Characterization and Expression of Senescence Marker in Prolonged Passages of Rat Bone Marrow‐Derived Mesenchymal Stem Cells

Abstract

The present study is aimed at optimizing the in vitro culture protocol for generation of rat bone marrow- (BM-) derived mesenchymal stem cells (MSCs) and characterizing the culture-mediated cellular senescence. The initial phase of generation and characterization was conducted using the adherent cells from Sprague Dawley (SD) rat's BM via morphological analysis, growth kinetics, colony forming unit capacity, immunophenotyping, and mesodermal lineage differentiation. Mesenchymal stem cells were successfully generated and characterized as delineated by the expressions of CD90.1, CD44H, CD29, and CD71 and lack of CD11b/c and CD45 markers. Upon induction, rBM-MSCs differentiated into osteocytes and adipocytes and expressed osteocytes and adipocytes genes. However, a decline in cell growth was observed at passage 4 onwards and it was further deciphered through apoptosis, cell cycle, and senescence assays. Despite the enhanced cell viability at later passages (P4-5), the expression of senescence marker, β-galactosidase, was significantly increased at passage 5. Furthermore, the cell cycle analysis has confirmed the in vitro culture-mediated cellular senescence where cells were arrested at the G0/G1 phase of cell cycle. Although the currently optimized protocols had successfully yielded rBM-MSCs, the culture-mediated cellular senescence limits the growth of rBM-MSCs and its potential use in rat-based MSC research.

Concurrent Isolation of 3 Distinct Cardiac Stem Cell Populations From a Single Human Heart Biopsy

“…The MSC population exhibited slowest proliferation rate (doubling time: 119±35 hours) in agreement with prior studies using mid-passage MSC populations 34–36 . CPCs and EPCs show markedly faster proliferation rates (doubling times: CPC, 33±2 hours; EPC, 35±7 hours) (Figure 3D and 3E).…”

“…Key factors for successful propagation and expansion ex vivo are culture conditions and growth medium. Each cell type has its own preferential plating density and depending on cell type, differentiation and/or senescence can occur with prolonged culture 15, 34 or due to cell-cell contact if cultured to confluency 42, 43 . CPCs and EPCs are successfully cultured at 50–70% confluency.…”

Downregulation of MicroRNA-206 Alleviates the Sublethal Oxidative Stress-Induced Premature Senescence and Dysfunction in Mesenchymal Stem Cells via Targeting Alpl

“…However, replicative senescence and pathological conditions, such as serum deprivation, hypoxia, and oxidative stress inevitably compromised the therapeutic potential of MSCs by inducing the cells into senescence or apoptosis. For example, researchers have revealed that, caused by the occurrence of replicative senescence, MSCs in early passages showed enhancer active proliferation and shorter doubling time compared to MSCs in late passages [23][24][25]. Besides, Palumbo et al have confirmed that oxidative stress-induced senescence could reduce the regenerative potential of MSCs and lead to increased sensitivity of senescent MSCs to apoptotic cell death [26].…”

Characteristics of Full-Term Amniotic Fluid-Derived Mesenchymal Stem Cells in Different Culture Media

“…It could be possible that the lack of optimized serum batch selection for FBS and HS that support MSC colony formation and expansion rendered the observed non-conducive proliferation. The selection of serum batch for a particular cell type, especially stem cells, is crucial since the halted cellular expansion is often noticed due to senescence [15].…”