Abstract
Mouse embryonic stem cells (ESC) make cell fate decisions based on intrinsic and extrinsic factors. The decision of ESC to differentiate to multiple lineages in vitro occurs during the formation of embryoid bodies (EB) and is influenced by cell-environment interactions. However, molecular mechanisms underlying cell-environmental modulation of ESC fate decisions are incompletely understood. Since adhesion molecules (AM) influence proliferation and differentiation in developing and adult tissues, we hypothesized that specific AM interactions influence ESC commitment toward hematopoietic and endothelial lineages. Expression of AM in the adherens, tight and gap junction pathways in ESC subpopulations were quantified. E-cadherin (E-cad), Claudin-4 (Cldn4), Connexin-43 (Cx43), Zona Occludens-1 (ZO-1) and Zona Occludens-2 (ZO-2) transcript levels were differentially expressed during early stages of hematopoietic/endothelial commitment. Stable ESC lines were generated with reduced expression of E-cad, Cldn4, Cx43, ZO-1 and ZO-2 using shRNA technology. Functional and phenotypic consequences of modulating AM expression were assessed using hematopoietic colony forming assays, endothelial sprouting assays and surface protein expression. A decrease in E-cad, Cldn4, Cx43 and ZO-1 expression was associated with less commitment to the hematopoietic lineage and increased endothelial differentiation as evidenced by functional and phenotypic analysis. A reduction in ZO-2 expression did not influence endothelial differentiation, but decreased hematopoietic commitment two-fold. These data indicate that a subset of AM influence ESC decisions to commit to endothelial and hematopoietic lineages. Furthermore, differentially expressed AM may provide novel markers to delineate early stages of ESC commitment to hematopoietic/endothelial lineages.
Highlights
Stem cells from multiple sources are used for transplantation therapy and tissue regeneration
adhesion molecules (AM) are differentially expressed during embryoid bodies (EB) development Gene expression profiles of a panel of AM were measured in developing EB (Table S1)
Committed mesoderm GFP+ cells in Bry-GFP EB were sorted at day 4, Flk-1+ cells undergoing early hematopoietic and endothelial differentiation were sorted from day 5 EB generated from D3-Embryonic stem cells (ESC), and Sclexpressing subpopulations, containing cells that are committed to hematopoiesis, but have not fully differentiated, were sorted from day 6 EB generated from Scl-LacZ ESC
Summary
Stem cells from multiple sources are used for transplantation therapy and tissue regeneration. Endothelial progenitor cells (EPC) are used to treat tissue ischemia, repair blood vessels and relieve pulmonary hypertension in diabetes, vascular and kidney diseases [1]. Hematopoietic stem cells (HSC) have been used to treat blood disorders and influence immunological tolerance in graft versus host disease [2]. The ability to reliably guide ESC toward hematopoietic or endothelial lineages is complicated by a lack of understanding of key regulatory signals/pathways involved in their proliferation and differentiation decisions. Increased understanding of factors that guide early stages of ESC commitment decisions towards hematopoietic and endothelial lineages is an important step in developing strategies to direct differentiation
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