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1.  A Modified Aggregate Culture for Chondrogenesis of Human Adipose-Derived Stem Cells Genetically Modified with Growth and Differentiation Factor 5 
BioResearch Open Access  2013;2(4):258-265.
Adipose-derived stem cells (ADSCs) are an attractive cell source for tissue engineering, and recently a modified aggregate culture of human ADSCs (hADSCs) was established based on preparation of three-dimensional (3D) cell aggregates in growth factor–enriched low serum medium using the hanging droplet method. Growth and differentiation factor 5 (GDF5) plays a critical role in chondrogenesis and cartilage development. In the present study, we examine (1) whether the modified aggregate culture is feasible for chondrogenic induction of hADSCs, (2) whether overexpressed GDF5 can promote chondrogenesis, and (3) the gene expression profile during chondrogenesis in this aggregate culture. hADSCs were infected with an adenovirus carrying the GDF5 gene (Ad-GDF5). Cells were cultured with chondrogenic media either in a modified aggregate culture or in an attached micromass culture that served as a control. The chondrogenic phenotype was assessed by morphology (n=8), biochemistry (n=3), and histology (n=2). Expression of 12 genes was determined by quantitative real-time polymerase chain reaction (n=3). We found that ADSCs cultured in the modified aggregates exhibited denser pellets and higher content of sulfated glycosaminoglycan (sGAG) compared with those cultured in the micromass. Infection of cells with Ad-GDF5 increased the aggregate size and sGAG content. It also up-regulated expression of GDF5, aggrecan, and leptin and down-regulated expression of COL I, while expression of COL II and COL 10 remained unchanged. We concluded that the modified aggregate culture is feasible for chondrogenic induction of human ADSCs. Infection with Ad-GDF5 appears to promote the chondrogenesis. These findings suggest that genetic modification of ADSCs with GDF5 in the modified aggregate culture could be useful for treating diseases with cartilage defects.
PMCID: PMC3731687  PMID: 23914332
aggregate culture; cartilage regeneration; chondrogenesis; GDF5; genetic modification; human adipose-derived stem cell
2.  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.
PMCID: PMC3264753  PMID: 22185824
Adipose derived stem cell; Mesenchymal Stem Cell Transplantation/toxicity
3.  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.
PMCID: PMC2952117  PMID: 20038211
4.  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.
PMCID: PMC2829884  PMID: 18362576
Adipose-derived stromal cells; hypoxia; liposuction; parallel plate flow chamber; adhesion cascade
5.  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.
PMCID: PMC2672107  PMID: 18436860
adipose-derived stromal cells; microcirculation; pericyte; angiogenesis
6.  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.
PMCID: PMC2636895  PMID: 19247427

Results 1-6 (6)