title: Formation of well-defined embryoid bodies from dissociated human induced pluripotent stem cells using microfabricated cell-repellent microwell arrays.
abstract: A simple, scalable, and reproducible technology that allows direct formation of large numbers of homogeneous and synchronized embryoid bodies (EBs) of defined sizes from dissociated human induced pluripotent stem cells (hiPSCs) was developed. Non-cell-adhesive hydrogels were used to create round-bottom microwells to host dissociated hiPSCs. No Rho-associated kinase inhibitor (ROCK-i), or centrifugation was needed and the side effects of ROCK-i can be avoided. The key requirement for the successful EB formation in addition to the non-cell-adhesive round-bottom microwells is the input cell density per microwell. Too few or too many cells loaded into the microwells will compromise the EB formation process. In parallel, we have tested our microwell-based system for homogeneous hEB formation from dissociated human embryonic stem cells (hESCs). Successful production of homogeneous hEBs from dissociated hESCs in the absence of ROCK-i and centrifugation was achieved within an optimal range of input cell density per microwell. Both the hiPSC- and hESC-derived hEBs expressed key proteins characteristic of all the three developmental germ layers, confirming their EB identity. This novel EB production technology may represent a versatile platform for the production of homogeneous EBs from dissociated human pluripotent stem cells (hPSCs).
authors.affiliation: Institute for Engineering and Medicine, Department of Biomedical Engineering, Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, 23284 USA.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pettinato
authors.affiliation: Institute for Engineering and Medicine, Department of Biomedical Engineering, Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, 23284 USA.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Wen
authors.affiliation: Institute for Engineering and Medicine, Department of Biomedical Engineering, Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, 23284 USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Zhang
mesh_terms: D000943:Antigens, Differentiation / Q000096:biosynthesis*; D018929:Cell Culture Techniques / Q000379:methods*; D058732:Embryoid Bodies / Q000166:cytology / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D046888:Tissue Array Analysis / Q000379:methods*
chemical_list: D000943:Antigens, Differentiation