Influencing Hematopoietic Differentiation of Mouse Embryonic Stem Cells using Soluble Heparin and Heparan Sulfate Saccharides

Abstract

Heparan sulfate proteoglycans (HSPG) encompass some of the most abundant macromolecules on the surface of almost every cell type. Heparan sulfate (HS) chains provide a key interaction surface for the binding of numerous proteins such as growth factors and morphogens, helping to define the ability of a cell to respond selectively to environmental cues. The specificity of HSprotein interactions are governed predominantly by the order and positioning of sulfate groups, with distinct cell types expressing unique sets of HS epitopes. Embryos deficient in HS-synthesis (Ext1 ؊/؊ ) exhibit pre-gastrulation lethality and lack recognizable organized mesoderm and extraembryonic tissues. Here we demonstrate that embryonic stem cells (ESCs) derived from Ext1 ؊/؊ embryos are unable to differentiate into hematopoietic lineages, instead retaining ESC marker expression throughout embryoid body (EB) culture. However hematopoietic differentiation can be restored by the addition of soluble heparin. Consistent with specific size and composition requirements for HS:growth factor signaling, chains measuring at least 12 saccharides were required for partial rescue of hematopoiesis with longer chains (18 saccharides or more) required for complete rescue. Critically N-and 6-O-sulfate groups were essential for rescue. Heparin addition restored the activity of multiple signaling pathways including bone morphogenic protein (BMP) with activation of phosphoSMADs re-established by the addition of heparin. Heparin addition to wild-type cultures also altered the outcome of differentiation, promoting hematopoiesis at low concentrations, yet inhibiting blood formation at high concentrations. Thus altering the levels of HS and HS sulfation within differentiating ESC cultures provides an attractive and accessible mechanism for influencing cell fate.Cells have adopted elaborate systems to identify growth factor and morphogen cues in order to elicit an appropriate response throughout embryonic development and adulthood. A key component of this process is the sugar polymer heparan sulfate (HS), 2 which coats the outer surface of almost every cell within the body enabling the selective detection of environmental signals. HS polymerization is catalyzed by a heterodimer consisting of EXT1 and EXT2 enzymes resulting in the addition of alternating glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) residues. This backbone is then enzymatically modified by de-N-acetylation and N-sulfation of selected GlcNAc residues to form N-sulfoglucosamine (GlcNS), epimerization of a subset of GlcA residues to iduronic acid (IdoUA) and the addition of sulfate groups at the 2-O-position of GlcA/IdoUA and 6-O-and rarely 3-O-position of GlcNS/GlcNAc. Modification is highly regulated and not complete, forming a cell-type specific pattern of highly sulfated domains (S-domains) interspersed by unmodified regions with the length, position and patterning of sulfate groups ultimately determining HS:ligand binding. The related GAG heparin is virtually fully sulfated along its l...

Specific Glycosaminoglycans Modulate Neural Specification of Mouse Embryonic Stem Cells

“…The loss of endogenous HS is not deleterious to self-renewal and pluripotency. However, HS is critical when cells are challenged to differentiate by at least three distinct protocols, in vitro neural differentiation, embryoid-body mediated hematopoiesis, and leukemia inhibitory factor (LIF) withdrawal [25,34,38]. One of the hallmarks of early differentiation is the response to autocrine signals, many of which are dependent on HS, for example, the FGF-mediated system described in this study.…”

“…The phenotypes of these cells, primarily their ability to maintain self-renewal, differ significantly [35,38]. Both the knockout Ext1 À/À ES cell lines (described previously [25,34] and here), the stably transfected RNAi line (this study) and the Ext1 flox/flox ES cell line [38], can self-renew and maintain pluripotency in contrast to previous reports of Ext1 kd cells generated by transient RNA interference [35]. The loss of endogenous HS is not deleterious to self-renewal and pluripotency.…”

“…The Ext1 À/À mouse ES cells (mES) used in this and other studies [25,34] contain a disruption of the Ext1 gene and were obtained by gene targeting and homologous recombination [14]. To confirm that the inability of Ext1 À/À ES cells to undergo neural specification was solely due to lack of HS, we used a stable plasmid-based shRNAi approach to knockdown Ext1 expression in the 46C line.…”

Function of Heparan Sulfate in Pluripotent Stem Cells

“…Indeed, in EXT1 or EXT2-deficient mice, On the other hand, other groups reported that EXT1-deficient ES cells, which have no HS chains, could not differentiate (36,(40)(41)(42). In the EXT1-deficient ES cells, inhibition of Nanog expression via FGF4 signaling was eliminated because FGF4 signaling, the differentiation trigger, could not work with deficient HS chains.…”

Heparan Sulfate Biosynthesis Enzymes in Embryonic Stem Cell Biology

“…In contrast, a total lack of Ext1 prevents both spontaneous differentiation as well as directed neural differentiation (Johnson et al 2007;Kraushaar et al 2010;Holley et al 2011). A recent study by Holley and coworkers (2011) showed that Ext1 -/-cells do form embryonic bodies (EBs), an in vitro three-dimensional (3D) model for ES cell differentiation, with a subsequent increase in early mesodermal markers.…”