Different isolation methods alter the gene expression profiling of adipose derived stem cells

Abstract

Human adipose stem cells (ASCs) has been in the limelight since its discovery as a suitable source of mesenchymal stem cells (MSCs) in regenerative medicine. Currently, two major techniques are used to isolate ASCs, namely liposuction and tissue biopsy. These two methods are relatively risk-free but the question as to which method could give a more efficient output remains unclear. Thus, this study was carried out to compare and contrast the output generated in regards to growth kinetics, differentiation capabilities in vitro, and gene expression profiling. It was found that ASCs from both isolation methods were comparable in terms of growth kinetics and tri-lineage differentiation. Furthermore, ASCs from both populations were reported as CD44(+), CD73(+), CD90(+), CD166(+), CD34(-), CD45(-) and HLA-DR(-). However, in regards to gene expression, a group of overlapping genes as well as distinct genes were observed. Distinct gene expressions indicated that ASCs (liposuction) has endoderm lineage propensity whereas ASCs (biopsy) has a tendency towards mesoderm/ectoderm lineage. This information suggests involvement in different functional activity in accordance to isolation method. In conclusion, future studies to better understand these gene functions should be carried out in order to contribute in the applicability of each respective cells in regenerative therapy.

Keywords: Ingenuity Pathway Analysis.; TLDA; biopsy; liposuction; mesenchymal.

Figure 1

Schematic representation of the isolation methods employed to obtain ASCs (biopsy) and ASCs (liposuction) as previously described. Both ASCs were treated with collagenase before filtering via cell strainer and seeded in tissue culture flasks.

Figure 2

Basic characterisations of ASCs. (A) Morphological features of BM-MSCs, ASCs (liposuction) and ASCs (biopsy) at 10X magnification, via phase contrast microscope; (B) SVF count for both ASCs (n=5); (C) and (D) Cell count and Cumulative Population Doubling (CPD), for ASCs (liposuction) and ASCs (biopsy), against BM-MSCs as control (* indicates p-value < 0.05); (E) Senescence assay to evaluate the activity of β-galactosidase in cells of BM-MSCs, ASCs (liposuction), and ASCs (biopsy); and (F) Cell cycle analysis describing the DNA content in cells from BM-MSCs, ASCs (liposuction) and ASCs (biopsy). Bar = 100 µm.

Figure 3

Immunophenotyping and trillineage differentiation. (A) Colony-forming unit (CFU) of both ASCs (liposuction) and ASCs (biopsy). Pictures represent triplicates taken at 4x magnification. (B) Fluorescence-activated cell sorting (FACS) analysis describing presence or absence of cell surface markers in both ASCs, with BM-MSCs as control. Cells were tested against human antigens CD34, CD44, CD45, CD73, CD90, CD166, and HLA-DR. 7-Amino-actinomycin D (7-AAD) was used to check for cell viability. CD, cluster of differentiations. (C) Mesoderm differential potentiality of ASCs in vitro. Adipogenesis was detected by neutral oil droplet formation stained with Oil red O at day 21 in ASCs (liposuction) and ASCs (biopsy). Osteogenesis was confirmed by mineralized matrix deposition stained with von Kossa staining at day 21 in ASCs (liposuction) and ASCs (biopsy). Chondrogenesis was detected by the presence of proteoglycans stained with Alcian blue at day 21 in ASCs (liposuction) and ASCs (biopsy). All experiments were conducted at sub-culture 4. Results represent average of 5 culture replicates. Bar = 100 µm. (D) The gene expression profile for mesoderm differentiation capacity using real-time as well as reverse-transcription PCR (* indicates p-value < 0.05).

Figure 4

Diagram summarizing gene expression patterns of ASCs (liposuction) and ASCs (biopsy). (A) Heat map defining gene expressions via Taqman® Low Density Array (TLDA) of both ASCs (liposuction) and ASCs (biopsy) with BM-MSCs as control. The gene expression fold change from both sources with BM-MSCs as control is depicted from lowest (in green) to highest (in red). (B) and (C) Venn diagram describing number of up-regulated as well as down-regulated genes with BM-MSCs as control.

