Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency

摘要

class="head no_bottom_margin" id="sec1title">IntroductionEmbryonic stem cells (ESCs) are karyotypically normal, self-renewing cell lines, derived from the inner cell mass (ICM) of the pre-implantation embryo (, ). ESCs can be derived and expanded using a variety of conditions, including culture with the cytokine leukemia inhibitory factor (LIF) in the presence of serum (, ), in serum-free medium with two small-molecule inhibitors (2i) (), or with knockout serum replacement (KOSR) (). ESCs can be maintained indefinitely in vitro, while retaining the capacity to participate in development and generate all cell types of the embryo including the germ cells (, , , , ). They are therefore said to be pluripotent.Although the first ESCs were derived more than 30 years ago, a number of fundamental questions remain unanswered. At the embryonic stages from which ESCs are derived, the blastocyst is composed of several cell types, the epiblast (Epi), primitive endoderm (PrE), and trophoblast, and, during ESC derivation, subpopulations of embryo-derived cells are selected to expand. While these populations are not the same as the parental embryonic cells from which they are derived (), to what degree do they represent embryonic development? ESC cultures are also heterogeneous (, , , href="#bib19" rid="bib19" class=" bibr popnode">Kobayashi et al., 2009, href="#bib38" rid="bib38" class=" bibr popnode">Singh et al., 2007, href="#bib44" rid="bib44" class=" bibr popnode">Toyooka et al., 2008) and this heterogeneity is dynamic, perhaps more dynamic than the blastocyst from which they are derived. However, does this heterogeneity reflect the endogenous cell populations that arise in normal blastocyst development?The functional potential of ESCs can be assessed using a number of different approaches including in vitro differentiation, teratoma formation, and chimera generation (href="#bib3" rid="bib3" class=" bibr popnode">Beddington and Robertson, 1989, href="#bib31" rid="bib31" class=" bibr popnode">Poueymirou et al., 2007, href="#bib33" rid="bib33" class=" bibr popnode">Robertson et al., 1986, href="#bib36" rid="bib36" class=" bibr popnode">Saburi et al., 1997). Nevertheless, as ESCs are heterogeneous and chimeras are routinely generated by injecting 10–15 ESCs into morula or blastocyst-stage embryos (href="#bib5" rid="bib5" class=" bibr popnode">Bradley et al., 1984, href="#bib20" rid="bib20" class=" bibr popnode">Lallemand and Brulet, 1990) it is difficult to discern the functional properties of individual ESCs or specific ESC subpopulations. Based on the prospective isolation of ESC subpopulations, it has been shown that ESCs cultured in serum and LIF contain dynamic populations of PrE- and Epi-biased cells (href="#bib7" rid="bib7" class=" bibr popnode">Canham et al., 2010). However, these cells are clearly different from the blastocyst from which they are derived, as the PrE-primed cells express elevated levels of PrE RNA, but not protein. ESCs cultured under these conditions also contain a subpopulation that expresses 2-cell embryo (2C)-specific genes (href="#bib11" rid="bib11" class=" bibr popnode">Falco et al., 2007, href="#bib23" rid="bib23" class=" bibr popnode">Macfarlan et al., 2012). Similarly, culture of ESCs in 2i supports a totipotent population of cells that coexpress Epi determinants such as Nanog and the RNA for extraembryonic genes such as Gata6 or Hhex (href="#bib26" rid="bib26" class=" bibr popnode">Morgani et al., 2013).So, how do the conditions used to maintain ESCs influence the gene-expression state and populations contained within the culture? In this paper we explore this question by testing the impact of culture and derivation conditions on ESC populations, comparing ESC gene expression and heterogeneity, and the capacity of individual ESCs to contribute to full-term embryonic development. We found that ESCs maintained in standard serum culture conditions were comparable to populations of the late blastocyst (embryonic day 4.5 [E4.5]) ICM, at which point cells are specified and restricted in their functional potential. Conversely, ESCs cultured in 2i or KOSR showed a correlation with embryos from as early as the 2C stage, when cells are unrestricted and highly plastic. Consistent with expression data, we observed that single 2i and KOSR, but not serum, cultured ESCs could generate high-level chimeras when injected into either morulae or 2C embryos. This suggests that different ESC culture conditions support the expansion of populations reminiscent of different embryonic stages with distinct functional potentials. We found that populations induced during derivation could be “reprogrammed” by transferring ESCs to a different culture condition.