Project description:Heterogeneity among iPSC lines with regard to their gene expression profile and differentiation potential has been described and has been at least partly linked to the tissue of origin. We generated iPSCs from primitive (linneg) and non-adherent differentiated (linpos) bone marrow cells (BM-iPSC), and compared their differentiation potential to that of fibroblast-derived iPSCs (Fib-iPSC) and ESCs. In the undifferentiated state, individual iPSC clones but also ESCs proved remarkably similar when analyzed for alkaline phosphatase and SSEA-1 staining, endogenous expression of the pluripotency genes Nanog, Oct4, and Sox2, or global gene expression profiles. However, substantial differences between iPSC clones were observed after induction of differentiation, which became most obvious upon cytokine-mediated instruction towards the hematopoietic lineage. All three BM-iPSC lines derived from undifferentiated cells yielded high proportions of cells expressing the hematopoietic differentiation marker CD41, and in two of these lines, high proportions of CD41+/CD45+ cells were detected. In contrast, little hematopoiesis-specific surface marker expression was detected in linpos BM-iPSC and FIB-iPSC lines. These results were corroborated by functional studies demonstrating robust colony outgrowth from hematopoietic progenitors in two of the linneg BM-iPSCs only. Thus, in summary our data demonstrate efficient generation of iPSCs from primitive hematopoietic tissue as well as efficient hematopoietic redifferentiation for linneg BM-iPSC lines, thereby further supporting the notion of an epigenetic memory in iPSCs. Murine embryonic fibroblasts (MEFs) from C3H mice were cultured in low-glucose DMEM supplemented with 10% heat-inactivated fetal calf serum gold (PAA, Pasching, Austria), penicillin-streptomycin, 1 mM L-glutamine and 0.05 mM beta-mercaptoethanol on gelatine-coated dishes. C3H MEFs were grown to confluence, inactivated with 10 ug/ml Mitomycin C (Sigma) and used as feeder layers. Virus production was performed in a four plasmid-manner. Briefly, 3.5x10^6 293T cells were seeded 24h prior to transfection in 10 cm dishes. 293T cells were cultivated in high-glucose DMEM (Gibco) supplemented with 10% heat-inactivated FCS, penicillin-streptomycin and 1 mM L-glutamine. Cells were transfected with 5 ug lentiviral vector, 8 ug pcDNA3.GP.4xCTE (expressing HIV-1 gag/pol), 5 ug pRSV-Rev and 2 ug pMD.G (encoding the VSV glycoprotein) using the calcium phosphate method in the presence of HEPES and chloroquine. Supernatants were harvested 48h and 72h after transfection, filtered and subsequently 50x concentrated by ultracentrifugation. Titers determined based on real-time PCR, were in the range of 1-5x10^7/ml. For iPSC generation, bone marrow cells were isolated from femurs and tibias of Oct4-GFP transgenic mice (OG2) and immunomagnetically separated into lineage negative (Lin-) and lineage positive (Lin+) populations using the mouse lineage depletion kit (Miltenyi Biotec). Lin- cells were cultivated in serum-free StemSpan medium (Stem Cell Technology) supplemented with 2 mM L-glutamine, penicillin-streptomycin, 10 ng/ml mSCF, 20 ng/ml mTPO, 20 ng/ml, 20 ng/ml IGF-2 and 10 ng/ml FGF-1 (all Peprotech). Lin+ cells were cultivated in Iscove's modified eagle medium (IMDM), supplemented with 15% heat-inactivated FCS, 1 mM L-glutamine, penicillin-streptomycin, 100 ng/ml mSCF, 100 ng/ml mFLT3-L, 10 ng/ml hIL-3 and 100 ng/ml hIL-11. Both Lin- and Lin+ cells were pre-stimulated in the aforementioned media for 48 h. Thereafter, 2x10^5 Lin- and and Lin+ bone marrow cells were transduced on Retronection-coated plates (Takara) with lentiviral vectors encoding for human Oct4, Sox2, Klf4 and c-Myc using a multiplicity of infection (MOI) of 50 per virus. Twenty-four hours after transduction, media were supplemented with 2 mM valproic acid. Transduced bone marrow cells were kept in hematopoietic medium until 5 or 7 days post transduction (p.t.) and then transferred onto Mitomycin C-treated MEF feeders on gelatine-coated dishes. Henceforward, cells were cultivated in ES cell medium (knockout DMEM (Gibco), 15% ES-tested FCS, 1 mM L-glutamine, 0.1 mM non-essential amino acids (Gibco), 100 uM beta-mercaptoethanol (Sigma), penicillin-streptomycin and 103 units/ml leukemia inhibitory factor (LIF, provided by the Max-Planck-Institute, Munster, Germany). Upon appearance of GFP-positive ESC-like colonies, single colonies were picked based on morphology and GFP expression. Murine ESCs and iPSCs were cultured on Mitomycin C-treated MEF feeders in the aforementioned ES medium. Murine ESCs and iPSCs were passaged every 2-3 days. The murine embryonic fibroblast-derived iPSC lines (MEF-iPS, 3FLV2, 4FLV1) were generated by transduction of OG2-MEFs with the same lentiviral vector constructs using standard technology. For iPSC lines 3FLV2 and 4FLV1, complete reprogramming was demonstrated by alkaline phosphatase and SSEA1-staining, pluripotency factor expression and teratoma formation.
2012-08-10 | GSE29635 | GEO
Mario Fernandez 
