Apoptosis In Embryonic Stem Cells Research Articles

Linc-NSC affects cell differentiation, apoptosis and proliferation in mouse neural stem cells and embryonic stem cells in vitro and in vivo

BackgroundStem cell therapy is a promising therapeutic strategy. In a previous study, we evaluated tumorigenicity by the stereotactic transplantation of neural stem cells (NSCs) and embryonic stem cells (ESCs) from experimental mice. Twenty-eight days later, there was no evidence of tumor formation or long-term engraftment in the NSCs transplantation group. In contrast, the transplantation of ESCs caused tumor formation; this was due to their high proliferative capacity. Based on transcriptome sequencing, we found that a long intergenic non-coding RNA (named linc-NSC) with unknown structure and function was expressed at 1100-fold higher levels in NSCs than in ESCs. This finding suggested that linc-NSC is negatively correlated with stem cell pluripotency and tumor development, but positively correlated with neurogenesis. In the present study, we investigated the specific role of linc-NSC in NSCs/ESCs in tumor formation and neurogenesis.MethodsWhole transcriptome profiling by RNA sequencing and bioinformatics was used to predict lncRNAs that are widely associated with enhanced tumorigenicity. The expression of linc-NSC was assessed by quantitative real-time PCR. We also performed a number of in vitro methods, including cell proliferation assays, differentiation assays, immunofluorescence assays, flow cytometry, along with in vivo survival and immunofluorescence assays to investigate the impacts of linc-NSC on tumor formation and neurogenesis in NSCs and ESCs.ResultsFollowing the knockdown of linc-NSC in NSCs, NSCs cultured in vitro and those transplanted into the cortex of mice showed stronger survival ability (P < 0.0001), enhanced proliferation(P < 0.001), and reduced apoptosis (P < 0.05); the opposite results were observed when linc-NSC was overexpressed in ESCs. Furthermore, the overexpression of linc-NSC in ECSs induced enhanced apoptosis (P < 0.001) and differentiation (P < 0.01), inhibited tumorigenesis (P < 0.05) in vivo, and led to a reduction in tumor weight (P < 0.0001).ConclusionsOur analyses demonstrated that linc-NSC, a promising gene-edited target, may promote the differentiation of mouse NSCs and inhibit tumorigenesis in mouse ESCs. The knockdown of linc-NSC inhibited the apoptosis in NSCs both in vitro and in vivo, and prevented tumor formation, revealing a new dimension into the effect of lncRNA on low survival NSCs and providing a prospective gene manipulation target prior to transplantation. In parallel, the overexpression of linc-NSC induced apoptosis in ESCs both in vitro and in vivo and attenuated the tumorigenicity of ESCs in vivo, but did not completely prevent tumor formation.

Microgravity Inhibits Cell Proliferation and Promotes Senescence and Apoptosis in Embryonic Stem Cells

With advancements in deep space exploration missions, long-term spaceflights pose potential hazards to the reproductive and developmental functions of astronauts. Embryonic stem cells (ESCs), which are crucial to the development and growth of individual organisms, are observably altered by a microgravity environment. However, the role and mechanisms of microgravity in other activities of ESCs are still unclear. Here, mouse embryonic stem cells (mESCs) were used to investigate and understand the effect of microgravity on their activities. Combined with the SJ-10 satellite and random position machine, which were utilized for spaceflight and microgravity simulation, respectively, the bioinformatic tools were also used to assess the effect that microgravity might have on mESC activities. Based on differentially expressed genes (DEGs) analysis, 114 DEGs were significantly up-regulated and 859 DEGs were significantly down-regulated in mESCs after being subjected to spaceflight. The activities, such as cell proliferation, senescence, and apoptosis, were selected and confirmed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. It demonstrated a reduced proliferation capability of mESCs but increased the number of senescent and apoptotic cells after being subjected to simulated microgravity. Real-time polymerase chain reaction analysis of the screened activity-related DEGs demonstrated approximately consistent trends of these gene expressions in both spaceflight and simulated microgravity, as was predicted by bioinformatics analysis. Overall, these findings suggest that microgravity inhibits the proliferation of mESCs and induces senescence and apoptosis, shedding light on the impact of microgravity on the fundamental functions of mESCs in reproductive and embryonic development.

COL1A1 regulates the apoptosis of embryonic stem cells by mediating the PITX1/TBX4 signaling.

