We propose a new methodology to enhance the vascular differentiation of human embryonic stem cells (hESCs) by encapsulation in a bioactive hydrogel. hESCs were encapsulated in a dextran-based hydrogel with or without immobilized regulatory factors: a tethered RGD peptide and microencapsulated VEGF 165 . The fraction of cells expressing vascular endothelial growth factor (VEGF) receptor KDR/Flk-1, a vascular marker, increased up to 20-fold, as compared to spontaneously differentiated embryoid bodies (EBs). The percentage of encapsulated cells in hydrogels with regulatory factors expressing ectodermal markers including nestin or endodermal markers including α-fetoprotein decreased 2 or 3 fold, respectively, as compared to EBs. When the cells were removed from these networks and cultured in media conditions conducive for further vascular differentiation, the number of vascular cells was higher than the number obtained through EBs, using the same media conditions. Functionalized dextran-based hydrogels could thus enable derivation of vascular cells in large quantities, particularly endothelial cells, for potential application in tissue engineering and regenerative medicine.
1-INTRODUCTIONDuring normal embryogenesis, human embryonic stem cells (hESCs) differentiate along different lineages in the context of complex three-dimensional tissue structures, where the extracellular matrix (ECM) and different growth factors play an important role in this process. The three-dimensional ECM provides structural support for higher level of tissue organization and remodelling [1]. Significant differences were found in the differentiation profile of ESCs when cultured in a three-dimensional (3D) versus two-dimensional (2D) system [2,3] or 3D scaffold system versus embryoid body (EB) system [4,5]. In this last case, mouse ESCs cultured within tantalum scaffolds differentiate at higher extent into hematopoietic cells than EBs [4], while hESCs cultured in alginate scaffolds express significantly more vascular markers than EBs [5]. Moreover, the culture of hESCs within 3D poly(α-hydroxy esters) scaffolds with media containing different growth factors induced their differentiation into 3D structures with || To whom reprint requests should be addressed at: Department of Chemical Engineering, E25-342 Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. E-mail: rlanger@mit.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [1,6]. However, these growth factors were supplied from outside the scaffold, and thus their activity may be affected by diffusion lim...