Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells

Abstract

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated β-galactosidase (SA-β-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72 h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity.

Fig. 1

Mild (45 °C, 10 min) and sublethal heat shock (45 °C, 30 min) did not induce apoptosis in human MSCs. a Flow cytometry assay of apoptosis with annexin V/PI. b Apoptosis measured by counting cells with fragmented nuclei

Fig. 2

Heat shock (45 °C, 30 min) induced MSC premature senescence. a Growth curves of heated and unheated cells. MSCs exposed to heat shock stop division. b Immunofluorescence assay of cell proliferation with anti-Ki-67antibodies. Only single cells are Ki-67-positive 72 h after heat shock. c FACS assay of cell cycle distribution. MSCs that were heated at 45 °C for 30 min and returned to the normal culture conditions exhibited cell cycle arrest in G₂/M phases. d SA-β-X-gal staining during cell recovery from sublethal HS. The number of X-gal-positive cell increased. e Quantitative assay of X-gal-positive cells. f Expression of cyclin-dependent kinase inhibitor p21 in MSCs exposed to HS. p21 expression is increased along with the augment in X-gal-positive cells (d)

Fig. 3

Expression of heat shock proteins in MSCs subjected to heat shock. Cells were exposed to heat shock, allowed to recover for indicated time, and assayed by PCR (a), immunoblotting (b), or immunofluorescence (c). Densitometry data below each band represent “fold change” after normalization with a loading control (a, b). Hsp70 induced in MSCs by heat shock is localized mostly in the nuclei (c)

Fig. 4

MSCs that survived sublethal heat shock exhibit replicative senescence (a, b) and retain normal karyotype (c). MSCs at the seventh passage were heated (45 °C, 30 min) and returned to standard culture conditions. a X-gal staining of parental cells. b X-gal staining of cells that survived heat shock

Fig. 5

MSCs that survived sublethal heat shock display multilineage potential. Survived cells were assayed for MSC CD marker expression (a) and capacity for differentiation into adipocytes (c), osteoblasts (d), and neural cells (e). b Initial (control) cells were not subjected to differentiation stimuli