Senescent Growth Arrest in Mesenchymal Stem Cells Is Bypassed by Wip1-Mediated Downregulation of Intrinsic Stress Signaling Pathways

Abstract

Human mesenchymal stem cells (hMSCs) have been widely studied as a source of primary adult stem cells for cell therapy because of their multidifferentiation potential; however, the growth arrest (also known as ''premature senescence'') often found in hMSCs cultured in vitro has been a major obstacle to the in-depth characterization of these cells. In addition, the inability to maintain constant cell growth hampers the development of additional genetic modifications aimed at achieving desired levels of differentiation to specific tissues; however, the molecular mechanisms that govern this phenomenon remain unclear, with the exception of a few studies demonstrating that induction of p16INK4a is responsible for this senescence-like event. Here, we observed that the premature growth arrest in hMSCs occurs in parallel with the induction of p16INK4a, following abrogation of inhibitory phosphorylation of retinoblastoma protein. These stress responses were concurrent with increased formation of reactive oxygen species (ROSs) from mitochondria and increased p38 mitogen-activated protein kinase (MAPK) activity. The introduction of Wip1 (wildtype p53 inducible phosphatase-1), a well-studied stress modulator, significantly lowered p16INK4a expression and led to p38 MAPK inactivation, although it failed to affect the levels of ROSs. Moreover, the suppression of stress responses by Wip1 apparently extended the life span of hMSCs, compared with control conditions, while maintaining their multilineage differentiation potential. Based on these results, we suggest that senescent growth arrest in hMSCs may result from activation of stress signaling pathways and consequent onset of stress responses, due in part to ROS production during prolonged in vitro culture.

Umbilical Cord Mesenchymal Stromal Cells Affected by Gestational Diabetes Mellitus Display Premature Aging and Mitochondrial Dysfunction

“…Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was performed with Power SYBR Ò Green PCR Master Mix (4367659; Applied Biosystems, Foster City, CA) on a MyiQ Single Color RealTime PCR Detection System (Bio-Rad, Hercules, CA) using the following primer sets: CD105, 5¢-GCCAGCATTG TCT CACTTCA TG-3¢ and 5¢-GCAACAAGCT CTTTCTTTAG TACCA-3¢; CD90, TCAGGAAATG GCTTTTCCCA and TCCTCAATGA GATGCCATAA GCT; CD73, CGCAA CAATG GCACAATTAC and CAGGTTTTCG GGAAA GATCA; PPARg, GCAGTGGGGA TGTCTCATAA TGC and CAGGGGGGTG ATGTGTTTGA A; alkaline phosphatase (ALP), GGACATGCAG TACGAGCTGA and TGTCTTCCGA GGAGGTCAAG; osteocalcin (OC), CAC TCCTCGC CCTATTGGC and CCCTCCTGCT TGGACA CAAA G; collagen type 1 alpha 1 (Col1a1), TAGGGTC TAG ACATGTTCAG CTTTGT and CCGTTCTGTA CGCAGGTGAT T; p16, GCCTTTTCAC TGTGTTGGAG and TGCCATTTGC TAGCAGTGTG [22]; p21, ACTT CGCCTG GGAGCGTGTG and CGTTTGGAGT GGTA GAAATC TGTCAT; p27, CGGCTAACTC TGAGGAC ACG C and ATTTGGGGAA CCGTCTGAAA CATT [23]; and GAPDH, GAAGGTGAAG GTCGGAGTC and GAA GATGGTG ATGGGATTTC. The thermal cycling conditions consisted of an initial denaturation step at 95°C for 5 min, then 50 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by a final extension step at 72°C for 10 min.…”

H2O2Accumulation Mediates Differentiation Capacity Alteration, But Not Proliferative Decline, in Senescent Human Fetal Mesenchymal Stem Cells

“…However, ex vivo expansion to satisfy the immense cell numbers required for clinical use often results in senescence (27,31). We found that PDMCs, a population of multilineage MSCs derived from fetal placental tissue, are far more proliferative in vitro than adult BM-MSCs.…”

“…Many reports have shown that ROS accumulate in senescence cells and accompany cell cycle arrest (27). We therefore first analyzed the ROS levels of L-PDMCs by staining for 2¢,7¢-dichlorodihydrofluorescein diacetate (DCF-DA), which detects hydrogen peroxide (H 2 O 2 ) and superoxide anion levels, and hydroethidine (HE), which stains for superoxide anion.…”

Down-regulation of Wild-type p53-induced Phosphatase 1 (Wip1) Plays a Critical Role in Regulating Several p53-dependent Functions in Premature Senescent Tumor Cells

“…In fact, although normal senescent cells arrest with a G 1 DNA content, premature senescent tumor cells characteristically arrest in the G 2 phase of the cell cycle (29), likely due to defective G 1 checkpoint of cancer cells (43). Accordingly, it has been shown that introduction of Wip1 in normal human mesenchymal stem cells (G 1 arrested) allows bypass of senescence and extends cellular life span (44).…”