Dynamic Migration and Cell-Cell Interactions of Early Reprogramming Revealed by High-Resolution Time-Lapse Imaging

Abstract

Discovery of the cellular and molecular mechanisms of induced pluripotency has been hampered by its low efficiency and slow kinetics. Here, we report an experimental system with multi-color time-lapse microscopy that permits direct observation of pluripotency induction at single cell resolution, with temporal intervals as short as five minutes. Using granulocyte-monocyte progenitors as source cells, we visualized nascent pluripotent cells emerge from a hematopoietic state. We engineered a suite of image processing and analysis software to annotate the behaviors of the reprogramming cells, which revealed the highly dynamic cell-cell interactions associated with early reprogramming. We observed frequent cell migration, which can lead to sister colonies, satellite colonies and colonies of mixed genetic makeup. In addition, we discovered a previously unknown morphologically distinct 2-cell intermediate of reprogramming, which occurs prior to other reprogramming landmarks. By directly visualizing the reprogramming process with E-cadherin inhibition, we demonstrate the requirement of E-cadherin for proper cellular interactions from an early stage of reprogramming, including the 2-cell intermediate. The detailed cell-cell interactions revealed by this imaging platform shed light on previously unappreciated early reprogramming dynamics. This experimental system could serve as a powerful tool to dissect the complex mechanisms of early reprogramming by focusing on the relevant but rare cells with superb temporal and spatial resolution.

Nonstochastic Reprogramming from a Privileged Somatic Cell State

“…The majority of wells (97%) positive for Oct4:GFP+ colonies did not contain noticeable numbers of cells bearing hematopoietic morphology (Figure 2B), confirming our observation that most progeny became reprogrammed using the imaging approach (Movie S1). Many of these wells contained multiple sister colonies with small numbers of round-shaped cells (also Oct4:GFP+) in close proximity to the larger colonies (Figure 2B), which we have described previously to result from the dynamic migrative behavior associated with pluripotency (Megyola et al, 2013). To ensure all GMP-derived cells were scored, we repeated single cell reprogramming with GMPs from a transgenic mouse line that expresses an H2B-GFP fusion protein (Hadjantonakis and Papaioannou, 2004), so that all progeny could be identified based on their expression of H2B-GFP (Figure S1C).…”

“…To identify the existence of privileged somatic cells, we first took a live-cell imaging approach, with which the behaviors of single cells can be faithfully tracked with high resolution (Megyola et al, 2013). We focused on the well-defined granulocyte monocyte progenitors (GMP) since they support rapid and efficient reprogramming (Eminli et al, 2009; Megyola et al, 2013), and are more likely to contain privileged cells.…”

“…We focused on the well-defined granulocyte monocyte progenitors (GMP) since they support rapid and efficient reprogramming (Eminli et al, 2009; Megyola et al, 2013), and are more likely to contain privileged cells. Specifically, GMPs from mice that carry both Rosa26:rtTA and Oct4:GFP alleles were used as source cells for reprogramming (FACS-sorting scheme in Figure S1B), so that activation of endogenous Oct4 locus can be detected as green fluorescence in live cells.…”

“…The reprogramming cultures were imaged at 5–15 minute intervals for ~five days, when Oct4:GFP+ cells display typical features of mouse pluripotent cells. These Oct4:GFP+ cells, although still Dox-dependent, progress with reprogramming highly efficiently (Table S1) without any obvious bottleneck restrictions, reaching a pluripotent state that can support chimeric mice formation and germline transmission (Megyola et al, 2013). …”

3D-Technology of the Formation and Maintenance of Single Dormant Microspheres from 2000 Human Somatic Cells and Their Reactivation In Vitro

“…The use of this method provided unique information in modern cell biology. For instance, bivalent dumbbell-shaped cells, inductors of new subpopulation of pluripotent cells, and initiators of new immune memory T cells were thus identifi ed [6,8,21,23,28,32]. The dynamics of cell migration and 3D-interactions that allow histogenesis programming in the spheres of through of E-cadherin clusters and axial actin networks were studied [7,18].…”