A Bruno-like gene is required for stem cell maintenance in planarians

The regenerative abilities of freshwater planarians are based on neoblasts, stem cells maintained throughout the animal's life. We show that a member of the Bruno-like family of RNA binding proteins is critical for regulating neoblasts in the planarian Schmidtea mediterranea. Smed-bruno-like (bruli) mRNA and protein are expressed in neoblasts and the central nervous system. Following bruli RNAi, which eliminates detectable Bruli protein, planarians initiate the proliferative response to amputation and form small blastemas but then undergo tissue regression and lysis. We characterize the neoblast population by using antibodies recognizing SMEDWI-1 and Histone H4 (monomethyl-K20) and cell-cycle markers to label subsets of neoblasts and their progeny. bruli knockdown results in a dramatic reduction/elimination of neoblasts. Our analyses indicate that neoblasts lacking Bruli can respond to wound stimuli and generate progeny that can form blastemas and differentiate; yet, they are unable to self-renew. These results suggest that Bruli is required for stem cell maintenance.     

Histone deacetylase activity is required for embryonic stem cell differentiation

Mammalian development requires commitment of cells to restricted lineages, which requires epigenetic regulation of chromatin structure. Epigenetic modifications were examined during in vitro differentiation of murine embryonic stem (ES) cells. Global histone acetylation, a euchromatin marker, declines dramatically within 1 day of differentiation induction and partially rebounds by day 2. Histone H3-Lys9 methylation, a heterochromatin marker, increases during in vitro differentiation. Conversely, the euchromatin marker H3-Lys4 methylation transiently decreases, then increases to undifferentiated levels by day 4, and decreases by day 6. Global cytosine methylation, another heterochromatin marker, increases slightly during ES cell differentiation. Chromatin structure of the Oct4 and Brachyury gene promoters is modulated in concert with their pattern of expression during ES cell differentiation. Importantly, prevention of global histone deacetylation by treatment with trichostatin A prevents ES cell differentiation. Hence, ES cells undergo functionally important global and gene-specific remodeling of chromatin structure during in vitro differentiation. genesis 38:32-38, 2004.     

Nuclear reprogramming by human embryonic stem cells

Embryonic stem cells have two unique properties. They are capable of indefinite self-renewal and, being pluripotent, they can differentiate into all possible cell types, including germ cells. A new study by Cowan et al. (2005) published in Science shows that human embryonic stem cells are able to reprogram the nuclei of fully differentiated human somatic cells, apparently conferring on them a pluripotent state.     

Brg1 is required for murine neural stem cell maintenance and gliogenesis

Epigenetic alterations in cell-type-specific gene expression control the transition of neural stem cells (NSCs) from predominantly neurogenic to predominantly gliogenic phases of differentiation, but how this switch occurs is unclear. Here, we show that brahma-related gene 1 (Brg1), an ATP-dependent chromatin remodeling factor, is required for the repression of neuronal commitment and the maintenance of NSCs in a state that permits them to respond to gliogenic signals. Loss of Brg1 in NSCs in conditional brg1 mutant mice results in precocious neuronal differentiation, such that cells in the ventricular zone differentiate into post-mitotic neurons before the onset of gliogenesis. As a result, there is a dramatic failure of astrocyte and oligodendrocyte differentiation in these animals. The ablation of brg1 in gliogenic progenitors in vitro also prevents growth-factor-induced astrocyte differentiation. Furthermore, proteins implicated in the maintenance of stem cells, including Sox1, Pax6 and Musashi-1, are dramatically reduced in the ventricular zones of brg1 mutant mice. We conclude that Brg1 is required to repress neuronal differentiation in NSCs as a means of permitting glial cell differentiation in response to gliogenic signals, suggesting that Brg1 regulates the switch from neurogenesis to gliogenesis.     

Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches

To directly test the requirement for hedgehog signaling in the telencephalon from early neurogenesis, we examined conditional null alleles of both the Sonic hedgehog and Smoothened genes. While the removal of Shh signaling in these animals resulted in only minor patterning abnormalities, the number of neural progenitors in both the postnatal subventricular zone and hippocampus was dramatically reduced. In the subventricular zone, this was partially attributable to a marked increase in programmed cell death. Consistent with Hedgehog signaling being required for the maintenance of stem cell niches in the adult brain, progenitors from the subventricular zone of floxed Smo animals formed significantly fewer neurospheres. The loss of hedgehog signaling also resulted in abnormalities in the dentate gyrus and olfactory bulb. Furthermore, stimulation of the hedgehog pathway in the mature brain resulted in elevated proliferation in telencephalic progenitors. These results suggest that hedgehog signaling is required to maintain progenitor cells in the postnatal telencephalon.     

