Journal: Stem cells (Dayton, Ohio)

Article Title: Improved safety of hematopoietic transplantation with monkey embryonic stem cells in the allogeneic setting.

doi: 10.1634/stemcells.2005-0391

Figure Lengend Snippet: Figure 1. Flow cytometric analysis during the in vitro differentiation of cyESCs. Undifferentiated cyESCs expressing green fluorescent protein were cultured on OP9 cells with multiple cytokines (see Materials and Methods). (A): Cells on day 0 are shown in bright (left) and dark (right) fields. (B): Cells on day 6 are shown in bright (left) and dark (right) fields. (C): Cells on days 0, 4, 6, 12, and 18 were stained for CD34. (D): Cells on days 0, 4, 6, 12, and 18 were stained for CD31. (E): Cells on days 0, 4, 6, 12, and 18 were stained for VE-cadherin. (F): Cells on days 0, 4, 6, 12, and 18 were stained for CD45. The vertical axis shows the fraction (percentage) of cells that were stained positive. (C–F): Results of two or three independent experiments are shown. (G): Although cells on day 0 already express low levels of VEGFR-2, a VEGFR-2high population did not emerge until day 6. (H): Dot-plot profiles for VEGFR-2 and VE-cadherin expression indicate that cells positive for both VEGFR-2 and VE-cadherin emerged until day 6. (G, H): Representative results from three independent experiments are shown. (I): The Scl gene expression was upregulated on day 6 to a level similar to that in the cynomolgus fetal liver as assessed by RNA polymerase chain reaction. Day-6 cells (putative hematopoietic precursors) were used for transplantation. Abbreviations: cyESC, cynomolgus embryonic stem cell; cyFL, cynomolgus fetal liver; DW, distilled water; VE, vascular endothelial; VEGFR, vascular endothelial growth factor receptor.

Article Snippet: Briefly, undifferentiated cyESCs were transferred onto mitomycin Ctreated confluent OP9 cells and cultured for 6 days in Iscove’s modified Dulbecco’s medium (Invitrogen) supplemented with 8% FCS, 8% horse serum (Invitrogen), 5 10 6 M hydrocortisone (Sigma, St. Louis, http://www.sigmaaldrich.com), and multiple cytokines, including 20 ng/ml recombinant human (rh) bone morphogenetic protein-4 (R&D Systems, Minneapolis, http://www.rndsystems.com), 20 ng/ml rh stem cell factor (Biosource, Camarillo, CA, http://www.biosource.com), 20 ng/ml rh vascular endothelial growth factor (VEGF; R&D Systems), 20 ng/ml rh Flt-3 ligand (PeproTech, Rocky Hill, NJ, http://www. peprotech.com), 20 ng/ml rh interleukin-3 (PeproTech), 10 ng/ml rh interleukin-6 (PeproTech), 20 ng/ml rh granulocyte colony-stimulating factor (PeproTech), and 2 IU/ml rh erythropoietin (Roche, Basel, Switzerland, http://www.roche.com).

Techniques: In Vitro, Expressing, Cell Culture, Staining, Gene Expression, Polymerase Chain Reaction, Transplantation Assay