat full-thickness dermal wounds in leptin receptor-deficient (db/db) mice. Cells were delivered either as multicellular aggregates or as cell suspensions to determine the impact of cell formulation and delivery methods on biological activity and in vivo therapeutic effect. After treatment with ASCs that were formulated as multicellular aggregates, diabetic wounds experienced a significant increase in the rate of wound closure compared to wounds treated with an equal number of ASCs delivered in suspension. Analysis of culture supernatant and gene arrays indicated that ASCs formulated as three-dimensional aggregates produce significantly more extracellular matrix proteins (e.g., tenascin C, collagen VI α3, and fibronectin) and secreted soluble factors (e.g., hepatocyte growth factor, matrix metalloproteinase-2, and matrix metalloproteinase-14) compared to monolayer culture. From these results, it is clear that cell culture, formulation, and delivery method have a large impact on the in vitro and in vivo biology of ASCs.
doi:10.1089/ten.tea.2009.0616
PMCID: PMC2952117  PMID: 20038211

Results 1-10 (10)