Within 1h after mitosis, neither p220NPAT nor LSM10 exhibits focal organization in early G1 cells, and there is no evidence of association with histone gene loci in iPS cells at this time point. rapidly after mitosis (within0.5 to 1 1.5 h). Thus, reprogrammed iPS cells have cell cycle kinetics and dynamic subnuclear organization of regulatory machinery that are principal properties of pluripotent hES cells. Our findings support the concept that the abbreviated cell cycle of hES and iPS cells is functionally linked to pluripotency. strong class=”kwd-title” Keywords: human induced pluripotent stem (iPS) cells, human embryonic stem (hES) cells, cell cycle, histone, p220NPAT, HiNF-P, Histone Locus Body (HLB) body, LSM10, FLASH, 6p21 Introduction Programming of embryonic cells occurs concomitant with phenotype-commitment during mammalian development. Reprogramming of differentiated cells to a pluripotent state can be achieved by somatic cell nuclear transfer (Jaenisch and Young, 2008; Blelloch et al, 2006), or expression of defined sets of transcription factors (e.g. Oct4, Sox2, KLF4 and cMyc) (Hockemeyer et al, 2008; Takahashi and Yamanaka, 2006; Brambrink et al, 2008; Maherali and Hochedlinger, 2008; Park et al, 2008; Maherali et al, 2007; Wernig et al, 2007; Okita et al, 2007; Takahashi et al, 2007; Yu et al, 2007). However, there are biological variations in established iPS lines with respect to marker gene expression, colony morphology, cell culture parameters, teratoma formation and ability to differentiate (Chin et al, 2009; Daley et al, 2009; Meissner et al, 2008). This biological variation may relate to differences in epigenetic modifications (e.g., CpG methylation), reprogramming procedures, as well as variations in cell culture conditions and laboratory environment. In addition, embryonicstem cells may have a na?ve pluripotent phenotype or primed pluripotent state (Hanna et al, 2010; Nichols and Smith, 2009; Tesar et al, 2007; Brons et al, 2007). Therefore, stringent functional tests and standards have been established that characterize the multi-lineage potential of hES and induced pluripotent stem (iPS) cells (Daley et al, 2009; Maherali and Hochedlinger, 2008). Human embryonic stem (hES) cells have an abbreviated cell cycle of 16C18 hours with a very short G1 phase (2C3 hours)(Becker et al, 2006; Becker et al, 2007). Considering the plasticity of iPS and hES cell phenotypes, it is important to determine whether fidelity of pluripotent cell cycle control has been re-established in iPS cells. Unlike normal somatic cells, hES cells are competent to initiate two consecutive S phases in the absence of external growth factors and sustain an abbreviated cell cycle through autocrine mechanisms (Becker et al, 2010b; Becker et al, 2010a). Thus, hES cells are pre-mitotically committed to initiate a new round of cell division. Differentiation of hES cells alters cell cycle Mephenytoin kinetics by lengthening G1 within 72 h suggesting that the shortened G1 phase of the hES cell cycle is functionally coupled with the pluripotent state (Becker et al, 2010b; Becker et al, 2010a). We propose that reprogramming should not only involve the resetting of signaling pathways and epigenetic modifications to a basal state that supports pluripotency, but should also re-establish the unique abbreviated Agt cell cycle that characterizes pluripotent hES cells. Indeed, incomplete reprogramming at imprinted loci has been observed in iPS lines (Stadtfeld et al, 2010) and assessment of cell cycle kinetics may represent a Mephenytoin useful proxy for successful reprogramming. As in somatic cells, histone H4 gene expression is cell cycle regulated in hES cells. In both cell types, key histone gene regulatory factors (e.g., Nuclear Protein, ataxia-telangiectasia locusp220NPAT) are organized in a limited number (two or four depending on the stage of cell cycle) of subnuclear microenvironments (foci) designated Histone Locus Bodies (HLBs) (Becker et al, 2007; Ghule et al, 2007; Ghule et al, 2008; Bongiorno-Borbone et al, 2008). HLBs architecturally integrate transcriptional initiation and 3 end processing of histone gene transcripts. Assembly is carefully orchestrated with recruitment and/or CDK mediated phosphorylation of HLB components at sequential sub-stages of G1. Because G1 is shortened in hES cells, HLBs organize rapidly following mitotic division to achieve the necessary competency for expression of histone genes during S phase (Becker et al, 2007; Ghule Mephenytoin et al, 2007; Ghule et al, 2008). The accelerated and cell cycle dependent organization of HLBs provides a key spatio-temporal parameter that is characteristic of the abbreviated G1 of pluripotent hES cells. Here, we establish that, consistent with fidelity of reprogramming to pluripotency,.
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