The purpose of this study is to explore the regulatory function of COL1A1 against the apoptosis of embryonic stem cells (ESCs) and the potential function in congenital talipes equinovarus (CTEV). Muscle tissues were collected from 20 children with CTEV and 20 children without CTEV, followed by detecting the expression of COL1A1 using the RT-PCR method. COL1A1 was knocked down in H1 and H9 human ESCs using the RNA interference technology, followed by determining the level of COL1A1, PITX1, TBX4, HOXD10, Fas, FasL, and Bax using the Western blotting assay. COL1A1 was found markedly upregulated in muscle tissues of CTEV children. In H1 and H9 human ESCs, compared to the empty vector, COL1A1, PITX1, TBX4, HOXD10, Fas, FasL, and Bax were found notably downregulated after transfected with the siRNA targeting COL1A1. COL1A1 induced the apoptosis of ESCs by mediating the PITX1/TBX4 signaling and might be a potential target for treating CTEV.

Developmental Acquisition of p53 Functions.

Remarkably, the p53 transcription factor, referred to as “the guardian of the genome”, is not essential for mammalian development. Moreover, efforts to identify p53-dependent developmental events have produced contradictory conclusions. Given the importance of pluripotent stem cells as models of mammalian development, and their applications in regenerative medicine and disease, resolving these conflicts is essential. Here we attempt to reconcile disparate data into justifiable conclusions predicated on reports that p53-dependent transcription is first detected in late mouse blastocysts, that p53 activity first becomes potentially lethal during gastrulation, and that apoptosis does not depend on p53. Furthermore, p53 does not regulate expression of genes required for pluripotency in embryonic stem cells (ESCs); it contributes to ESC genomic stability and differentiation. Depending on conditions, p53 accelerates initiation of apoptosis in ESCs in response to DNA damage, but cell cycle arrest as well as the rate and extent of apoptosis in ESCs are p53-independent. In embryonic fibroblasts, p53 induces cell cycle arrest to allow repair of DNA damage, and cell senescence to prevent proliferation of cells with extensive damage.

Tumor Necrosis Factor Alpha Signaling and Organogenesis.

Tumor necrosis factor alpha (TNF-α) plays important roles in processes such as immunomodulation, fever, inflammatory response, inhibition of tumor formation, and inhibition of viral replication. TNF-α and its receptors are ubiquitously expressed in developing organs and they regulate the survival, proliferation, and apoptosis of embryonic stem cells (ESCs) and progenitor cells. TNF-α is an important inflammatory factor that also regulates the inflammatory response during organogenesis, and its cytotoxic effects can interfere with normal developmental processes, even leading to the onset of diseases. This review summarizes the various roles of TNF-α in organogenesis in terms of its secreting pattern, concentration-dependent activities, and interactions with other signaling pathways. We also explored new potential functions of TNF-α.

Resveratrol-induced p53 activation is associated with autophagy in mouse embryonic stem cells

Resveratrol is a natural polyphenol with several therapeutic effects, in particular, inducing p53-dependent cell cycle arrest and/or apoptosis in tumor cells. Resveratrol-induced p53 activation may trigger differentiation and apoptosis in embryonic stem cells (ESCs). We show that resveratrol activates p53 that is negatively regulated by SIRT1 deacetylation on Lys379 and positively by AMPK phosphorylation on Ser15 in mouse ESCs (mESCs). Surprisingly, the resveratrol-activated p53 is not associated with either G1/S cell cycle checkpoint or apoptosis in mESCs. Instead, it stimulates autophagy in a transcriptional-dependent manner involving up-regulation of dram1 gene expression. This study demonstrates a novel mechanism of resveratrol-dependent p53 activation in mESCs.

Transplantation of CREG modified embryonic stem cells improves cardiac function after myocardial infarction in mice

