Pluripotent stem cell marker analysis iPSCOCT express plurip

Pluripotent stem cell marker analysis: iPSCOCT4 express pluripotent markers SSEA3, TRA-1-60, OCT4, NANOG and SOX2 as measured by flow cytometry (Fig. 1E). Pluripotent functionality: iPSCOCT4 are capable of three germ layer differentiation capacity as assessed by in vivo teratoma assay (Fig. 1F).
Materials and methods Methodology used for cell line generation: Human adult dermal fibroblasts (104 adherent cells per well of a 12-well plate seeded on Matrigel the day before transduction) were incubated for 24h with concentrated pSIN-EF1α-OCT4-Puro lentiviral vector in 0.5mL hFib medium in the presence of 8μg/mL polybrene (Sigma Aldrich). After 24h of incubation, 2mL of reprogramming medium (RM) [DMEM/F12 (Gibco) with 20% Knockout Serum Replacement (Gibco), 100μM β-mercaptoethanol, 100μM nonessential amino lgk974 (Gibco), 1mM L-glutamine (Gibco) supplemented with 16ng/mL basic fibroblast growth factor (bFGF) and 30ng/mL insulin-like growth factor (IGFII)] were added to the well. Following a further 24h of incubation, lentiviral transduction conditions were replaced entirely with RM. Cells were maintained in this condition for prolonged culture with media changes and removal of areas of overgrown fibroblasts as necessary. iPSC colonies emerged between days 45 and 93, and were mechanically isolated and further expanded in mouse embryonic fibroblast conditioned media.
Flow cytometry Human Fib iPSCOCT4 were dissociated to single cell suspensions using TrypLE Express and were analyzed for the presence of SSEA3-PE and TRA-1-60-Alexa Fluor 647 (BD Pharmingen) cell surface markers. For detection of intracellular pluripotent transcription factors cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit. Cells were incubated overnight at 4°C with conjugated antibodies OCT4-Alexa Fluor 488, SOX2-Alexa Fluor 647 and NANOG-PE (BD Pharmingen). Flow cytometric analysis was performed using the BD LSRII Flow Cytometer with BD FACSDiva software and analyzed with FlowJo software (Tree Star Inc).
Teratoma formation To assess the development potential of hFib iPSCOCT4in vivo, cells were collected by collagenase IV treatment and injected into the left testicle of NOD/SCID mice (approximately one well of a 6 well plate of 80% confluence for each mouse). At eight to ten weeks, teratomas were harvested, dissected and fixed with 4% paraformaldehyde. Samples were embedded in paraffin and processed with hematoxylin and eosin staining.
Verification and authentication Karyotype: iPSCOCT4 lines derived from three independent fibroblast sources were analyzed by array comparative genomic hybridization (aCGH) to assess karyotype at a genomic resolution greater than that obtained using standard cytogenetic procedures. One iPSC line possessed normal karyotype, while the others possessed two and five amplifications, the majority of which are found in regions/chromosomes that are recurrently altered in PSCs (Fig. 1G) (Baker et al., 2007; Martins-Taylor et al., 2011; Taapken et al., 2011). iPS line identity: Matching RFLP signatures with starting fibroblast cells confirms the origins of iPSCOCT4 (Fig. 1D).
Resource table. Resource details Primary fibroblasts were grown from the skin biopsy donated by a healthy 83-year old male volunteer donor.
Materials and methods Cell culture and reprogramming. Dermal fibroblasts from healthy 83-year old male donor were derived from a skin biopsy and cultured in DMEM (Sigma) supplemented with 10% FBS (Life Technologies) and GlutaMAX (Life Technologies). Protocol for fibroblast derivation can be found http://research.med.helsinki.fi/neuro/Otonkoski/protocols. Fibroblasts were reprogrammed using CytoTuneTM-iPS Sendai Reprogramming Kit (https://www.lifetechnologies.com/order/catalog/product/A1378001) using the method described (Trokovic et al.,in press-a,b). To enhance the reprogramming of fibroblasts 0.25mM sodium butyrate (NaB; Sigma, B5887) was added to all reprogramming experiments. HEL47.2 was cultured in hESC medium: DMEM/F12 with GlutaMAX (Life Technologies), 10% KnockOut Serum Replacement (Life Technologies), 0.1mM 2-mercaptoethanol (Life Technologies), 1× Non-Essential Amino Acids (Life Technologies), and 6ng/ml bFGF (Sigma) and routinely propagated with combination of collagenase IV treatment and mechanic dissociation. HEL47.2 was subsequently adapted and culture in a feeder free conditions on matrigel in the presence of E8 medium (Life Technologies). iPSC lines were routinely split using 0.5mM EDTA and thawed in the presence of Rho-kinase inhibitor (Y-27632, Sigma) during 24h after thawing.