It is imperative that we

It is imperative that we develop efficient culture methods that can overcome the obstacles found in the current culture systems. Moreover, new culture methods should be optimized for production of clinically relevant cell numbers, tissue morphogenesis, high-throughput drug discovery, and efficient assays of cellular stress. A comprehensive analysis of different cell culture protocols for hPSC is available in a recent review (Chen et al., 2014). Here, we will focus on the fundamental principles of developmental biology and core signaling pathways that underlie different growth methods. We will further discuss the relationship between the development of early stage embryos and the mechanisms underlying hESC heterogeneity and chromosomal instability. Finally, we will provide new insights into the development of optimal growth models for hPSCs.
Human pluripotent stem cell colonies and CX5461 recapitulate some properties of early-stage embryo development Unlike mouse embryonic stem cells (mESCs), which possess high single-cell plating efficiency under dissociated conditions, both hESCs and iPSCs cannot grow as single cells under commonly used culture conditions. Human pluripotent stem cells are conventionally cultured as condensed colonies and must be passaged as cell clumps to avoid cumulative apoptotic and differentiation stress. These cells are routinely differentiated toward derivatives of the three germ layers through three-dimensional (3D) EBs. In general, colony-type and aggregated cultures often produce heterogeneous colonies and EBs. Hence, we frequently observe more apoptotic activity in the center of a colony (with more spontaneously differentiated cells at the periphery) (Figs. 1 and 2) and complicated cystic structures after EB agglomeration (Fig. 3). The unique structures of hESC colonies and EBs are reminiscent of the inner cell mass (ICM) and post-implantation blastocysts observed during early embryo development. In the following sections, we will discuss the links between the structures of hPSC colonies and EBs and their developmental counterparts.
Core signaling pathways depict multiple EMT phases and cellular heterogeneity in human pluripotent stem cells The molecular mechanisms that maintain the epithelial identity of hPSCs depend on apical ligand-receptor signal transductions, intercellular adhesion (mediated by molecules such as E-cad-conjugated β-catenin), and basement membrane and ECM interactions (Figs. 2A, B). With regard to intercellular connections, glycogen synthase kinase-3 (GSK-3) is a potent regulator of E-cad and plays a pivotal role in EMT and in cell fate commitment. In general, GSK-3 phosphorylates Snail and initiates its proteasomal degradation, hence upregulating E-cad and concurrently inhibiting EMT (Doble and Woodgett, 2007). However, in the case of hESCs, when GSK-3 inhibitor (6-bromoindirubin-3′-oxime) is added as a supplement to hESC medium to support hESC self-renewal, EMT is inhibited, likely through activation of the Wnt pathway (Sato et al., 2004; Ullmann et al., 2008). Nevertheless, the role of Wnt/β-catenin in the maintenance of undifferentiated hESCs has been questioned by a recent study in which the authors revealed that Wnt/β-catenin signaling actually promotes hESC differentiation, not self-renewal (Davidson et al., 2012). In general, concurrent upregulation of E-cad and down-regulation of metallopeptidases (such as MMP2 and MMP9), N-cadherin, vimentin, Snail, and Slug may be essential to inhibit EMT and differentiation (Fig. 2C) (Ullmann et al., 2008). Interestingly, E-cad may coordinate with many molecular events to reduce the motility and the occurrence of the fibroblast-like cells at the periphery of hESC colonies in various growth conditions (Ullmann et al., 2008; Ullmann et al., 2007). E-cad may be also associated with the arrangements of the core actin cytoskeleton and the EMT phenotype. This E-cad-EMT reverse correlation may be assayed by the presence of the membrane oncofetal protein 5T4, which is triggered by the addition of anti-adhesive antibody SHE78.7 that disrupts E-cad (Eastham et al., 2007). These data suggest that loss of E-cad and its regulators is a critical step that initiates EMT in hESCs.