Abstract
Embryonic stem cells (ESCs) possess remarkable abilities, as they can differentiate into all cell types (pluripotency) and be self-renewing, giving rise to two identical cells. These characteristics make ESCs a powerful research tool in fundamental embryogenesis as well as candidates for use in regenerative medicine. Significant efforts have been devoted to developing protocols to control ESC fate, including soluble and complex cocktails of growth factors and small molecules seeking to activate/inhibit key signaling pathways for the maintenance of pluripotency states or activate differentiation. Here we describe a novel method for the effective maintenance of mouse ESCs, avoiding the supplementation of complex inhibitory cocktails or cytokines, e.g., LIF. We show that the addition of zinc to ESC cultures leads to a stable pluripotent state that shares biochemical, transcriptional and karyotypic features with the classical LIF treatment. We demonstrate for the first time that ESCs maintained in long-term cultures with added zinc, are capable of sustaining a stable ESCs pluripotent phenotype, as well as differentiating efficiently upon external stimulation. We show that zinc promotes long-term ESC self-renewal (>30 days) via activation of ZIP7 and AKT signaling pathways. Furthermore, the combination of zinc with LIF results in a synergistic effect that enhances LIF effects, increases AKT and STAT3 activity, promotes the expression of pluripotency regulators and avoids the expression of differentiation markers.
Highlights
Embryonic stem cells (ESCs) are the main cellular source during embryogenesis in multicellular organisms, giving rise to the three embryonic germ layers which are the precursors required for the development of functional tissues (Niwa, 2007; Ohtsuka and Dalton, 2008; Evans, 2011)
For embryoid bodies (EBs), ESCs previously grown for 30 days (30d-ESC) in basal medium (BM) (30d-BM) BM supplemented with Zn 100 μM (30d-Zn) or Leukemia inhibitory factor (LIF) (30d-LIF) were cultured in hanging drops (1,000 cells/20 μL drop)
To determine zinc concentrations that are toxic for ESCs, cell viability was analyzed using concentrations of Zn2+ from 40 to 240 μM at 1, 4, and 7 days (Supplementary Figure 1)
Summary
Embryonic stem cells (ESCs) are the main cellular source during embryogenesis in multicellular organisms, giving rise to the three embryonic germ layers (mesoderm, endoderm and ectoderm) which are the precursors required for the development of functional tissues (Niwa, 2007; Ohtsuka and Dalton, 2008; Evans, 2011). The most common method of maintaining undifferentiated ESC phenotypes in vitro is their co-culture onto a feeder layer of inactivated mouse embryonic fibroblasts (MEF) (Tamm et al, 2013; Llames et al, 2015), which provides ESC paracrine factors for stemness maintenance (Llames et al, 2015). These feeder cells exhibit a heterogeneous population with different surface markers and phenotypes (Singhal et al, 2016) which entail a source of variability during ESC culture
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