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

Rebecca J Holley,Claire E Pickford,Graham Rushton,Georges Lacaud,John T Gallagher,Valerie Kouskoff,Catherine L.R Merry

doi:10.1074/jbc.m110.178483

Rebecca J Holley, Claire E Pickford + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110.178483

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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 HS-protein 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 phospho-SMADs 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.

Highlights

Other groups report that addition of Wnt3a and Activin A can induce primitive streak formation marked by Brachyury expression, with inhibitor studies suggesting Activin A, Wnt3a and BMP4 are all required for Flk1ϩ mesoderm formation [12, 17]
Ext1Ϫ/Ϫ embryonic stem cells (ESCs) Lack Cell Surface heparan sulfate (HS) Chains—Ext1Ϫ/Ϫ ESCs were readily maintained in culture forming optically bright colonies with typical ES morphology, colonies phenotypically appeared more compact than wild-type, with little visible differentiation (Fig. 1A)
The flow cytometry profile of Ext1ϩ/Ϫ ESCs stained with 10E4 was intermediate between that of wild-type and Ext1Ϫ/Ϫ, suggesting a decrease in the number of 10E4-reactive epitopes (Fig. 1C). 3G10 binding following heparinase digest showed that a similar number of chains to wild-type are present, as only one 3G10-reactive epitope is produced by heparinase activity per chain (Fig. 1D)

Summary

Introduction

Increasing concentrations of heparin were added to Ext1Ϫ/Ϫ cells from day 0, and EB cells stained with differentiation markers or replated under hematopoietic-colony forming conditions (with heparin addition) at day 6 (Fig. 3, A and B). Wild-type cells progressively down-regulate ESC-marker Oct4, show transient expression of Brachyury and Flk1 from days 2 to 3 and express hematopoietic transcription factors SCL, GATA1, and ␤-Major from days 3 to 4 (Fig. 3F), as seen previously [10].

Results

Conclusion