UCB-stem cell Class A (Profile-1) transcription factors.

1<p>ES, enrichment score of TFs identified by GSEA using three TF gene sets (TF activity, TF complex and DNA binding). A positive ES indicates the gene expression correlated with a Profile-1 pattern (decreased expression from day 21 to day 56).</p>2<p>Gene Rank, gene rank list generated by GSEA using 3786 TFs with >1.5-fold changes in expression from day 21 to day 56 in culture.</p><p>UCB-stem cell Class A (Profile-1) transcription factors.</p

TF networks identified in erythroid progenitors generated from adult stem cells.

<p>A) Profile-1 (3142) and Profile-2 (5517) genes generated by PCA of data generated from adult stem cells. We compared day 7 to day 28 for GSEA analysis to produce ES and gene rank list as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107133#pone-0107133-g004" target="_blank">Figure 4A</a>. We identified 18 Profile-2 (Class B) and 20 Profile-1 (Class A) TFs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107133#pone.0107133.s007" target="_blank">Table S7</a>). B) Hierarchical clustering analysis was performed for TFs identified by GSEA. The same color code was used as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107133#pone-0107133-g004" target="_blank">Figure 4B</a>. C) Shown is a major TFN generated by Cytoscape analysis of Profile-1 genes. The key is included for interpretation of predicted regulatory interactions. D) Shown is a major TFN generated by Cytoscape analysis of Profile-2 genes. The interaction key is the same as in panel C.</p

Erythroid progenitor characterization in the one-phase culture system.

<p>A) Cells were harvested every 7–14 days and cell counts and viability were performed by trypan blue exclusion. Over 10 billion cells were generated by day 56 in culture. **p<0.01 B) Shown is the cellular morphology demonstrated by Giemsa stain (40X magnification). C) Shown is a summary of the erythroid progenitors counted at the different days after Giemsa stain. At least 500 cells were counted per slide in triplicate. Abbreviation: MNC, mononuclear cells; BE, basophilic erythrpoblast, PE, polychromatophilic erythroblast, OE, orthochromatophilic erythroblast. D) Changes in cell surfaced biomarkers that occurred during fetal erythroid differentiation were measured by cytospin preparation and immunohistochemical stain (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107133#s2" target="_blank">Materials and Methods</a>). *p<0.05.</p

GSEA Analysis.

<p>A) Profile-1 (2568) and Profile-2 (2458) genes generated by PCA comparing day 21 to day 56 gene expression levels, were analyzed by GSEA to generate enrichment scores (ES) and a gene rank list. (Top) The top portion of the plot shows the ES for the different TFs as the analysis walks down the ranked gene list. The scores at the peak of the plot (the scores furthest from 0.0) are shown. TFs with a peak at the beginning or end of the ES plot are the most interesting. (Middle) This portion of the figure shows the position of the TFs relative to the ranked list of genes. (Bottom) The bottom portion of the plot shows the value of the ranking metric which measures a TFs' correlation with differential expression on day 21 and day 56. The Class A (Profile-1) genes have positive ES values and Class B (Profile-2) genes correlate with a negative ES value. B) Hierarchical clustering was performed for Profile-1 and Profile-2 genes. Culture day (columns) and genes (rows) were clustered by log-transformed intensity values using correlation distance with similarity metric and centroid linkage. Up-regulated genes are shown in red, repressed genes in green and genes with no change in expression in black. C) Shown is a major TFN generated by Cytoscape analysis of Profile-1 genes. The key is included for interpretation of predicted regulatory interactions. D) Shown is a major TFN generated by Cytoscape analysis of Profile-2 genes. The interaction key is the same as in panel C.</p

TESS/TFSEARCH predicted binding motifs across the β-globin locus.

1<p>Microarray data shown as fold change in expression from day 21 to day 56;</p>2<p>Nucleotide abbreviations: N = G, A or T; K = G or T, Y = T or C, M = A or C, R = A or G;</p>3<p>The Log-likelihood scores is a statistical measure representing the probability that a TF binding site exists in the region analyzed; we used a cutoff ≥7.0. The higher the score the more likely the predicted sequence binds the target TF indicated.</p>4<p>Number of binding sites identified in the different regions in the β-locus. For example there are 15, 21 and 10 predicted GATA1 binding sites in the LCR, γ-globin and β-globin regions respectively.</p><p>TESS/TFSEARCH predicted binding motifs across the β-globin locus.</p

The γ/β-globin switch is recapitulated in UCB-stem cells.

<p>A) RT-qPCR analysis was performed at the days shown to determine changes in γ-globin and β-globin mRNA levels after normalization by the internal control GAPDH. B) RT-qPCR was performed with Gγ- and Aγ-globin specific primers to determine the Gγ/Aγ mRNA ratio during erythropoiesis. A representative gel is shown. Gγ-globin expression was normalized to one (black bars) and the relative changes in Aγ-globin mRNA are shown in the gray bars. C) Progenitors were induced on day 42 or day 56 with 50 µM hemin (HE) for 72 hr and then RT-qPCR was completed to measure γ-globin and β-globin mRNA levels.</p

shRNA treatment of lead TFs augments γ-globin expression.

<p>KU812 cells were treated with shRNA lentiviral particles and selected with puromycin for 5 days followed by RT-qPCR and western blot analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107133#s2" target="_blank">Materials and Methods</a>). A) Shown is the fold change in γ-globin mRNA after treatment with the shRNA viral particles. γ-Globin expression was normalized by the percent of green fluorescent protein (GFP) positive cells representing transduction efficiency. B) RT-qPCR analysis was performed to confirm target gene silencing after shRNA treatment. ***p<0.0001 C) Western blot analysis was performed to confirm target gene silencing. D) RT-qPCR analysis of gene expression in shKLF1-treated KU812 cells to determine potential downstream gene targets. E) Erythroid progenitors generated from UCB-SC were transduced with shRNA lentiviral particles and selected with puromycin for 5 days followed by RT-qPCR and FACS analysis. Shown is the level of γ/γ+β-globin ratio after shRNA treatments. F) Target gene silencing was quantified after shRNA transductions.</p

UCB-stem cell Class B (Profile-2) transcription factors.

1<p>ES, enrichment score of TFs identified by GSEA using three TF gene sets (TF activity, TF complex and DNA binding). A negative ES indicates genes with a negative correlation to Profile-1 (i.e. Profile-2 gene with increased expression from day 21 to day 56).</p>2<p>Gene Rank, gene rank list generated by GSEA using 3786 TFs with >1.5-fold changes in expression from day 21 to day 56 in culture.</p><p>UCB-stem cell Class B (Profile-2) transcription factors.</p

PCA defines major gene profiles during erythropoiesis.

<p>A) PCA was performed for gene subsets with >1.5-fold change in expression from day 21 to day 56. The results are shown for the two major gene profiles generated by PCA. The gray line represents the mean value of time course changes in gene expression. B) Microarray data for two known γ-globin regulators were confirmed by RT-qPCR. C) Microarray data for three Profile-2 genes were confirmed by RT-qPCR.</p