Time-Lapse Analysis of Human Embryonic Stem Cells Reveals Multiple Bottlenecks Restricting Colony Formation and Their Relief upon Culture Adaptation

Ivana Barbaric,Veronica Biga,Paul J Gokhale,Mark Jones,Dylan Stavish,Adam Glen,Daniel Coca,Peter W Andrews

doi:10.1016/j.stemcr.2014.05.006

Abstract

SummaryUsing time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns.

Highlights

At odds with their indefinite self-renewing capability in vitro, human embryonic stem cells display a high death rate in culture, contributing to the problems of efficient mass culture
Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells and that are relieved by karyotypically abnormal variants that are selected by prolonged culture
Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal human embryonic stem cells (hESCs)

Summary

Introduction

At odds with their indefinite self-renewing capability in vitro, human embryonic stem cells (hESCs) display a high death rate in culture, contributing to the problems of efficient mass culture. The low cloning efficiency is at least partly due to an excessive apoptosis of cells upon dissociation (Chen et al, 2010; Ohgushi et al, 2010), but the discrepancy in the number of cells surviving the initial plating and the overall cloning efficiency suggests that critical restriction points exist between initial plating and when robust colony formation is established The nature of these further restrictions remains unknown, we have previously posited that cell:cell contact provides crucial signals, perhaps mediated by the NOTCH system, for the survival and proliferation of undifferentiated human pluripotent stem cells (Andrews et al, 1982; Fox et al, 2008). We have used a combination of cell sorting, timelapse video microscopy, single-cell tracking, and modeling techniques to characterize the bottlenecks that prevent clonal expansion of normal hESCs and elucidate how these are overcome by adapted cells

Results

Discussion

Conclusion