Directed Differentiation of Human Embryonic Stem Cells as Spin Embryoid Bodies and a Description of the Hematopoietic Blast Colony Forming Assay

Abstract

This unit describes a protocol for the differentiation of human embryonic stem cells (hESCs). To generate spin embryoid bodies (EBs), known numbers of hESCs are deposited into low‐attachment, round‐bottomed 96‐well plates in a serum‐free medium supplemented with growth factors. The cells are then aggregated by centrifugation, initiating formation of EBs of uniform size. The spin EBs generated using this technique differentiate efficiently and synchronously along the lineages preferentially induced by the combinations of growth factors to which the cells are exposed. The 96‐well format permits an assessment of the effects of different combinations of growth factors in the same experiment, facilitating the optimization of differentiation conditions for any given cell type. Up to 40 plates can be set up within a couple of hours by one experimenter, and aliquots of the differentiating EBs can be harvested at intervals and subjected to analyses using a variety of techniques. Curr. Protoc. Stem Cell Biol. 4:1D.3.1‐1D.3.23. © 2008 by John Wiley & Sons, Inc.

Induced pluripotent stem cells offer new approach to therapy in thalassemia and sickle cell anemia and option in prenatal diagnosis in genetic diseases

“…After washing, the cells were resuspended in the first differentiation medium, which is the basal medium, SFM supplemented with human recombinant bone morphogenetic protein 4, human VEGF (hVEGF), human stem cell factor (hSCF), human Flt3, human IL3, human IGF-II, and TPO (22). Embryoid bodies (EBs) were made by plating these cells in a V-bottom 96-well plate and centrifuged according to the method of Ng et al (23,24). Initially, we optimized the formation of EB by plating 5,000, 10,000, and 20,000 cells in the wells.…”

Functionality of Endothelial Cells and Pericytes From Human Pluripotent Stem Cells Demonstrated in Cultured Vascular Plexus and Zebrafish Xenografts

“…36 This also might not be convenient if working with the multiple diseased hiPSC lines at the same time. We also noticed rather high line-to-line variation in the spin EB system when using hiPSC (Orlova, unpublished).…”

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“…The Ednrb Kik -or Ret TGMpositive cells were isolated by flow cytometry (MoFlo; Beckman Coulter) and sorted into round-bottomed, low-attachment, sterile 96-well plates (Corning, Costar) at a density of 10,000 cells/well (or 50,000 cells/ml). The cells were aggregated by centrifugation at 480 g for 3 minutes at 4°C, as described previously (73) and then were cultured in DMEM/F12 (GIBCO, Invitrogen) containing 1% l-glutamine (Sigma-Aldrich), 1% penicillin/ streptomycin, 1× B-27 supplement (GIBCO, Invitrogen), 1× N-2 supplement (GIBCO, Invitrogen), and 20 ng/ml EGF and bFGF in a humidified incubator at 37°C and 5% CO2 for 7 days to allow for NS formation.…”