Three-Dimensional Porous Alginate Scaffolds Provide a Conducive Environment for Generation of Well-Vascularized Embryoid Bodies From Human Embryonic Stem Cells Sharon Gerecht-Nir, 1,2 Smadar Cohen, 3 Anna Ziskind, 2,4 Joseph Itskovitz-Eldor 2,4 1 Biotechnology Interdisciplinary Unit, Technion – Israel Institute of Technology, Haifa, Israel 2 Department of Obstetrics and Gynecology, Rambam Medical Center, POB 9602, Haifa 31096, Israel; telephone: +972-4-854-3236; fax: +972-4-854-2579; e-mail: Itskovitz @rambam.health.gov.il 3 Department of Biotechnology Engineering and the Institute for Applied Biosciences, Ben-Gurion University of the Negev, Beer Sheva, Israel 4 Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel Received 19 December 2003; accepted 18 March 2004 Published online 12 October 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20248 Abstract: Differentiation of human embryonic stem cells (hESCs) can be instigated through the formation of embryo-like aggregates in suspension, termed human embryoid bodies (hEBs). Controlling cell aggregation and agglomeration during hEBs formation has a profound ef- fect on the extent of cell proliferation and differentiation. In a previous work, we showed that control over hEBs formation and differentiation can be achieved via cultiva- tion of hESC suspensions in a rotating bioreactor system. We now report that hEBs can be generated directly from hESC suspensions within three-dimensional (3D) porous alginate scaffolds. The confining environments of the algi- nate scaffold pores enabled efficient formation of hEBs with a relatively high degree of cell proliferation and dif- ferentiation; encouraged round, small-sized hEBs; and in- duced vasculogenesis in the forming hEBs to a greater extent than in static or rotating cultures. We therefore conclude that differentiation of hEBs can be induced and directed by physical constraints in addition to chemical cues. B 2004 Wiley Periodicals, Inc. Keywords: alginate scaffold; human embryonic stem cells; differentiation; agglomeration INTRODUCTION Human embryonic stem cells (hESCs) provide a unique resource for numerous types of differentiated cells for transplantation in human therapy. Differentiation of hESCs can be instigated in cell suspensions via the formation of human embryoid bodies (hEBs), in which various differ- entiation events in the developing embryo yield diverse cells that constitute the human body (Itskovitz-Eldor et al., 2000). Recent data have shown that spontaneous neo- vasculogenesis occurs in differentiating hEBs (Gerecht-Nir et al., 2003; Levenberg et al., 2002). Endothelial cells were observed from day 4 of differentiation, whereas vascular arrangements and networks have been observed from day 10 of differentiation (Gerecht-Nir et al., 2003; Levenberg et al., 2002). We recently reported the formation of hEBs in dynamic culture using the rotating cell culture system (Gerecht-Nir et al., 2004). The slow-turning lateral vessel (STLV) of a specific bioreactor significantly enhances the formation and differentiation of hEBs, as compared with conven- tional Petri dishes. These studies showed that, by con- trolling cell aggregation via manipulation of the fluid dynamics in the vessel, formation of hEBs was more ef- ficient, yielding highly proliferating hEBs with enhanced differentiation capabilities. We now report the formation of hEBs within three- dimensional (3D) porous alginate scaffolds. We hypothe- sized that the confined environment of the pore structure in the scaffold would enable the formation of a more homog- eneous population of hEBs, minimize agglomeration of hEBs, and eventually lead to efficient cell proliferation and differentiation. Alginate scaffolds are characterized by a macromolecular structure resembling the extracellular matrix; their hydrogel nature enables efficient cell seeding and tissue engineering (Glicklis et al., 2000; Leor et al., 2000; Shapiro and Cohen, 1997). The porosity of the alginate scaffolds can be controlled during fabrication, yielding sponge-like material with >90% porosity, con- necting pore structures and pore sizes ranging from 50 to B 2004 Wiley Periodicals, Inc. Correspondence to: J. Itskovitz-Eldor Contract grant sponsor: National Institutes of Health; Technion Re- search and Development Foundation, Ltd. Contract grant number: 1RO1Hl73798-01