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Comparative Study
, 3 (2), 206-17

Comparison of Human Adipose-Derived Stem Cells Isolated From Subcutaneous, Omental, and Intrathoracic Adipose Tissue Depots for Regenerative Applications

Affiliations
Comparative Study

Comparison of Human Adipose-Derived Stem Cells Isolated From Subcutaneous, Omental, and Intrathoracic Adipose Tissue Depots for Regenerative Applications

Valerio Russo et al. Stem Cells Transl Med.

Abstract

Adipose tissue is an abundant source of multipotent progenitor cells that have shown promise in regenerative medicine. In humans, fat is primarily distributed in the subcutaneous and visceral depots, which have varying biochemical and functional properties. In most studies to date, subcutaneous adipose tissue has been investigated as the adipose-derived stem cell (ASC) source. In this study, we sought to develop a broader understanding of the influence of specific adipose tissue depots on the isolated ASC populations through a systematic comparison of donor-matched abdominal subcutaneous fat and omentum, and donor-matched pericardial adipose tissue and thymic remnant samples. We found depot-dependent and donor-dependent variability in the yield, viability, immunophenotype, clonogenic potential, doubling time, and adipogenic and osteogenic differentiation capacities of the ASC populations. More specifically, ASCs isolated from both intrathoracic depots had a longer average doubling time and a significantly higher proportion of CD34(+) cells at passage 2, as compared with cells isolated from subcutaneous fat or the omentum. Furthermore, ASCs from subcutaneous and pericardial adipose tissue demonstrated enhanced adipogenic differentiation capacity, whereas ASCs isolated from the omentum displayed the highest levels of osteogenic markers in culture. Through cell culture analysis under hypoxic (5% O(2)) conditions, oxygen tension was shown to be a key mediator of colony-forming unit-fibroblast number and osteogenesis for all depots. Overall, our results suggest that depot selection is an important factor to consider when applying ASCs in tissue-specific cell-based regenerative therapies, and also highlight pericardial adipose tissue as a potential new ASC source.

Keywords: Adipose tissue; Adult stem cells; Cellular therapy; Differentiation; Hypoxia; Matched-pair analysis.

