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

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.     

Molecular basis for premature senescence induced by surfactants in normal human cells

Sublethal doses of surfactants as exemplified by NP-40 clearly induce premature senescence in normal human cells. To understand molecular basis for this phenomenon, we tried to suppress it with use of various inhibitors. An inhibitor of p38 of the MAPK family almost completely suppressed growth arrest and morphological changes induced by surfactants; however, other inhibitors tested had no effect. Oleic acid, a weak inducer of premature senescence, was found to suppress the effect of NP-40. Fluorescein-labeled oleic acid rapidly bound to the cell surface, and this binding was clearly blocked by pre-treatment with surfactants, suggesting that surfactants and oleic acid compete for binding to the cell surface. Moderate concentrations of cycloheximide, an inhibitor of protein synthesis, also suppressed the senescent features induced by NP-40. These results suggest that surfactants activate p38 signaling pathway by binding to the cell surface, and induce cellular senescence.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.     

Yeast models of human mitochondrial diseases: from molecular mechanisms to drug screening

Mitochondrial diseases are rare diseases most often linked to energy in the form of ATP-depletion. The high number of nuclear- and mitochondrial-DNA-encoded proteins (>500), required for ATP production and other crucial mitochondrial functions such as NADH re-oxidation, explains the increasing number of reported disorders. In recent years, yeast has revealed to be a powerful model to identify responsible genes, to study primary effects of pathogenic mutations and to determine the molecular mechanisms leading to mitochondrial disorders. However, the clinical management of patients with mitochondrial disorders is still essentially supportive. Here we review some of the most fruitful yeast mitochondrial disorder models and propose to subject these models to highthroughput chemical library screening to prospect new therapeutic drugs against mitochondrial diseases.     

ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence

DNA DSBs are induced by IR or radiomimetic drugs such as doxorubicin. It has been indicated that cells from ataxia-telangiectasia patients are highly sensitive to radiation due to defects in DNA repair, but whether they have impairment in apoptosis has not been fully elucidated. A-T cells showed increased sensitivity to high levels of DNA damage, however, they were more resistant to low doses. Normal cells treated with combination of KU55933, a specific ATM kinase inhibitor, and doxorubicin showed increased resistance as they do in a similar manner to A-T cells. A-T cells have higher viability but more DNA breaks, in addition, the activations of p53 and apoptotic proteins (Bax and caspase-3) were deficient, but Akt expression was enhanced. A-T cells subsequently underwent premature senescence after treatment with a low dose of doxorubicin, which was confirmed by G2 accumulation, senescent morphology, and SA-β-gal positive until 15 days repair incubation. Finally, A-T cells are radio-resistant at low doses due to its defectiveness in detecting DNA damage and apoptosis, but the accumulation of DNA damage leads cells to premature senescence.     

In vitro strategies for human gametes production from stem cells

Embryonic stem cells (ESC) are self-renewal and pluripotent cells that are able to differentiate in vitro into several cell types in favourable conditions. Technical protocols for in vitro gametes production have been developed in mice and human species. The functionality of such differentiated cells is not always analysed and an early meiotic arrest is a current observation. These kinds of experimentations have also been tested from human induced pluripotent stem cells (IPSC). However, differentiation ends shortly at the primordial germ cell stage.     

Liposomal daunorubicin overcomes drug resistance in human breast,ovarian and lung carcinoma cells

Multi-drug resistance due in part to membrane pumps such as P-glycoprotein (Pgp) is a major clinical problem in human cancers. We tested the ability of liposomally-encapsulated daunorubicin (DR) to overcome resistance to this drug. A widely used breast carcinoma cell line originally selected for resistance in doxorubicin (MCF7ADR) was 4-fold resistant to DR compared to the parent MCF7 cells (IC50 79 nM vs. 20 nM). Ovarian carcinoma cells (SKOV3) were made resistant by retroviral transduction of MDR1 cDNA and selection in vinblastine. The resulting SKOV3MGP1 cells were 130-fold resistant to DR compared to parent cells (IC50 5700 nM vs. 44 nM). Small-cell lung carcinoma cells (H69VP) originally selected for resistance to etoposide were 6-fold resistant to DR compared to H69 parent cells (IC50 180 nM vs. 30 nM). In all three cases, encapsulation of DR in liposomes as Daunoxome (Gilead) did not change the IC50 of parent cells relative to free DR. However, liposomal DR overcame resistance in MCF7ADR breast carcinoma cells (IC50 20 nM), SKOV3MGP1 ovarian carcinoma cells (IC50 237 nM) and H69VP small-cell lung carcinoma cells (IC50 27 nM). Empty liposomes did not affect the IC50 for free DR in the three resistant cell lines, nor did empty liposomes affect the IC50 for other drugs that are part of the multi-drug resistance phenotype (etoposide, vincristine) in lung carcinoma cells. These data indicate the possible value of liposomal DR in overcoming Pgp-mediated drug resistance in human cancer.     

