3C

3C. proliferate. Summary PrP+ cells isolated from EB included undifferentiated cells in day time 21. PrP+/SSEA1C cells included cardiomyoctes, suggesting PrP and SSEA1 may be useful as markers to enrich the portion of cardiomyocytes. suggesting that PrP+ cells can proliferate and form tumors after transplantation.7, 9, 21 This might indicate the harmfulness of PrP+ cells like a cell resource for transplantation. However, it has never been tested whether PrP+ cells from EB include undifferentiated cells. In the present study, we attempted to characterize PrP+ cells derived from EB created by mouse Sera cells. We found that PrP+ cells from EB of days 21, but not those of day time 7 and 14, indicated pluripotency markers BX-912 and were capable of proliferation. Combining the PrP with stage specific embryo antigen 1 (SSEA1) as the second marker enabled us to enrich the portion of cardiomyocytes that do not proliferate. MATERIALS AND METHODS Cell tradition and differentiation Abdominal1 Sera cells derived from 129SV/EV mice were kindly provided by Dr. Shimotsuke (Riken CDB, Kobe, Japan). They were cultured on SNL feeder cells treated with mitomycin C (Sigma-Aldrich, St Louis, MO). SNL cells were derived from STO mouse embryonic fibroblasts having a pressured manifestation of (LIF) and in one of the loci.22 Derived from the ht7 cells, hcgp7 (in one of the loci.23 BX-912 Both ht7 and hcgp7 cells were produced and managed on gelatin-coated IL-1A dishes in Glasgow minimum essential medium (GMEM; Wako Pure Chemical) supplemented with 10% heat-inactivated FBS (Corning), 1 penicillin-streptomycin-L-glutamine answer (Wako Pure Chemical), 1 MEM non-essential amino acid answer (Wako Pure Chemical), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 1 mM sodium pyruvate (Wako Pure Chemical), and 1,000 models/mL LIF (Merck KGaA), without feeder cells. Differentiation of Sera cells into cardiac progenitors was induced via formation of EB. Briefly, EB were generated by plating 20 L of cell suspension (2.5C10 104 cells/mL) in DMEM (Wako Pure Chemical) supplemented with 10C20% heat-inactivated FBS (Corning), 1 penicillin-streptomycin-L-glutamine solution (Wako Pule Chemical), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich) (EB medium) within the lid of a dish, followed by incubation in hanging drops for 2 days. EB were transferred into the medium and cultured as floating EB or attached out-growth cells for indicated days until analysis. Circulation cytometry Cells were dissociated from EB at day time 7 1, 14 1 and 21 1 by Collagenase type (Worthington, Lakewood, NJ) with mild pipetting, followed by a treatment with Cell Dissociation Buffer (enzyme-free, Hanks-based; Thermo Fisher Scientific, Waltham, MA) for 5C8 min. Cells were stained with phycoerythrin (PE) -conjugated anti-PrP (mouse monoclonal clone SAF83; Funakoshi, Tokyo, Japan) labeled with the PE Labeling Kit-NH2 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturers instructions. Dead cells were excluded with Draq7 (Biostatus, Shepshed, England). The percentage of cells positive for PrP or GFP was determined by circulation cytometry (BD FACS Canto II; BD, Franklin Lakes, NJ). They were resuspended in Hanks balanced salt answer (HBSS, Wako Pure Chemical) comprising 2% FBS and Draq7, diluted 100 occasions, and subjected to cell sorting (Moflo XDP, Beckman Coulter, Brea, CA) with Summit software to collect either PrP+ or GFP+ cells.20 Clonogenic cell assay BX-912 PrP+ cells were isolated from EB at day time 7, 14 and 21 by FACS. 1,000 or 10,000 cells were seeded on gelatin-coated dish and cultured in EB medium for 7 to 17 days. Colonies fixed with 100% ethyl alcohol were stained with Giemsa. Reverse transcriptase-polymerase chain reaction Total RNA was isolated BX-912 from EB using an RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturers instructions. RNA samples were treated with DNaseI (Promega Corporation, Fitchburg, WI) to remove genomic DNA and cDNA was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, Kusatsu, Japan). PCR amplifications were performed using Emerald Amp Maximum polymerase (Takara Bio) with primers outlined in Table 1. Table 1. Primer list in gene manifestation analysis = 5.8. = 3), 13.7, (= 1.9. = 3) and 18.3, (= 12.7. = 3) (%) in EB of day time 7, 14 and 21, respectively. PrP+ cells were not recognized in EB before day time 4 of differentiation induction.