Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies

Abstract

The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (P = 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 +/- 1.1%, mean +/- SEM) and hip/thigh region (1.2 +/- 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.

Figures

Fig. 1

Effect of adipose-tissue-harvesting site on the frequency of adipose-derived stem cells (ASCs) in the stromal vascular fraction (SVF). After isolation of the SVF from adipose tissue of both tissue-harvesting sites, the frequency of ASCs in the SVF isolates was determined by using: (a) a limiting dilution assay (LD) and (b) a colony-forming unit (CFU) assay (CFU-F), with similar results. When combined (c), a significant difference was detected in ASC frequency between adipose tissue harvested from the abdomen and adipose tissue harvested from the hip/thigh region (P-values: a limiting dilution: P = 0.003, b colony-forming unit: P = 0.05, c combined: P = 0.0009)

Fig. 2

Effect of adipose-tissue-harvesting site on the osteogenic diffentiation capacity of CFU from the abdomen or hip/thigh regions. No significant difference is apparent in the CFU-alkaline phosphatase (CFU-ALP) frequency from the abdomen and hip/thigh region when corrected for CFU-fibroblast (CFU-F).

Fig. 3

Effect of the adipose-tissue-harvesting site on growth kinetics of ASCs in vitro. a Growth kinetics of ASCs of a representative donor when adipose tissue was harvested from the abdomen. b Growth kinetics of ASCs when adipose tissue was harvested from the hip/thigh region. c Population doubling time was calculated from the exponential growing phase of the cells. There was no significant difference in population doubling time of ASCs from the abdomen and hip/thigh region (P = 0.78, independent Student t-test).

Fig. 4

Effect of adipose-tissue-harvesting site on the osteogenic differentiation of cultured ASCs in vitro. a–cRUNX-2 (runt-related transcription factor 2; P = 0.002), COL1α1 (collagen type Ia; P = 0.024), and OPN (osteopontin; P = 0.38) gene expression was measured after 0, 4, and 7 days (d) of osteogenic induction, by using quantitative real-time polymerase chain reaction (qRT-PCR). No significant differences were detected in osteogenic gene expression between ASCs derived from the abdomen and ASCs derived from the hip/thigh region, at all three time points tested. d ALP activity was significantly increased after 14 days in the osteogenically stimulated cells (stim) compared with that in unstimulated cells (con; P = 0.047). No statistically significant difference was apparent in ALP activity between ASCs derived from abdominal fat and ASCs from hip/thigh fat. e–g Von Kossa staining of ASCs from abdomen (e) and hip/thigh region (f) after 21 days of culture in osteogenic medium and in control medium (g), showing mineralized matrix visible as black spots.

Fig. 5

Effect of the tissue-harvesting site on the chondrogenic differentiation of cultured ASCs in vitro. a Both abdomen (lane 1) and hip/thigh region (lane 2) display COL2B mRNA. Under nonchondrogenic conditions, no COL2B could be detected (lane 3). b, c Aggrecan (AGG; P = 0.041) and collagen 10A (Col10a; P = 0.024) gene expression, respectively, was up-regulated after 7 days (d), as measured by qRT-PCR. No significant differences were detected in chondrogenic gene expression between ASCs derived from the abdomen and ASCs derived from the hip/thigh region, at all three time points tested. d, f Cartilaginous matrix expression was visualized in both tissue-harvesting sites by staining proteoglycans (Alcian blue) and COL2 (Col2-II6B3 antibody), respectively. e At higher magnification, the ASC nodules resembled cartilage-like tissue, composed of spherical cells surrounded by lacunae and lying in a proteoglycan-rich extracellular matrix.