Anna M. Parker

Adipose-derived stem cells for the regeneration of damaged tissues.

As the promise of stem cell-based therapies begins to be realised, and efforts to bring advances to the clinic mount, the source of these cells is increasingly important. The morbidity associated with harvesting stem cells from solid organs and the invasive nature of bone marrow biopsies may limit their practicality for wider clinical applications. An emerging body of literature suggests that adipose tissue may provide an abundant, readily accessible source of cells with similar potential to that described of other adult stem cells. This review will address advances in the use of adipose stem cells in fields as divergent as soft tissue reconstruction and cerebral infarction recovery. Numerous challenges will also be discussed; however, rapidly accumulating advances suggest that adipose stem cells may be as effective as they are abundant.

Low serum and serum-free culture of multipotential human adipose stem cells

BACKGROUND Adipose tissue provides an easily accessible and abundant source of putative stem cells for translational clinical research. Currently prevalent culture techniques include the use of FBS, a highly variable and undefined component, which brings with it the potential for adverse patient reactions. In an effort to eliminate the use of animal products in human adipose stem cell (ASC) cultures, we have developed two new culture methods, a very low human serum expansion medium and a completely serum-free medium. METHODS Through serial testing, a highly enriched medium formulation was developed for use with and without the addition of 0.5% human serum, an amount easily obtainable from autologous blood draws. RESULTS Very low-serum culture yielded population-doubling times averaging 1.86 days in early passage, while the serum-free formulation was associated with less robust cell growth, with doubling times averaging 5.79 days. ASC in both conditions maintained its ability to differentiate into adipo-, chondro- and osteogenic lineages in vitro, despite lower expression of CD34 in early passage. Expression of ALDH, HLA, CD133, CD184, and CD31 was comparable with that seen in cells cultured in 10% FBS. DISCUSSION These newly developed culture conditions provide a unique environment within which to study ASCs without the interference of animal serum, and allow for the rapid expansion of autologous ASCs in culture in an animal product-free environment for use in human clinical trials.

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. Copyright

Long-term In-Vivo Tumorigenic Assessment of Human Culture-expanded Adipose Stromal/Stem Cells

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 hosts immune system. In this study, a high dose of culture expanded ASCs was 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.