Gene expression-based enrichment of live cells from adipose tissue produces subpopulations with improved osteogenic potential

Marble et al. Stem Cell Research & Therapy 2014, 5:145 http://stemcellres.com/content/5/5/145 RESEARCH Open Access Gene expression-based enrichment of live cells from adipose tissue produces subpopulations with improved osteogenic potential Hetal D Marble1, Bryan A Sutermaster1,2, Manisha Kanthilal1,2, Vera C Fonseca1 and Eric M Darling1,2,3* Abstract Introduction: Mesenchymal stem cells have been increasingly used for cell-based therapies. Adipose-derived stem/stromal cells (ASCs) from the stromal vascular fraction (SVF) of fat tissue are a particularly attractive option for cell based therapy given their accessibility and relative abundance. However, their application in both clinical and basic science investigations is complicated by the isolation of differentiable cells within the SVF. Current enrichment strategies, such as monolayer passaging and surface marker-based sorting, can be time-consuming or overly stringent. Ideally, a population of cells with great regenerative capacity could be isolated with high yields so that extensive in vitro manipulation is not necessary. The objective of this study was to determine whether SVF cells sorted based on expression of alkaline phosphatase liver/bone/kidney (ALPL) resulted in populations with increased osteogenic differentiation potential. Methods: SVF samples were obtained from four, human donors and processed to isolate initial, heterogeneous cell populations. These SVF cells underwent a four day osteogenic priming period, after which they were treated with a fluorescent, oligodeoxynucleotide molecular beacon probe specific for ALPL mRNA. Cells were separated into positive and negative groups using fluorescence-activated cell sorting (FACS) then differentiated down the osteogenic lineage. Differentiation was assessed by measuring calcified matrix production in each sample. Results: Cells positive for ALPL expression (ALPL+) represented approximately 34% of the gated population, while cells negative for ALPL expression (ALPL-) represented approximately 18%. ALPL+ cells produced 3.7-fold and 2.1-fold more calcified matrix than ALPL- and unsorted SVF cells, respectively, indicating a significant improvement in osteogenic differentiation. Further, ALPL+ cells showed increases in metabolite production for both adipogenesis and chondrogenesis, suggesting that the enrichment process yields an enhanced multipotent phenotype. Osteogenic differentiation response and cell yields for ALPL+ cells were markedly improved over surface marker-sorted samples. Conclusion: This study demonstrates a novel method to enrich heterogeneous SVF cells for increased osteogenic potential. The procedure requires less time and results in higher yields of therapeutically useful cells than other existing approaches. Gene expression-based sorting of MSCs is a potentially paradigm-shifting approach that could benefit applications spanning from basic science to clinical therapy. * Correspondence: 1 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI 02912, USA 2 Center for Biomedical Engineering, Brown University, Providence, RI 02912, USA Full list of author information is available at the end of the article © 2014 Marble et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Marble et al. Stem Cell Research & Therapy 2014, 5:145 http://stemcellres.com/content/5/5/145 Box 1. About Eric Darling EMD is the Manning Assistant Professor of Medical Science, Orthopaedics, and Engineering in the Department of Molecular Pharmacology, Physiology, & Biotechnology at Brown University. He also currently serves as the Graduate Program Director for the Center for Biomedical Engineering. He received a BS in engineering from Harvey Mudd College, a PhD in bioengineering from Rice University, and postdoctoral training in orthopedic research at Duke University. His research area focuses on understanding the relationship between the mechanical and biological characteristics of cells and tissues, with emphasis on the musculoskeletal system. He is specifically interested in understanding heterogeneity in adult stem cell populations and developing approaches to identify tissue-specific cells for regenerative medicine and disease diagnostics. Recent work in his group has focused on two, novel cellular characteristics: single-cell mechanical biomarkers and live-cell gene expressions. Introduction Cell enrichment or purification is often a necessary first step for clinical, cell-based therapies as well as basic science investigations into homogeneous subpopulations. Adult mesenchymal stem cells (MSCs) are one type of cell for which this is of great importance. As our understanding of MSCs improves, their use in regenerative medicine becomes ever more promising. This has been especially true for musculoskeletal tissues, with researchers proposing many possibilities using MSCs for the treatment of orthopedic ailments [1-7]. Autologous stem Page 2 of 13 cell transplantation therapies have been proposed for their potential therapeutic versatility and low immunogenicity [8,9]. However, many of these proposed therapies rely on prior in vitro expansion of the cell populations, which is a slow process and can negatively affect cell phenotype [10]. Single-surgery therapies, where autologous MSCs are isolated and reintroduced into the site of injury in short succession, have the potential to save on both costs and rehabilitation time. Human adipose-derived stem/stromal cells (ASCs), isolated from the stromal vascular fraction (SVF) of lipoaspirate, may be particularly well suited for such single-surgery strategies due to their accessibility and relative abundance in fat tissue, as well as their ability to differentiate down the osteogenic, adipogenic, and chondrogenic lineages [11]. While prolonged culture and passaging is an effective method for isolating ASCs from SVF populations, this process can take weeks to complete. A more rapid approach for the isolation of regeneratively advantageous cells from other cell types contained within the SVF would be enormously beneficial. Traditional cell enrichment strategies have found limited success purifying MSCs due to the lack of a universal surface marker profile [12]. This approach is further complicated by the temporal variability of surface antigens, which can change over time with passaging [13]. Additionally, reported surface marker profiles often result in very low cell yields, necessitating post-sort expansion to obtain sufficient cell numbers for (...truncated)