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

Abstract

IntroductionMesenchymal 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.MethodsSVF 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.ResultsCells 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.ConclusionThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/scrt502) contains supplementary material, which is available to authorized users.

Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells

“…Novel markers are already available that allow one to discriminate self‐renewing from differentiation‐prone PSCs, in vitro, suggesting the existence within this cell compartment of a developmental genealogy similar to that encountered in hematopoietic and epithelial systems (Khan et al, unpublished results). Such innate characteristics, if they exist among subpopulations of perivascular cells, might recommend the use of one subset as more efficacious than another in particular types of tissue regeneration, as has been demonstrated for adipose derived stem cells expressing higher levels of alkaline phosphatase in improving osteogenic potential , and it may help to explain the relative participation and contribution of these perivascular cells in endogenous tissue turnover and repair . Furthermore, such a partition of PSCs into discrete functional subsets should be of high medical relevance in the perspective of a therapeutic use thereof.…”

On the origin and impact of mesenchymal stem cell heterogeneity: new insights and emerging tools for single cell analysis

“…These methods are used to monitor gene expression in live MSCs undergoing osteogenesis, and can be combined with FACS techniques to sort individual cells on the basis of their gene expression (Fig. 9) (Li et al, 2016;Marble et al, 2014).…”

“…There is also active development surrounding single cell mass spectrometry, which was recently used to quantify the abundance of thousands of proteins at the single cell level during embryonic stem cell differentiation (Budnik et al, 2017 -non-peer reviewed e-publication). (Marble et al 2014), b) spherical nucleic acids (Li et al 2016) and c) single molecule RNA FISH (Cote et al 2016) have been used to query the single cell abundance of various differentiation markers (ALPL, Runx2 and ACAN, respectively). Subfigures reproduced under the terms of the Creative Commons Attribution License.…”

Variation in Osteogenic Differentiation Capacities of Adiposederived Stromal Cells by Anatomic Depot

“…In 2001, Zuk et al described this population of culture-derived, multipotential MSC from subcutaneous human white adipose tissue, termed adipose-derived stromal cells (or ASC) [3] . Since this time, ASC have been of great interest to investigators due to their relative abundance and accessibility being derived from adiposetissue, in contrast to BMSC [3][4][5][6][7] . Unlike BMSC, ASC are also advantageous in that they present minimal morbidity [1] .…”