Generating human intestinal tissue from pluripotent stem cells in vitro

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1-3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.     

Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines - a report on the Vitrocellomics EU-project

Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance.     

Microencapsulated multicellular tumor spheroids as a novel in vitro model for drug screening

To generate multicellular tumor spheroids (MTS) based on human breast adenocarcinoma MCF-7 cells and to study them as a novel in vitro model for anticancer drug screening, a technique for cell microencapsulation in biocompatible alginate-chitosan microcapsules has been used in this study. Using the MTS based on the MCF-7 cells methotrexate (MTX) cytotoxicity has been investigated. A set of MTS with an average size of 150, 200 and 300 μm was prepared as a function of cultivation time. Cell viability was evaluated after MTS incubation in cultivation medium containing various MTX concentrations (1, 2, 10, 50 and 100 nM) for 48 h. MTS were shown to be markedly more resistant to MTX than the monolayer culture. The increase of the spheroid size was in correlation with the enhanced MTS resistance to MTX. Thus, at 100 nM MTX a number of viable cells in MTS with the size of 300 μm was 2.5-fold higher than that in the monolayer culture. It is suggested that the cells microencapsulated into MTS can better mimic cell behavior in small solid tumors compared to the monolayer culture. In the future MTS could be proposed as a novel in vitro model for anticancer drug screening.     

Inhibition of Mcl-1 promotes senescence in cancer cells: implications for preventing tumor growth and chemotherapy resistance

Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS). Overexpression of Mcl-1 in p53⁺ cell lines inhibits CIS. Conversely, downregulation of Mcl-1 makes cells sensitive to CIS. Surprisingly, downregulation of Mcl-1 in p53⁻ cells restored CIS to similar levels as p53⁺ cells. In all cases where senescence can be induced, we observed increased p21 expression. Moreover, we show that the domain of Mcl-1 responsible for its antisenescent effects is distinct from that known to confer its antiapoptotic qualities. In vivo we observe that downregulation of Mcl-1 can almost retard tumor growth regardless of p53 status, while overexpression of Mcl-1 in p53⁺ cells conferred resistance to CIS and promoted tumor outgrowth. In summary, our data reveal that Mcl-1 can inhibit CIS in both a p53-dependent and -independent manner in vitro and in vivo and that this Mcl-1-mediated inhibition can enhance tumor growth in vivo.     

Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO₂) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO₂) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.     

Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells

Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and aging-associated diseases. Senescence is also recognized as a mechanism for limiting the regenerative potential of stem cells and to protect cells from cancer development. The senescence program is realized through autocrine/paracrine pathways based on the activation of a peculiar senescence-associated secretory phenotype (SASP). We show here that conditioned media (CM) of senescent mesenchymal stem cells (MSCs) contain a set of secreted factors that are able to induce a full senescence response in young cells. To delineate a hallmark of stem cells SASP, we have characterized the factors secreted by senescent MSC identifying insulin-like growth factor binding proteins 4 and 7 (IGFBP4 and IGFBP7) as key components needed for triggering senescence in young MSC. The pro-senescent effects of IGFBP4 and IGFBP7 are reversed by single or simultaneous immunodepletion of either proteins from senescent-CM. The blocking of IGFBP4/7 also reduces apoptosis and promotes cell growth, suggesting that they may have a pleiotropic effect on MSC biology. Furthermore, the simultaneous addition of rIGFBP4/7 increased senescence and induced apoptosis in young MSC. Collectively, these results suggest the occurrence of novel-secreted factors regulating MSC cellular senescence of potential importance for regenerative medicine and cancer therapy.     

In vitro assays for new drug screening: comparison of a thymidine incorporation assay with the human tumor colony-forming assay

Because of technical limitations with the human tumor colony-forming assay (HTCFA), we determined the feasibility of using a thymidine incorporation assay (TIA) for new drug screening. Concordance between the TIA and the HTCFA was obtained (r = 0.840) with twenty coded compounds. Toxic agents without proven clinical efficacy were active in both assays, while non-toxic substances were inactive. Growth rates were higher with the TIA (75%) than with the HTCFA (45%), and the TIA could be completed in 6 days compared to 14-21 days with the HTCFA. We concluded that the TIA is a useful adjunct to in vivo tumor models for screening new anticancer agents.     

Cancer stem cells and tumor metastasis: first steps into uncharted territory

In several forms of human cancer, only a phenotypic subset of cancer cells, usually termed "cancer stem cells" (CSC), can initiate tumor growth when transplanted. In this issue of Cell Stem Cell, Hermann et al. (2007) analyze the relationship between CSC and tumor metastasis.