p53 is required for etoposide-induced apoptosis of human embryonic stem cells

The molecular mechanisms controlling DNA-damage-induced apoptosis of human embryonic stem cells (hESC) are poorly understood. Here we investigate the role of p53 in etoposide-induced apoptosis. We show that p53 is constitutively expressed at high levels in the cytoplasm of hESC. Etoposide treatment results in a rapid and extensive induction of apoptosis and leads to a further increase in p53 and PUMA expression as well as Bax processing. p53 both translocates to the nucleus and associates with the mitochondria, accompanied by colocalization of Bax with Mcl1. hESC stably transduced with p53 shRNA display 80% reduction of endogenous p53 and exhibit an 80% reduction in etoposide-induced apoptosis accompanied by constitutive downregulation of Bax and an attenuated upregulation of PUMA. Our data further show that undifferentiated hESC that express Oct4 are much more sensitive to etoposide-induced apoptosis than their more differentiated progeny. Our study demonstrates that p53 is required for etoposide-induced apoptosis of hESC and reveals, at least in part, the molecular mechanism of DNA-damage-induced apoptosis in hESC.     

Establishment and maintenance of three Chinese human embryonic stem cell lines

To establish a potential resource for cell therapy and a developmental model for human diseases, we had isolated three Chinese human embryonic stem cell lines from the inner cell mass of human blastocysts in 2002. All the three cell lines were grown on mouse embryonic fibroblasts as feeder cells; one of these cell lines, chHES-3, has maintained its normal karyotype even after being cultured in vitro for more than 100 passages, after the standardization of mouse feeder preparation. Each hES cell line has been completely characterized. All the three cell lines expressed hES-specific markers and pluripotency-related genes. These cells maintained their normal karyotype during long-term culture and displayed a high telomerase activity. When differentiated in vivo and in vitro, the derivatives representing the three germ layers could be observed. Human leukocyte antigen, ABO blood type, and DNA fingerprinting were also performed to provide a unique identity to each cell line. By establishing these hES cell lines, we provide an appropriate in vitro model to study human development and regeneration. All the three cell lines can be obtained for research purposes by placing a request at our website at www.hescbank.cn.     

APC is required for muscle stem cell proliferation and skeletal muscle tissue repair

The tumor suppressor adenomatous polyposis coli (APC) is a crucial regulator of many stem cell types. In constantly cycling stem cells of fast turnover tissues, APC loss results in the constitutive activation of a Wnt target gene program that massively increases proliferation and leads to malignant transformation. However, APC function in skeletal muscle, a tissue with a low turnover rate, has never been investigated. Here we show that conditional genetic disruption of APC in adult muscle stem cells results in the abrogation of adult muscle regenerative potential. We demonstrate that APC removal in adult muscle stem cells abolishes cell cycle entry and leads to cell death. By using double knockout strategies, we further prove that this phenotype is attributable to overactivation of β-catenin signaling. Our results demonstrate that in muscle stem cells, APC dampens canonical Wnt signaling to allow cell cycle progression and radically diverge from previous observations concerning stem cells in actively self-renewing tissues.     

Staufen1 is expressed in preimplantation mouse embryos and is required for embryonic stem cell differentiation

Pluripotent mouse embryonic stem (mES) cells derived from the blastocyst of the preimplantation embryo can be induced to differentiate in vitro along different cell lineages. However the molecular and cellular factors that signal and/or determine the expression of key genes, and the localisation of the encoded proteins, during the differentiation events are poorly understood. One common mechanism by which proteins can be targeted to specific regions of the cell is through the asymmetric localisation of mRNAs and Staufen, a double-stranded RNA binding protein, is known to play a direct role in mRNA transport and localisation. The aims of the present study were to describe the expression of Staufen in preimplantation embryos and mES cells and to use RNA interference (RNAi) to investigate the roles of Staufen1 in mES cell lineage differentiation. Western blotting and immunocytochemistry demonstrated that Staufen is present in the preimplantation mouse embryo, pluripotent mES cells and mES cells stimulated to differentiate into embryoid bodies, but the Staufen staining patterns did not support asymmetric distribution of the protein. Knockdown of Staufen1 gene expression in differentiating mES cells reduced the synthesis of lineage-specific markers including Brachyury, alpha-fetoprotein (AFP), PAX-6, and Vasa. There was however no significant change in either the gene expression of Nanog and Oct4, or in the synthesis of SSEA-1, all of which are key markers of pluripotency. These data indicate that inhibition of Staufen1 gene expression by RNAi affects an early step in mES cell differentiation and suggest a key role for Staufen in the cell lineage differentiation of mES cells.