Keeping an Eye on Retinoblastoma Control of Human Embryonic Stem Cells

Department of Pediatrics, Stanford Medical School, Stanford, California 94305, USA.
Journal of Cellular Biochemistry (Impact Factor: 3.26). 12/2009; 108(5):1023-30. DOI: 10.1002/jcb.22342
Source: PubMed


Human embryonic stem cells (hESCs) hold great promise in regenerative medicine. However, before the full potential of these cells is achieved, major basic biological questions need to be addressed. In particular, there are still gaps in our knowledge of the molecular mechanisms underlying the derivation of hESCs from blastocysts, the regulation of the undifferentiated, pluripotent state, and the control of differentiation into specific lineages. Furthermore, we still do not fully understand the tumorigenic potential of hESCs, limiting their use in regenerative medicine. The RB pathway is a key signaling module that controls cellular proliferation, cell survival, chromatin structure, and cellular differentiation in mammalian cells. Members of the RB pathway are important regulators of hESC biology and manipulation of the activity of this pathway may provide novel means to control the fate of hESCs. Here we review what is known about the expression and function of members of the RB pathway in hESCs and discuss areas of interest in this field.

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Available from: Julien Sage
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    • "Notably, no significant change in the growth rate of the 4F-infected MEFs was observed upon Rb knockdown (Figure 2A), as was previously observed in Rb-deficient MEFs (Dannenberg et al., 2000; Sage et al., 2000) (see below). Similarly, there was no change in the S phase fraction in the shRb-infected MEFs, although, at day 6 there was a trend to a longer G2 and shorter G1 (Figures 2B and 2C, Figure S2A), possibly a consequence of increased reprogramming because a shorter G1 correlates with the acquisition of pluripotency (Conklin and Sage, 2009). Annexin V staining did not show a significant difference in apoptosis (Figure 2D, Figure S2B). "
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    ABSTRACT: Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.
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    • "Increasing evidence has implicated this pathway in stemness modulation, initially in plants (Ebel et al., 2004; Wildwater et al., 2005) and subsequently in animals (Liu et al., 2009). Accumulating evidence reinforces the role of pRb in stem cell homeostasis (Conklin and Sage, 2009). "
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    • "Loss of RB1 function is associated with a variety of human cancers, while inactivation of the RB1 tumor suppressor gene has been reported in several human malignancies in addition to RB [6], such as cancers of the breast [7,8], prostate [9], and lung [10]. Furthermore, the RB1 gene family is intimately involved in the control of cellular proliferation, survival, and differentiation pathways in many mammalian cells [11]. "
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    ABSTRACT: Retinoblastoma (RB), an intraocular tumor of childhood, is commonly associated with mutations in the RB1 gene. RB116 is a novel, early passage RB cell line that has not been previously characterized. In this study, we examined RB116 for the expression of RB1 and tested the hypothesis that RB116 cells would express stem cell markers as well as retinal progenitor cell markers. We compared RB116 cells with other well known RB cell lines, including Y79 and WERI-RB27. We evaluated expression of RB1 in RB116 cells by sequencing, multiplex ligation-dependent probe amplification, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), western immunoblot, and immunocytochemistry. Next, RB116 cells, along with Y79 and WERI-RB27 cells, were examined for expression of stem cell markers (ABCG2, Nanog, Oct3/4, ALDH1A1) and retinal progenitor markers (PAX6, CHX10) by quantitative immunocytochemistry. Immunocytochemical findings were accompanied by PCR analysis. RB116 cells expressed RB1 at the mRNA and protein levels, with no mutations detected by either sequencing analysis, or gene dosage abnormalities detected by multiplex ligation-dependent probe amplification. The RB1 protein was immunoreactive in RB116 cells with an atypical perinuclear localization. RB116 cells also expressed stem cell markers, with 3%-5% of cells immunopositive for ABCG2, Oct3/4 and ALDH1A1, with at least 18% of cells immunoreactive to Nanog. These findings were confirmed by RT-PCR. Small percentages of RB116 cells also exhibited immunoreactivity to retinal progenitor markers PAX6 (9.8%) and CHX10 (1.2%). Expression of mRNAs for these markers was confirmed by qRT-PCR. RB116 cells demonstrate RB1 expression accompanied by atypical perinuclear localization. RB116 cells also express primitive stem cell and retinal progenitor cell markers. Further studies on the phenotypes of both RB1-positive and RB1-negative human RB cells may be important in assessing differentiation potential of these cells, as well as designing targeted differentiation therapies.
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