Molecular Aspects of Adipose-Derived Stromal Cell Senescence in a Long-Term Culture: A Potential Role of Inflammatory Pathways

Abstract

Long-term culture of mesenchymal stromal/stem cells in vitro leads to their senescence. It is very important to define the maximal passage to which the mesenchymal stromal/stem cells maintain their regenerative properties and can be used for cellular therapies and construction of neo-organs for clinical application. Adipose-derived stromal/stem cells were isolated from porcine adipose tissue. Immunophenotype, population doubling time, viability using bromodeoxyuridine assay, MTT assay, clonogencity, β-galactosidase activity, specific senescence-associated gene expression, apoptosis, and cell cycle of adipose-derived mesenchymal stromal/stem cells (AD-MSCs) were analyzed. All analyses were performed through 12 passages (P). Decreasing viability and proliferative potential of AD-MSCs with subsequent passages together with prolonged population doubling time were observed. Expression of β-galactosidase gradually increased after P6. Differentiation potential of AD-MSCs into adipogenic, chondrogenic, and osteogenic lineages decreased at the end of culture (P10). No changes in the cell cycle, the number of apoptotic cells and expression of specific AD-MSC markers during the long-term culture were revealed. Molecular analysis showed increased expression of genes involved in activation of inflammatory response. AD-MSCs can be cultured for in vivo applications without loss of their properties up to P6.

Evaluation of Fat Accumulation and Adipokine Production during the Long-Term Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Study of the Influence of Immunobiotics

“…The progressive death of adipocytes may lead to the release of DAMPs, which may subsequently induce a low-grade inflammatory response. On the other hand, the long-term culture of adipocyte progenitors such as preadipocytes can induce senescence of the growing cells, which function as mediators of inflammation associated with obesity [ 51 , 52 ]. It was reported that in addition to no longer undertaking their original function effectively, senescent cells can induce the secretion of proinflammatory factors in the tissue in which they reside [ 53 ].…”

“…It was reported that in addition to no longer undertaking their original function effectively, senescent cells can induce the secretion of proinflammatory factors in the tissue in which they reside [ 53 ]. Cellular senescence in adipocytes attenuates the physiological resolution of inflammation leading to a low-grade chronic inflammation, insulin resistance, type-2 diabetes, and companion senescence-associated secretory phenotype in the obese individual [ 51 , 52 , 53 ], indicating that these phenotypes are associated with impaired adipokine production. A detailed evaluation of senescence, cell death and DAMPs-mediated signaling pathway in PIP cells during LTAD would be of great value to further characterize this process in vitro and establish in parallel with the in vivo physiology.…”

Phenotypic and functional properties of dedifferentiated fat cells derived from infrapatellar fat pad

“…All samples were measured in triplicate. Population doubling time of the cultured cells was determined as described previously [ 25 ].…”

Stem Cell Therapy – When We Can Use Such Terminology?

“…The other issue is a danger of not obtaining necessary cell number, which is associated with cell senescence. Cells should be passaged maximally to 6th passage in order to maintain their regenerative properties, in some cases that time point may be not enough to obtain appropriate cell number, especially if the tissue source for cell isolation is limited [7,8]. In exchange for such restricted procedure we receive product which consists almost entirely of MSCs (>95%).…”

“…However compared to heterogeneous population of SVF and BMMCs, pure MSCs have greater regenerative potential. Beside paracrine effect they can differentiate into many cell types, osteogenic, adipogenic and chondrogenic potential of MSC is also one of minimal criteria defining MSCs [7]. Like mentioned above, both methods have pros and cons, however in order to use "real" stem cell therapies more often in clinical practice, additional improvements in cell preparation, reducing the cost of procedure, have to be performed.…”