Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells

doi:10.1073/pnas.0703723104

Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells

*Harvard–Massachusetts Institute of Technology Division of Health Sciences and Technology and
^‡Departments of Chemical Engineering and Bioengineering, Massachusetts Institute of Technology, Cambridge, MA 02139;
^§Center of Neurosciences and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal;
^¶Biocant Centro de Inovação em Biotecnologia, 3060-197 Cantanhede, Portugal;
^†Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104; and
**Department of Biomedical Engineering, Columbia University, New York, NY 10027

Contributed by Robert Langer, April 26, 2007 (received for review February 7, 2007)

Abstract

Control of self-renewal and differentiation of human ES cells (hESCs) remains a challenge. This is largely due to the use of culture systems that involve poorly defined animal products and do not mimic the normal developmental milieu. Routine protocols involve the propagation of hESCs on mouse fibroblast or human feeder layers, enzymatic cell removal, and spontaneous differentiation in cultures of embryoid bodies, and each of these steps involves significant variability of culture conditions. We report that a completely synthetic hydrogel matrix can support (i) long-term self-renewal of hESCs in the presence of conditioned medium from mouse embryonic fibroblast feeder layers, and (ii) direct cell differentiation. Hyaluronic acid (HA) hydrogels were selected because of the role of HA in early development and feeder layer cultures of hESCs and the controllability of hydrogel architecture, mechanics, and degradation. When encapsulated in 3D HA hydrogels (but not within other hydrogels or in monolayer cultures on HA), hESCs maintained their undifferentiated state, preserved their normal karyotype, and maintained their full differentiation capacity as indicated by embryoid body formation. Differentiation could be induced within the same hydrogel by simply altering soluble factors. We therefore propose that HA hydrogels, with their developmentally relevant composition and tunable physical properties, provide a unique microenvironment for the self-renewal and differentiation of hESCs.

Footnotes

^‖To whom correspondence may be addressed. E-mail: rlanger{at}mit.edu
^††To whom correspondence may be addressed at:
Department of Biomedical Engi neering, Columbia University, William Black Research Building 1605–1611, 650 West 168th Street, MC 104B, New York, NY 10032.
E-mail: gv2131{at}columbia.edu
Author contributions: S.G., J.A.B., S.A.T., R.L., and G.V.-N. designed research; S.G., L.S.F., and S.A.T. performed research; L.S.F. contributed new reagents/analytic tools; S.G., J.A.B., S.A.T., and G.V.-N. analyzed data; S.G., J.A.B., R.L., and G.V.-N. wrote the paper; and J.A.B. and L.S.F. designed hydrogels.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0703723104/DC1.
Abbreviations:

EB,

embryoid body;

HA,

hyaluronic acid;

FL-HA,

fluorescein-labeled HA;

MEF,

mouse embryonic fibroblast feeder layers;

XTT,

2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.