Engraftment of embryonic stem cells (ESC) has been proposed as a potential therapeutic approach for post-infarction cardiac dysfunction. However, only mild function improvement has been achieved due to low survival rate and paracrine dysfunction of transplanted stem cells. Cellular repressor of E1A stimulated genes (CREG) has been reported to be a secreted glycoprotein implicated in promoting survival and differentiation of many cell types. Therefore we hypothesized that transplantation of genetically modified ESC with CREG (CREG-ESC) can improve cardiac function after myocardial infarction in mice. A total of 2 × 105 CREG-ESC or EGFP-ESC were engrafted into the border zone in a myocardial infarction model in mice. Cardiac function, infarct size and fibrosis at 4 weeks, survival of transplanted ESC, apoptosis and cytokine level of heart tissue, and teratoma formation were assessed in vivo. Apoptosis of ESC under inflammatory stimuli and cardiac differentiation of ESC were investigated in vitro. After 4 weeks, we found transplantation of CREG-ESC could significantly improve cardiac function, ameliorate cardiac remodeling, and reduce infarct size and fibrosis area. Transplantation of CREG-ESC remarkably increased ESC survival in the border zone and inhibited apoptosis of cardiomyocytes. Furthermore, the decrease of inflammatory factors (IL-1β, IL-6 and TNF-α) and increase of anti-inflammatory factors (TGF-β, bFGF and VEGF165) in the border zone were higher in CREG-ESC transplanted hearts. Safety evaluation showed that all transplantation at 2 × 105 per heart dose produced no teratoma. Surprisingly, the mice with 3.0 × 106 CREG-ESC transplantation was demonstrated teratoma free without cardiac rhythm disturbances in contrast to 100% teratoma formation and rhythm abnormality for the same dose of EGFP-ESC transplantation. In addition, overexpression of CREG inhibits ESC apoptosis and enhanced their differentiation into cardiomyocytes in vitro. Transplantation of CREG-modified ESC exhibits a favorable survival pattern in infarcted hearts, which translates into a substantial preservation of cardiac function after acute myocardial infarction.

Akt3 is responsible for the survival and proliferation of embryonic stem cells.

ABSTRACTThe phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway plays an important role in regulating cell proliferation, metabolism, and survival. However, the distinct roles of Akt isoforms (Akt1, Akt2, and Akt3) in pluripotent stem cell maintenance are not fully defined. Using mouse embryonic stem cells (ESCs), we show that direct inhibition of Akt activity leads to ESC apoptosis. The Akt3, but not Akt1 or Akt2, activity specifically regulates this effect. Inhibiting Akt3 also leads to a cell cycle arrest at G1 phase. These regulatory roles of Akt3 are dependent on its kinase activity. Blocking the expression of Akt1 plus Akt2 in ESCs does not affect cell survival or proliferation, although blocking Akt1 aggravates the apoptotic effect induced by depletion of Akt3. We further show that blocking Akt3 in ESCs results in significant nuclear accumulation of p53, as well as the activation of its downstream targets, such as Mdm2, p21, and Fas. Inhibiting p53 and its downstream targets partially rescued the effects caused by Akt3-depletion. Our results revealed an Akt3 isoform-specific mechanism for ESC survival and proliferation involving the control of p53 activity.

An Apela RNA-Containing Negative Feedback Loop Regulates p53-Mediated Apoptosis in Embryonic Stem Cells

Maintaining genomic integrity is of paramount importance to embryonic stem cells (ESCs), as mutations are readily propagated to daughter cells. ESCs display hypersensitivity to DNA damage-induced apoptosis (DIA) to prevent such propagation, although the molecular mechanisms underlying this apoptotic response are unclear. Here, we report that the regulatory RNA Apela positively regulates p53-mediated DIA. Apela is highly expressed in mouse ESCs and is repressed by p53 activation, and Apela depletion compromises p53-dependent DIA. Although Apela contains a coding region, this coding ability is dispensable for Apela's role in p53-mediated DIA. Instead, Apela functions as a regulatory RNA and interacts with hnRNPL, which prevents the mitochondrial localization and activation of p53. Together, these results describe a tri-element negative feedback loop composed of p53, Apela, and hnRNPL that regulates p53-mediated DIA, and they further demonstrate that regulatory RNAs add a layer of complexity to the apoptotic response of ESCs after DNA damage.

Concise review: androgen receptor differential roles in stem/progenitor cells including prostate, embryonic, stromal, and hematopoietic lineages.

Stem/progenitor (S/P) cells are special types of cells that have the ability to generate tissues throughout their entire lifetime and play key roles in the developmental process. Androgen and the androgen receptor (AR) signals are the critical determinants in male gender development, suggesting that androgen and AR signals might modulate the behavior of S/P cells. In this review, we summarize the AR effects on the behavior of S/P cells, including self-renewal, proliferation, apoptosis, and differentiation in normal S/P cells, as well as proliferation, invasion, and self-renewal in prostate cancer S/P cells. AR plays a protective role in the oxidative stress-induced apoptosis in embryonic stem cells. AR inhibits the self-renewal of embryonic stem cells, bone marrow stromal cells, and prostate S/P cells, but promotes their differentiation except for adipogenesis. However, AR promotes the proliferation of hematopoietic S/P cells and stimulates hematopoietic lineage differentiation. In prostate cancer S/P cells, AR suppresses their self-renewal, metastasis, and invasion. Together, AR differentially influences the characteristics of normal S/P cells and prostate cancer S/P cells, and targeting AR might improve S/P cell transplantation therapy, especially in embryonic stem cells and bone marrow stromal cells.

