Age-Related Effects on the Potency of Human Adipose-Derived Stem Cells: Creation and Evaluation of Superlots and Implications for Musculoskeletal Tissue Engineering Applications

Abstract

Human adipose-derived stem cells (hASC) are now a prevalent source of adult stem cells for studies in tissue engineering and regenerative medicine. However, researchers utilizing hASC in their investigations often encounter high levels of donor-to-donor variability in hASC differentiation potential. Because of this, conducting studies with this primary cell type can require extensive resources to generate statistically significant data. We present a method to generate pooled donor cell populations, termed ''superlots,'' containing cell populations derived from four to five age-clustered donors. The goal of generating these superlots was to 1) increase experimental throughput, 2) to utilize assay resources more efficiently, and 3) to begin to establish global hASC differentiation behaviors that may be associated with donor age. With our superlot approach, we have validated that pooled donor cell populations exhibit proliferative activity representing the combined behavior of each individual donor cell line. Further, the superlots also exhibit differentiation levels roughly approximating the average combined differentiation levels of each individual donor cell line. We established that high donor-to-donor variability exists between the pre-, peri-, and postmenopausal age groupings and that proliferation and differentiation characteristics can vary widely, independent of age. Interestingly, we did observe that cell lines derived from postmenopausal donors demonstrated a relatively high proclivity for osteogenic differentiation and a relatively lowered proclivity for adipogenic differentiation as compared with cells derived from pre-and perimenopausal donors. In general, superlots effectively represented the average differentiation behavior of each of their contributing cell populations and could provide a powerful tool for increasing experimental throughput to more efficiently utilize resources when studying hASC differentiation.

Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source

“…15 Human ASCs were isolated from the tissue using a method described previously by our laboratory and others. 9,10,14,15,[29][30][31][32][33] At second passage, 100,000 cells of each cell line were seeded in a single T-75 flask in CGM comprised of alpha-modified minimal essential medium (a-MEM with l-glutamine; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Premium Select; Atlanta Biologicals, Lawrenceville GA), 200 mM l-glutamine, and 100 IU penicillin/100 mg/mL streptomycin (Mediatech, Herndon, VA). The cells were allowed to proliferate at 37°C in 5% carbon dioxide until reaching 80% confluency, and then passaged.…”

“…The amassed cells were then characterized for osteogenic and adipogenic potential, ensuring that the cells differentiated representative of an average of the five cell lines. 15 …”

“…Human adipose-derived stem cells (hASC) provide an abundant and easily accessible source of cells for tissue engineering, 13 and previous studies have shown the ability of these cells to differentiate into osteogenic or chondrogenic lineages under appropriate culture conditions. 10,14,15 Our laboratory has previously published the effects of elevating Ca 2 + levels on hASC osteogenesis, in both complete growth and osteogenic differentiation media, comparing mineralization and proliferation. 9,10,16 The use of elevated Ca 2 + to direct hASC osteogenic lineage specification is a clear advantage in bone tissue engineering.…”

Enhanced cellular infiltration of human adipose-derived stem cells in allograft menisci using a needle-punch method

“…Human ASC were isolated from liposuction aspirates of female patients undergoing voluntary liposuction procedures using an approved IRB protocol at the University of North Carolina at Chapel Hill (IRB 04-1622), as described previously by our laboratory [36]. CGM was used to expand hASC.…”

“…CGM was used to expand hASC. An age-grouped (age 24–36, five donors), hASC superlot was generated and verified as representative of individual donors, as described previously [36]. The hASC superlot was seeded at a density of 250,000 cells per gram of meniscal tissue.…”