br Introduction Stem cell divisions resulting

Introduction Stem cell divisions resulting in alternative pathways of self-renewal or differentiation require very distinctive epigenetic regulation of gene expression from the same genome. Where division has a symmetrical output of progeny cells, the assumption is that the molecular signatures derived from sister Z-IETD-FMK (daughter cells from a common parent cell) are identical. In this context, various types of markers and biological functions have been used to evaluate the symmetry of cell divisions (Beckmann et al., 2007; Huang et al., 1999; Muramoto et al., 2010; Punzel et al., 2002; Suda et al., 1983; Wu et al., 2007; Zwaka and Thomson, 2005). Although each of these studies addressed a particular biological question (e.g., similarity levels of transcriptional oscillation of a few genes between Dictyostelium sister cells [Muramoto et al., 2010]) and provided important information to relevant fields, the overall level of similarity between sister cells has not been thoroughly addressed. Human ESCs, for example, are considered to divide and differentiate “symmetrically” regardless of the cultural condition, but this assumption is based on the distribution of the expression of a single gene POU5F1 measured through the signal of highly stable protein, eGFP (Zwaka and Thomson, 2005). More comprehensive and sensitive approaches should be undertaken to evaluate the actual level of division symmetry. (In this report, the term “symmetric division” refers to the generation of two daughter cells that exhibit high-level similarities in cell fates, proliferative capacities, and/or the presence of biomarkers.) Although murine embryonic stem cells (ESCs) in culture look morphologically similar, a subset of genes is often differentially expressed within a population (Carter et al., 2008; Chambers et al., 2007; Hayashi et al., 2008; Kalmar et al., 2009; Payer et al., 2006; Singh et al., 2007). Nanog and Gata 6 proteins are expressed heterogeneously in both ESCs and the inner cell mass of E3.5 blastocysts, suggesting that the heterogeneity is not solely an in vitro phenomenon (Chazaud et al., 2006). These results raise the possibility that the fidelity of ESC “self-renewal” is less than one would predict. Here, we established a method to isolate single sister cells through microdissection to evaluate the symmetry of ESC divisions at molecular levels. High-throughput RT-PCR analyses using isolated single sister cells suggests that ESCs divided symmetrically at the ground state of pluripotency, which was induced by a 2i condition (Ying et al., 2008), whereas the symmetry was significantly declined in pluripotent (medium with LIF and BMP4) and differentiation states (medium without LIF and BMP4). We also found that the ground pluripotent state (medium with 2i, LIF, and BMP4) was accompanied by the reduction in the number of coregulating genes at single-cell levels. Importantly, we found that DNA methyltransferases 3a and 3b (Dnmt3a/3b) are downregulated in 2i-grown ESCs as reported recently (Leitch et al., 2013), and ESCs that are deficient for three DNA methyltransferases generated nearly identical sister cells, suggesting the link between epigenetic regulation and the fidelity of cell divisions. We believe that our systems will expand the capability of single-cell analyses and will help identifying mechanisms that cause cellular heterogeneity, which emerges as an important problem in stem cell biology, translational research, and effective cancer treatment.
Results
Discussion Although the similarity between sister cells can be evaluated through imaging analyses, they are often labor intensive and thus are not suitable to process a large number of cells using multiple marker probes. In order to obtain a high-throughput data set, we processed each of over 350 sister cells to a series of molecular reactions and were able to identify a possible cause of generating the diversity between sister cells. Importantly, the microdissection method can be combined with other single-cell methodologies including analyses for genomic DNA sequence (Zong et al., 2012), CpG methylation (Kantlehner et al., 2011), noncoding and coding RNA sequence (Tang et al., 2010; Tang et al., 2006), and protein analyses (Shi et al., 2012). Sister cells isolated with this method are also viable and, thus, can be used for a series of functional analyses, such as the evaluation of proliferation kinetics and differentiation potentials.