Functional Analysis of Tcl1 Using Tcl1-Deficient Mouse Embryonic Stem Cells

Tcl1 is highly expressed in embryonic stem (ES) cells, but its expression rapidly decreases following differentiation. To assess Tcl1’s roles in ES cells, we generated Tcl1-deficient and -overexpressing mouse ES cell lines. We found that Tcl1 was neither essential nor sufficient for maintaining the undifferentiated state. Tcl1 is reported to activate Akt and to enhance cell proliferation. We found that Tcl1 expression levels correlated positively with the proliferation rate and negatively with the apoptosis of ES cells, but did not affect Akt phosphorylation. On the other hand, the phosphorylation level of β-catenin decreased in response to Tcl1 overexpression. We measured the β-catenin activity using the TOPflash reporter assay, and found that wild-type ES cells had low activity, which Tcl1 overexpression enhanced 1.8-fold. When the canonical Wnt signaling is activated by β-catenin stabilization, it reportedly helps maintain ES cells in the undifferentiated state. We then performed DNA microarray analyses between the Tcl1-deficient and -expressing ES cells. The results revealed that Tcl1 expression downregulated a distinct group of genes, including Ndp52, whose expression is very high in blastocysts but reduced in the primitive ectoderm. Based on these results, we discuss the possible roles of Tcl1 in ES cells.

Delphinidin prevents hypoxia-induced mouse embryonic stem cell apoptosis through reduction of intracellular reactive oxygen species-mediated activation of JNK and NF-κB, and Akt inhibition

Delphinidin, gallic acid, betulinic acid, and ursolic acid, which are bio-active ingredients in a variety of fruits, vegetables, and herbs, have potent antioxidant activity and various biological activities. However, it is not clear whether these bio-active ingredients can significantly contribute to the protection of embryonic stem (ES) cells from hypoxia-induced apoptosis. In the present study, hypoxia-induced ES cells apoptosis with time, which were abrogated by pretreatment with all ingredients. Hypoxia-induced ROS generation was blocked by pretreatment with all ingredients in a dose-dependent manner, with the maximum ROS scavenging effect observed for delphinidin. Hypoxia increased phosphorylation of JNK and NF-κB were blocked by pretreatment of delphinidin as well as NAC. Hypoxia decreased phosphorylation of Akt(thr308) and (ser473); these decreases were reversed by pretreatment with delphinidin or NAC. However, Akt inhibition did not affect NF-κB phosphorylation. Delphinidin attenuated the hypoxia-induced increase in Bax, cleaved caspase-9, cleaved caspase-3, and decrease in Bcl-2, which were diminished by pretreatment of Akt inhibitor. Hypoxia induced Bax translocation from the cytosol to mitochondria. Furthermore, hypoxia induced mitochondria membrane potential loss and cytochrome c release in cytosol, which were blocked by delphinidin pretreatment. Hypoxia induced cleavage of procaspase-9 and procaspase-3 which were blocked by delphinidin or SP600125, but Akt inhibitor abolished the protection effect of delphinidin. Moreover, inhibition of JNK and NF-κB abolished hypoxia-induced ES cell apoptosis and inhibition of Akt attenuated delphinidin-induced blockage of apoptosis. The results indicate that delphinidin can prevent hypoxia-induced apoptosis of ES cells through the inhibition of JNK and NF-κB phosphorylation, and restoration of Akt phosphorylation.

Systems Biology Approach Identifies the Kinase Csnk1a1 as a Regulator of the DNA Damage Response in Embryonic Stem Cells

In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits β-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.