Figures

Figure 1.
Figure 1.
Anatomic location of the intrathoracic adipose tissue depots and histological overview of the tissue ultrastructure. (A): Schematic showing the anatomical position of the pericardial and thymic remnant adipose tissue depots. (B): Intraoperative image of the intrathoracic depots exposed through median sternotomy. (C): Representative Masson’s trichrome staining of subcutaneous, omentum, pericardial, and thymic remnant adipose tissue. Scale bars represent 100 μm. Abbreviations: OM, omentum; PF, pericardial fat; SC, subcutaneous; TH, thymic remnant.
Figure 2.
Figure 2.
Data summary for average values for all donors for each of the depots. All data are expressed as mean ± SD. ∗, Significantly different at (p < .05). (A): Viable cell yield per gram of digested tissue (n = 3, N = 7). (B): Percent viability in stromal vascular fraction (n = 3, N = 7). (C): In vitro clonogenic potential measured through the colony-forming unit-fibroblast assay at 14 days (n = 3, N = 3). (D): Doubling time of P2 ASCs (n = 3, N = 6). (E): GPDH enzyme activity at 7 days post-induction of adipogenic differentiation (n = 3, N = 6). (F): ALP enzyme activity at 28 days post-induction of osteogenic differentiation (n = 3, N = 6). Abbreviations: ALP, alkaline phosphatase; CFE, colony-forming efficiency; GPDH, glycerol-3-phosphate dehydrogenase; H, hypoxic (5% O2/90% N2/5% CO2); N, normoxic (95% air/5% CO2); OM, omentum; PF, pericardial fat; SC, subcutaneous; TH, thymic remnant.
Figure 3.
Figure 3.
Immunophenotype of P2 ASCs isolated from each of the depots. (A): Representative histograms of donor-matched subcutaneous/omentum samples and donor-matched pericardial fat/thymic remnant samples. (B): Percentage of positive cells for each of the markers analyzed. Data are presented as the mean ± SD (n = 3, N = 6). Interdepot differences in the expression were observed for CD34 and CD166. ∗, Statistically significant (p < .05). Abbreviations: OM, omentum; PF, pericardial fat; SC, subcutaneous; TH, thymic remnant.
Figure 4.
Figure 4.
Adipogenic differentiation of ASCs isolated from subcutaneous fat and the omentum. (A): GPDH enzyme activity of induced donor-matched P2 ASCs at 7 days. Data are expressed as mean ± SD (n = 3, N = 6). ∗, Significantly different (p < .05). (B): Representative oil red O staining at 7 days post-induction. Intracellular lipid accumulation was observed under both oxygen conditions for subcutaneous ASCs, but not ASCs derived from the omentum or the negative controls. Scale bars represent 100 μm. Relationship between GPDH activity and donor BMI for ASCs derived from subcutaneous adipose tissue (C) and omentum (D). Crossed values (x) indicate nonmatched donor samples not included in the comparative assessments. (E): Representative endpoint RT-PCR analysis of adipogenic gene expression at 7 and 14 days (n = 3, N = 2) with GAPDH as the housekeeping gene. Abbreviations: BMI, body mass index; GPDH, glycerol-3-phosphate dehydrogenase; H, hypoxic (5% O2/90% N2/5% CO2); N, normoxic (95% air/5% CO2); OM, omentum; SC, subcutaneous.
Figure 5.
Figure 5.
Adipogenic differentiation of ASCs isolated from pericardial and thymic remnant adipose tissue. (A): GPDH enzyme activity of induced donor-matched P2 ASCs at 7 days. Data are expressed as mean ± SD (n = 3, N = 6). ∗, Significantly different (p < .05). (B): Representative oil red O staining at 7 days post-induction. Scale bars represent 100 μm. Relationship between GPDH activity and donor BMI for ASCs derived from pericardial adipose tissue (C) and thymic remnant (D). (E): Representative endpoint RT-PCR analysis of adipogenic gene expression at 7 and 14 days (n = 3, N = 2) with GAPDH as the housekeeping gene. Abbreviations: BMI, body mass index; GPDH, glycerol-3-phosphate dehydrogenase; H, hypoxic (5% O2/90% N2/5% CO2); N, normoxic (95% air/5% CO2); PF, pericardial fat; TH, thymic remnant.
Figure 6.
Figure 6.
Osteogenic differentiation of ASCs isolated from subcutaneous fat and the omentum. (A): ALP enzyme activity of induced donor-matched P2 ASCs at 28 days. Data are expressed as mean ± SD (n = 3, N = 6). With the exception of donor 1 (SC), donor 5 (OM), and donor 6 (SC + OM), a significant difference was observed in ALP activity in both depots under normoxic versus hypoxic conditions. ∗, Significant difference between donor-matched subcutaneous and omentum ASCs (p < .05). (B): Representative von Kossa staining at 28 days post-induction. Matrix mineralization was observed in all induced samples, but was dramatically reduced under hypoxic conditions for both depots. Scale bars represent 100 μm. (C): In donors 2 and 5, matrix mineralization was observed in ASCs isolated from the omentum but not subcutaneous fat. Scale bars represent 100 μm. (D): Representative endpoint RT-PCR analysis of osteogenic gene expression at 21 and 28 days (n = 3, N = 2) with GAPDH as the housekeeping gene. Abbreviations: ALP, alkaline phosphatase; H, hypoxic (5% O2/90% N2/5% CO2); N, normoxic (95% air/5% CO2); OM, omentum; SC, subcutaneous.
Figure 7.
Figure 7.
Osteogenic differentiation of ASCs isolated from pericardial adipose tissue and the thymic remnant. (A): ALP enzyme activity of induced donor-matched P2 ASCs at 28 days. Data are expressed as mean ± SD (n = 3, N = 6). For all donors, a significant difference was observed for both depots under normoxic versus hypoxic conditions. ∗, Significant difference between donor-matched pericardial and thymic remnant ASCs (p < .05). (B): Representative von Kossa staining at 28 days post-induction. Matrix mineralization was observed in all induced samples, but was reduced under hypoxic conditions for both depots. Scale bars represent 100 μm. (C): Representative endpoint RT-PCR analysis of osteogenic gene expression at 21 and 28 days (n = 3, N = 2) with GAPDH as the housekeeping gene. Abbreviations: ALP, alkaline phosphatase; H, hypoxic (5% O2/90% N2/5% CO2); N, normoxic (95% air/5% CO2); PF, pericardial fat; TH, thymic remnant.

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