Figure 5

IPA pathway representation of the molecular relationships among differentially expressed genes and proteins of ASCs (liposuction). The network is displayed graphically as nodes (proteins) and edges (the biological relationships between the nodes). Various shapes of the nodes represent the functional class of the proteins. Edges are displayed with various labels that describe the nature of the relationship between the nodes. Names of proteins corresponding to the abbreviations are as follows: ACTC1, actin, alpha, cardiac muscle 1; ACTIN; AFP, alpha-fetoprotein; CD9, CD9 molecule; COL1A1, collagen, type I, alpha 1; Collagen type 1; CRABP2, cellular retinoic acid binding protein 2; CTNNB1, catenin (cadherin-associated protein), beta 1, 88kDa; DES, desmin; DNMT3B, DNA (cytosine-5-)-methyltransferase 3 beta; EEF1A1, eukaryotic translation elongation factor 1 alpha 1; F Actin; FGF, fibroblast growth factor; FN1, fibronectin 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; GRB7, growth factor receptor-bound protein 7; IAPP, islet amyloid polypeptide; IFITM1, interferon induced transmembrane protein 1; IFITM2, interferon induced transmembrane protein 2; IL6ST, interleukin 6 signal transducer (gp130, oncostatin M receptor); ISL1, ISL LIM homeobox 1; KIT, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; LAMA1, laminin, alpha 1; LAMB1, laminin, beta 1; LAMC1, laminin, gamma 1; Laminin; NANOG, Nanog homeobox; NES, nestin; NR6A1, nuclear receptor subfamily 6, group A, member 1; OLIG2, oligodendrocyte lineage transcription factor 2; PGF5, placental growth factor; PODXL, podocalyxin-like; POU5F1, POU class 5 homeobox 1; RAF1, v-raf-1 murine leukemia viral oncogene homolog 1; REST, RE1-silencing transcription factor; RUNX2, runt-related transcription factor 2; SEMA3A, sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A; SERPINA1, serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1; SFRP2, secreted frizzled-related protein 2; SOX17, SRY (sex determining region Y)-box 17; SYCP3, synaptonemal complex protein 3; T, brachyury homolog; TFCP2L1, transcription factor CP2-like 1; XIST, X (inactive)-specific transcript (non-protein coding); ZFP42, zinc finger protein 42 homolog.

Figure 6

IPA pathway representation of molecular relationships among differentially expressed genes and proteins of ASCs (biopsy). The network is displayed graphically as nodes (proteins) and edges (the biological relationships between the nodes). Various shapes of the nodes represent the functional class of the proteins. Edges are displayed with various labels that describe the nature of the relationship between the nodes. Names of proteins corresponding to the abbreviations are as follows: CD9, CD9 molecule; CDX2, caudal-related homeobox 2; CGB, chorionic gonadotropin, beta polypeptide; COL1A1, collagen, type I, alpha 1; COLLAGEN TYPE 1; CRABP2, cellular retinoic acid binding protein 2; DES, desmin; FN1, fibronectin 1; FOXA2, forkhead box A2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFITM2, interferon induced transmembrane protein 2; IFITM4, interferon induced transmembrane protein 4; IL6ST, interleukin 6 signal transducer (gp130, oncostatin M receptor); ISL1, ISL LIM homeobox 1; LAMA1, laminin, alpha 1; LAMININ; MNX1, motor neuron and pancreas homeobox 1; NANOG, Nanog homeobox; NES, nestin; OLIG2, oligodendrocyte lineage transcription factor 2; PAX4, paired box 4; POU5F1, POU class 5 homeobox 1; SFRP2, secreted frizzled-related protein 2; SYCP3, synaptonemal complex protein 3; T, brachyury homolog.

Figure 7

Expression profile of pluripotent and lineage-specific stem cell markers of MSCs. (A) The Ct value of genes analysed in the study by SYBR green-based qRT-PCR for BM-MSCs, ASCs (liposuction) and ASCs (biopsy). The lower a cycle threshold (CT) value, the more copies are present in the specific sample. Values are presented after normalization to 18s mRNA levels. (B) Semi-quantitative RT-PCR of selected genes for BM-MSCs, ASCs (liposuction) and ASCs (biopsy). Average of three replicates is displayed.