High sensitivity of embryonic stem cells to proteasome inhibitors correlates with low expression of heat shock protein and decrease of pluripotent cell marker expression

The ubiquitin-proteasome system is a major proteolytic system for nonlysosomal degradation of cellular proteins. Here, we investigated the response of mouse embryonic stem (ES) cells under proteotoxic stress. Proteasome inhibitors induced expression of heat shock protein 70 (HSP70) in a concentration- and time-dependent manner, and also induced apoptosis of ES cells. Importantly, more apoptotic cells were observed in ES cells compared with other somatic cells. To understand this phenomenon, we further investigated the expression of HSP70 and pluripotent cell markers. HSP70 expression was more significantly increased in somatic cells than in ES cells, and expression levels of pluripotent cell markers such as Oct4 and Nanog were decreased in ES cells. These results suggest that higher sensitivity of ES cells to proteotoxic stress may be related with lower capacity of HSP70 expression and decreased pluripotent cell marker expression, which is essential for the survival of ES cells.

Apoptosis: Reprogramming and the Fate of Mature Cells

Apoptosis is essential for embryogenesis, organ metamorphosis, and tissue homeostasis. In embryonic stem cells, self-renewal is balanced with proliferative potential, inhibition of differentiation, and prevention of senescence and apoptosis. Growing evidence supports the role of apoptosis in self-renewal, differentiation of pluripotent stem cells, and dedifferentiation (reprogramming) of somatic cells. In this paper we discuss the multiple roles of apoptosis in embryonic stem cells (ESCs) and reprogramming of differentiated cells to pluripotency. The role of caspases and p53 as key effectors in controlling the generation of iPSC is emphasized. Remarkably, the complication of apoptosis arising during reprogramming may provide insights into technical improvements for derivation of iPSC from senescent cells as a tool for modeling aging-related diseases.

Influence of nerve growth factor on proliferation, differentiation and apoptosis of neural stem cells

Objective To evaluate the influence of different doses of nerve growth factor (NGF) on the proliferation, differentiation and apoptosis of embryonic rat neural stem cells. Methods The neural stem cells were isolated from subventricular zone of rat embryonic brains and cultured in serum-free medium DMEM/F12 with B27. NGF with different doses of 10, 20, 50 μg/L were added into the different experimental groups. The proliferation ability of the cells was measured by methyl thiazol tetrazolium (MTT) assay at 10th day. All cells were collected in plates at 7th day. Streptvidin-peroxidase immunocytochemistry was used to detect the expression of neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) , and the number of NSE( + ) and GFAP( + ) cells was counted. The apoptosis rat at 5th day after culture was assessed. Results There was no significant difference in the the number of neurospheres between 10 μg/L NGF group (0. 150 ±0. 025) and positive control group (0. 158 ±0. 017), but there was significant difference between 10 μg/L NGF group and blank control group (0. 128 ±0. 015). In 10, 20,50 μg/L N GF groups, the rate of NSE ( + ) cells was ( 15. 21 ± 1.27) %, (20. 64 ± 2. 61 )%, (24. 20 ± 3. 10) %, and that of GFAP( + ) cells was (27.98 ± 2. 35 ) %, ( 28.94 ± 2. 84 ) %, ( 30. 79 ± 3.23 ) % respectively. The rate of cells differentiation was increased with the dose of NGF added[( FNsE (+ ) = 3.47,FGFAp( + ) = 3. 47 ,PGFAP( + ) ＜ 0. 05)]. Flow cytometry revealed that in 20, 50 μg/L NGF groups the apoptosis rate was significantly increased as compared with other groups. The proliferation index (PI) in 10 μg/L NGF group was higher than other groups. Conclusion NGF at a rational dose can promote the proliferation and differentiation of human NSC. Low dose of NGF mainly facilitates the proliferation of NSCs, and high dose of NGF shows obvious influence on proliferation of NSC. Key words: Neural growth factor; Neural stem cells; Proliferation; Differentiation

Essential and overlapping functions for mammalian Argonautes in microRNA silencing

MicroRNA (miRNA) silencing fine-tunes protein output and regulates diverse biological processes. Argonaute (Ago) proteins are the core effectors of the miRNA pathway. In lower organisms, multiple Agos have evolved specialized functions for distinct RNA silencing pathways. However, the roles of mammalian Agos have not been well characterized. Here we show that mouse embryonic stem (ES) cells deficient for Ago1-4 are completely defective in miRNA silencing and undergo apoptosis. In miRNA silencing-defective ES cells, the proapoptotic protein Bim, a miRNA target, is increased, and up-regulation of Bim is sufficient to induce ES cell apoptosis. Expression of activated Akt inhibits Bim expression and partially rescues the growth defect in Ago-deficient ES cells. Furthermore, reintroduction of any single Ago into Ago-deficient cells is able to rescue the endogenous miRNA silencing defect and apoptosis. Consistent with this, each Ago is functionally equivalent with bulged miRNA duplexes for translational repression, whereas Ago1 and Ago2 appear to be more effective at utilizing perfectly matched siRNAs. Thus, our results demonstrate that mammalian Agos all contribute to miRNA silencing, and individual Agos have largely overlapping functions in this process.

Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity

Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast growth factor 4 (FGF4) deprivation induces differentiation of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase 1 (CDK1), the enzyme required to enter mitosis, induced differentiation of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in embryonic stem cells, revealing that inactivation of CDK1 triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK1 inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p21/CIP1. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK1, while p21 suppressed expression of the checkpoint protein kinase CHK1, thereby preventing induction of apoptosis. Furthermore, Cdk2(-/-) TS cells revealed that CDK2 is required for endoreduplication when CDK1 is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK1 with concomitant suppression of the DNA damage response by p21.

Mouse Hematopoietic Stem Cells, Unlike Human and Mouse Embryonic Stem Cells, Exhibit Checkpoint-Apoptosis Coupling.

Cell cycle checkpoints guarantee that cells move through the events of the cell cycle in the appropriate manner. The mitotic spindle checkpoint, also known as the spindle assembly checkpoint (SAC), helps to ensure the proper segregation of chromosomes into daughter cells during mitosis. Our lab recently reported on the condition of the SAC in both mouse and human embryonic stem cells (ESCs). We found that ESCs do not initiate apoptosis when the SAC is activated, which allowed these cells to tolerate a polyploid state resulting from the aberrant mitosis (Mantel et al. Blood. 109: 4518–4527. 2007). These results lead us to conclude that the spindle checkpoint is uncoupled from apoptosis in ESCs. Knowing whether adult tissue specific stem/progenitor cells, such as hematopoietic stem cells (HSCs), have checkpoints which are uncoupled from apoptosis is extremely important information. If HSCs were to manifest such checkpoint uncoupling as that which we defined for ESCs, this might present a problem for the ex-vivo expansion and transplantation of HSCs. Using multiparametric permeablized cell flow cytometric analysis, we found the mitotic spindle checkpoint to be functional in primary murine sca 1+/c-kit+/lin- cells (LSK cells), a population highly enriched in primitive hematopoietic stem/progenitor cells. Using nocodazole, which exerts its affect by depolymerizing microtubules, we were able to activate the spindle checkpoint in low density mononuclear cells collected from murine bone marrow. Through flow cytometric analysis of the LSK cells in the mononuclear fraction, we were able to determine that spindle checkpoint activation in LSK cells resulted in a cell cycle arrest in mitosis, which was determined by DNA content of the cells, and eventually this arrest lead to cell death via apoptosis, as indicated by caspase-3 activation. This behavior is unlike that of ESCs, which exit mitosis and become polyploidy after prolonged spindle checkpoint activation. Thus the mitotic spindle checkpoint appears to be coupled to apoptosis in this particular set of tissue specific stem/progenitor cells, which lessens the possibility that ex-vivo expansion of hematopoietic stem cells will result in abnormalities to these cells that may give rise to disease initiation or progression after their transplantation.

Protective effects of resveratrol on ethanol-induced apoptosis in embryonic stem cells and disruption of embryonic development in mouse blastocysts

Previous studies have established that ethanol induces apoptosis, but the precise molecular mechanisms are currently unclear. Here, we show that 0.3–1.0% (w/v) ethanol induces apoptosis in mouse blastocysts and that resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties, prevents ethanol-induced apoptosis and inhibition of cell proliferation. Moreover, ethanol-treated blastocysts show normal levels of implantation on culture dishes in vitro but a reduced ability to reach the later stages of embryonic development. Pretreatment with resveratrol prevented ethanol-induced disruption of embryonic development in vitro and in vivo. In an in vitro cell-based assay, we further found that ethanol increases the production of reactive oxygen species in ESC-B5 embryonic stem cells, leading to an increase in the intracellular concentrations of cytoplasmic free Ca 2+ and NO, loss of mitochondrial membrane potential, mitochondrial release of cytochrome c, activation of caspase-9 and -3, and apoptosis. These changes were blocked by pretreatment with resveratrol. Based on these results, we propose a model for the protective effect of resveratrol on ethanol-induced cell injury in blastocysts and ESC-B5 cells.