Human Pluripotency Is Initiated and Preserved by a Unique Subset of Founder Cells

Abstract

The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD + ) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative singlecell analysis with pre-and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.

A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and their Differentiated Derivatives

“…Some hiPSCs located on the colony's edge could translocate interiorly while others remained at the edge during colony expansion ( Figure 4G). Tracking spatial locations of a cell within a colony can yield new information regarding geometric cues, as recent work identified a subpopulation of PSCs on colony edges that share hallmark properties with primitive endoderm, with distinct cell states compared to those in the interior (Yoney et al, 2018;Nakanishi et al, 2019). To understand the kinetics of PSC translocation from the perimeter to the interior, PSCs at the edge of colonies were tracked over a period of 12 hours and analyzed for position changes from the edge to the interior ( Figure 4H).…”

“…A new era of single cell analyses, such as single cell transcriptomics and single cell epigenomics assays, has highlighted the wide range of molecular profiles that were previously unappreciated in bulk population assays (Angermueller et al, 2016;Nestorowa et al, 2016;Cusanovich et al, 2018;Nakanishi et al, 2019). These findings have been of special relevance to stem cell biology and directed differentiation approaches as they discovered transcriptional and epigenetic heterogeneity that were inferred to affect cellular functions, despite homogeneous expression of cell-type specific markers within the populations (Angermueller et al, 2016;Kumar, Tan and Cahan, 2017;Friedman et al, 2018).…”

“…We found this reporter of single cell lineage and clonal expansion had broader utility and enabled longitudinal quantification of cell morphology and migration. Studies have recently identified a subset population of PSCs that reside on the perimeter of PSC colonies (Li et al, 2010;Warmflash et al, 2014;Rosowski et al, 2015;Yoney et al, 2018;Nakanishi et al, 2019).…”

“…These edge PSCs share hallmark properties with primitive endoderm and display distinct non-canonical Wnt signaling and gene expression that enhance their colony initiation capacity compared to PSCs localized in the colony interior (Yoney et al, 2018;Nakanishi et al, 2019).…”

“…Single cell RNA sequencing studies have recently demonstrated that individual PSCs within a culture express a wide range of transcriptional profiles despite uniformly expressing the same cell-type specific markers, such as Oct4 and Tra1 (Kumar, Tan and Cahan, 2017;Nakanishi et al, 2019). It has been challenging to determine whether this translates to functional heterogeneity among PSCs.…”