Ells/ cm2 in MSC medium containing 10 MSC-qualified FBS, 5 ng/ ml bFGF in low-glucose DMEM. MSCs had been split every single 4 days, along with a growth curve was constructed by direct cell 8-Hydroxy-DPAT In stock counting at each and every passage. For characterization of MSCs, we analyzed expression of MSC phenotype (CD73+, CD90+, CD105+, CD44+, CD166+, CD29+, CD34 CD45 CD14 CD19 and HLA-DR by flow cytometry. All antibodies and manage immunoglobulin G isotypes were purchased from BD Biosciences. BM-MSCs have been included as a good manage. To test multipotency, we differentiated MSCs to adipocytes, osteocytes, and chondrocytes by STEMPRO Adipogenesis, Osteogenesis, and Chondrogenesis Differentiation Media (Life Technologies). Adipocytes and osteocytes were stained with Nile Red and Alizarin Red S immediately after four and two weeks of differentiation, respectively. Cell aggregates of chondrocytes have been fixed and sectioned for Safranin O staining after two weeks of differentiation.EXPERIMENTAL PROCEDURESCell Lines and CulturesWe obtained WS and typical handle fibroblasts from Coriell Cell Repositories. Extra typical manage fibroblasts (BC, AN1, AN2, and AN3) have been obtained from healthier donors in the Clinical Center of National Institutes Overall health with written consent. hESC lines CT2 and ESI-053 were obtained from University of Connecticut Stem Cell Core and BioTime, respectively. Bone marrow MSCs (BM-MSCs) and ReNcell CX human NPCs (CX-NPCs) had been purchased from StemCell Technologies and Millipore, respectively. All cell cultures were maintained as recommended by the suppliers.Differentiation and Characterization of NPCs Derived from iPSCsTo derived NPCs, we formed embryoid bodies (EBs) from iPSCs cultured on Matrigel with mTeSR1 medium (StemCell Technologies) on AggreWell (Marchetto et al., 2010). EBs have been induced for542 Stem Cell Reports j Vol. 2 j 53446 j April eight, 2014 j 014 The AuthorsStem Cell ReportsTelomerase Protects against Lineage-Specific Agingneural differentiation by STEMdiff Neural Induction Medium (StemCell Technologies) for five days on AggreWell and then transferred to poly-L-ornithine (PLO)/laminin-coated plates for yet another 7 days. Neural rosettes have been collected by treating cell aggregates with Neural Rosette Selection Reagent then had been replated on PLO/laminin-coated plate. NPCs were allowed to grow to confluence from neural rosettes and thereafter passed just about every four days. NPCs have been cultured as monolayer on PLO/laminin-coated plates in N2B27 medium containing 0.five N2, 1 B27, EGF, and bFGF (20 ng/ml of every single) in DMEM/F12 with GlutaMAX (Life Technologies). For characterization of NPCs, we analyzed expression of neural stem markers NESTIN (Millipore) and SOX1 (BD Biosciences) by immunofluorescence staining. To test multipotency, we plated NPCs on Matrigel-coated plates and cultured them in N2B27 medium using the withdrawal of EGF and bFGF and inclusion of neurotrophic components BDNF, CNTF, GDNF, and IGF1 (ten ng/ml of every single) (Peprotech) and 1 mM of cAMP (Wang et al., 2013). Following 2 weeks of differentiation, cells had been examined for the expression of neuronal markers TUJ1, MAP2, and DCX and the astrocyte marker GFAP by immunofluorescence staining.and dropped on HCl-treated glass slides. To eliminate newly synthesized BrdU/BrdC-incorporated DNA strands, cells were stained with 0.5 mg/ml Hoechst 33258, exposed to UV light for half an hour, and after that digested by Exonuclease III (ten U/ml) for 10 min at space temperature. Hybridization was performed utilizing fluorescence-labeled PNA telomere probes (PNA Bio) for.