1. Volume 18, Issue 12, Pages 2173-2181 (December 2010)
Nodal/Activin Signaling Predicts Human Pluripotent Stem Cell Lines Prone to Differentiate Toward the Hematopoietic Lineage 
Veronica Ramos-Mejia, Gustavo J Melen, Laura Sanchez, Ivan Gutierrez-Aranda, Gertrudis Ligero, Jose L Cortes, Pedro J Real, Clara Bueno, Pablo Menendez 
Molecular Therapy 
Volume 18, Issue 12, Pages (December 2010)
DOI: /mt
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Comparative study revealing that hematopoietic differentiation potential varies among different human pluripotent stem cell (hPSC) lines. (a) Schematic of the hematopoietic differentiation protocol from human embryonic stem cell (hESC)/induced pluripotent stem cell (iPSC) lines and end point analyses. (b) Representative flow cytometry dot plots displaying how hemogenic progenitors (CD45−CD31+) and hematopoietic cells (CD45+) are identified and analyzed at day 10, 15, and 22 of embryoid body (EB) development. Differences between 13 hESC lines and one iPSC line regarding their propensity to differentiate into hemogenic progenitors (c) (CD45−CD31+), (d) primitive blood cells (CD45+CD34+), (e) mature blood cells (CD45+CD34−) and (f) differences in their colony-forming unit potential and type. Analysis was consistently performed at day 10, 15, and 22 of EB development. CFUs were plated from day 15 EBs. The horizontal dotted red lines indicate the mean value for all lines studied. All differentiation experiments were independently done at least three times. CFU, colony-forming unit.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Correlation between the total number of CFU and the percentage of embryonic hematopoietic cell subsets. (a) Hemogenic progenitors (CD45−CD31+), (b) primitive blood cells (CD45+CD34+), and (c) mature blood cells (CD45+CD34−). All CFU experiments were independently done at least two times. CFU, colony-forming unit.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Relative expression trends of hematopoietic transcription factors along hEB development. Gene expression kinetics of the mesendodermal (Brachyury and MIXL1) and hematopoietic transcription factors (SCL, HOXA9, GATA1 and PU.1) during a 15-day period of differentiating hEBs from human pluripotent stem cells displaying different hematopoietic potentials. Expression or early (SCL and HOXA9) and late (GATA1 and PU.1) hematopoietic transcription factors does not identify the human embryonic stem cell lines with good hematopoietic differentiation output analyzed by flow cytometry. hEB, human embryonic body.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Differential gene expression profile between human embryonic stem cell (hESC) lines with good versus poor blood differentiation potential. (a) Heatmap diagram summarizing microarray gene expression changes in hESC lines displaying good versus poor blood differentiation potential (left panel). Of note, genes involved in neural differentiation and function are upregulated in hESCs with poor hematopoietic potential while many key genes belonging to the Nodal/Activin pathway are highly upregulated in hESCs with hematopoietic differentiation propensity. Nodal genes are highlighted in red and those genes associated to neural development are shown in blue. The right panel shows in more detail the gene expression changes for each of the six hESC lines studied. (b) Real-time reverse transcriptase-PCR validating the microarray data displaying high expression levels of Nodal, Lefty1, and Lefty 2 in AND1, H1, and SHEF1 (good blood differentiation potential) as compared with H9, HS181, and VAL3 (poor-blood-differentiation potential). (c) Immunocytochemistry confirming expression of the Lefty2 protein in H1 but not in HS181. (d) Scheme of the Nodal/Activin signaling pathway highlighting those members whose expression was four- to ninefold upregulated (red), 1.5- to 2-fold upregulated (blue), or that showed no regulation (white) in hESCs displaying robust hematopoietic differentiation potential. (e) Flow cytometry expression of the early neuroectodermal marker A2B5 in H9 and AND1. Please note that the ability of hESC lines to differentiate toward ectodermal (A2B5+ cells) versus mesodermal (blood) lineages seems to be opposite.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Loss- and gain-of-function assays revealed that inhibition of Nodal/Activin signaling during undifferentiated human embryonic stem cell (hESC) culture abrogates hematopoietic specification whereas its activation augments the hematopoietic differentiation potential. (a) Inhibition of Nodal/Activin signaling during undifferentiated hESC culture (AND1 and H1) by treatment for 4 days with 10 µmol/l of SB , a potent and selective chemical inhibitor of Nodal/Activin signaling, resulted in up to 14-fold reduction of mature hematopoietic cells. (b) Flow cytometry dot plots displaying how these hematopoietic cell subsets are identified and analyzed in the presence and absence of SB treatment. (c) Four-day exogenous addition of 100 ng/ml of Activin-A to HS181 undifferentiated cultures augmented 3.5-fold times the differentiation into mature hematopoietic cells. (d) Flow cytometry dot plots depicting the effect of Activin-A treatment in the hematopoietic differentiation potential of HS181 human embryonic stem cell line.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions