Human ESC colony formation is dependent on interplay between self‐renewing hESCs and unique precursors responsible for niche generation

Abstract

Human embryonic stem cell (hESC) cultures are heterogeneous and constituting paracrine signals are required to maintain pluripotency. The cellular interplay and dynamic nature of this heterogeneity is not understood. Here, long-term hESC imaging and tracking revealed that hESC heterogeneity is dynamic and hESC self-renewal is dependent on colony-proximal distributions of paracrine signals. Tracking of hESCs forming colonies revealed that a biologically distinct cell type arises at the colony periphery in the absence of feeders. Higher rates of cell death occur in these hESC-derived cells, leading to clonal selection of colony reestablishing cells. hESC-derived feeders co-transferred during passaging promoted rapid colony recovery and expansion and reduced overall clonal selection of self-renewing hESCs. Our findings demonstrate that hESCderived feeders arise from a distinct subpopulation of hESCs that respond to paracrine cues at the colony periphery that are required to sustain and establish clonal hESC self-renewal. ' 2010 International Society for Advancement of Cytometry Key termslive cell imaging; cell tracking; lineage analysis; human embryonic stem cells; human embryonic stem cell niche STEM cell developmental potential is maintained by self-renewal, which is thought to be partially controlled in vivo through extrinsic signals that regulate stem cell survival, self-renewal, and differentiation. Similarly, recent evidence has demonstrated that human embryonic stem cell (hESCs) both create and are reliant on a supportive in vitro niche (1,2). Similar to in vivo stem cell niches, the hESC in vitro niche consists of supportive differentiated cells, including hESC-derived fibroblast-like cells (hdFs), paracrine signals, and interactions with extracellular matrix (1-3). Recently, multiple niche-independent hESC cultures have demonstrated a variety of features suggestive of early transformation events, including growth factor independence, increased proliferation, and dramatically reduced differentiation potential (4). These results illustrate the importance of the in vitro niche, and that niche components are defining factors regulating hESC fate within the culture. However, the cellular dynamics involved in hESC niche regulation are not well understood.Differentiation of hESCs to hdFs is observed in all hESC culture formats, but it is more prevalent as the culture becomes more defined and feeder layer-free (i.e. moving from mouse embryonic feeder (MEF) layers, to conditioned media, to defined media) (5). Repeated passaging for expansion of hESC cultures causes disruption of the hESC microenvironment and requires the reestablishment of the cellular and noncellular niche (such as ECM produced by hESCs) (6). To establish a cellular niche in the absence of MEFs, hESCs generate hdFs (1,7). This reestablishment period, in which niche signals for survival are suboptimal, appears to invoke significant cell death, based on observed cell debris following passage and the established disparity between hESC

Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation

“…However, this approach has not been efficiently implemented, due in part to currently used inefficient cell culture and differentiation strategies, which are heavily based on cell culture as colonies and aggregated embryoid bodies (EBs) (Chen et al, 2014; Mallon et al, 2006; Thomson et al, 1998; Xu et al, 2001). These methods have led to time-consuming production of relatively low numbers of cells (Hartung et al, 2010; Kehoe et al, 2010; Serra et al, 2012), heterogeneous cellular and molecular states (Bendall et al, 2007; Chen et al, 2012a; Moogk et al, 2010; Stewart et al, 2006), and frequent reports of chromosomal abnormalities (reviewed in Baker et al, 2007; Lee et al, 2013). Certain chromosomal alterations such as trisomies 12, 17, and 20 have the capacity to confer growth advantages as indicated by comparative genetic analyses (Amps et al, 2011; Catalina et al, 2008; Draper et al, 2004; Lefort et al, 2008).…”

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

“…The heterogeneity in hPSC culture is often induced by excessive apoptotic signals and spontaneous differentiation due to suboptimal growth conditions. Thus, in colony-type culture, the heterogeneous cells are often observed in the periphery of the colonies 7,8 . It has been also shown that the cells in human embryonic stem cell (hESC) colonies exhibit differential responses to signaling molecules such as BMP-4 9 .…”

Probing Embryonic Stem Cell Autocrine and Paracrine Signaling Using Microfluidics

“…This phenomenon was attributed to an unknown autocrine non-LIF gp130 ligand (24) (Figure 2C). Spatial variation was also found in hESC colonies in terms of Oct4 levels and Smad1 signaling (9), and cells at the periphery of hESC colonies have been shown to have distinct morphologies and different cell growth and death characteristics (48), all phenotypes that were attributed to the presence of autocrine and paracrine cues.…”