Journal: Nature communications

Article Title: Somatic transcriptome priming gates lineage-specific differentiation potential of human-induced pluripotent stem cell states.

doi: 10.1038/ncomms6605

Figure Lengend Snippet: Figure 3 | Distinct differentiation potential in hiPSCs derived from fibroblasts and cord blood. (a) Quantitative results of neurosphere expansion (n ¼ 14). Right panel shows the images of neurosphere generated at the end of the 2nd expansion cycle of hiPSCs. Scale bars, 200mm. (b) Quantification of the neurosphere generation capacity from 1 K numbers of cells (n ¼ 18). (c) Representative flow cytometry plots for neural stem cell marker nestin and the neural precursor marker A2B5 in neurosphere. Right panel shows total number of nestin- or A2B5-positive cells. (n ¼ 10). (d) Quantitative RT-PCR analysis of neural-specific genes from undifferentiated cells. Individual reactions were normalized to GAPDH. (n ¼ 6). Neural stem cells from hiPSCs were further differentiated into astrocytes as indicted by GFAP staining (e, top panel), oligodendrocytes as indicated by O4 staining (f, top panel) and neurons as indicated by b-III-tubulin staining (g, top panel). Scale bars, 50mm (e,f) and 100 mm (g). Bottom panels for e–g are graphs for quantification of total number of differentiated cells of respective neural lineages (n ¼ 10). (h) GSEA plot of gene signatures related to neural lineage (right) and heat map of genes (left). (i) Representative fluorescence-activated cell sorting plot for the expression of primitive blood cells markers, CD34 and CD45, of hiPSCs hEBs after 15 days hEB culture. (j) Total number of differentiated primitive blood cells and mature blood cells in hEBs from hiPSCs over days 7, 10 and 15 (n ¼ 14). (k) Total number of differentiated CD34 þCD45 þ cells from hiPSCs (n ¼ 10). (l) Total number of hematopoietic CFUs and number of colony subtypes generated from day 15 hEBs. Inset shows hematopoietic progenitor capacity of hEBs. (n ¼ 10). (m) Distribution of colony subtypes derived from hiPSC lines. (n) Quantitative RT-PCR analysis for blood-specific genes. Samples are purified from undifferentiated cells and individual reactions were normalized to GAPDH. (n ¼ 6). (o) GSEA plot of enrichment of gene signature related to hematopoietic lineage (right) and heat map of genes (left). (p) Quantification of expression of neural stem cell marker nestin during neural differentiation (left). Quantitative results of CD45 þ-positive cells during hematopoietic differentiation (right). All bars indicate average of all clones. All data are represented as mean±s.e.m. *Po0.05, **Po0.01, Student’s t-test.

Article Snippet: Single-cell suspensions were stained using the following antibodies: CD31-PE (1:100) (BD Pharmingen), CD34-PE or -FITC (1:100) (Miltenyi Biotech, Bergisch Gladbach, Germany), CD45-APC (1:100) (Miltenyi Biotech), SSEA-1 (1:100), SSEA-3 (1:100), SSEA-4 (1:100) (Developmental Studies Hybridoma Bank), TRA1-60-Alexa Fluor 647 (1:500), TRA1-85-APC (1:500), OCT4 (1:200) (BD Biosciences), A2B5 (1:100), Nestin (1:500), Oligodendrocyte Marker O4 (1:200), Neuron-specific beta-III Tubulin (1:200) (R&D Systems), GFAP (1:200) (Sigma) and Haemoglobin b-PerCP (1:500) (Santa Cruz).

Techniques: Derivative Assay, Generated, Cytometry, Marker, Quantitative RT-PCR, Staining, FACS, Expressing, Clone Assay