The synergistic effect of Lhx2 expression on colony formation is growth factor-specific. A.

<p>Frequency of definitive CFCs in day 6 EB cells generated from iLhx2 ES cells responding to the indicated growth factors/growth factor combinations in clonal assays performed in the absence (−Dox) or presence (+Dox) of dox. B. Frequency of definitive hematopoietic progenitor cells in day 6 EB cells responding to the indicated growth factor combinations in clonal assays in the absence (−Dox) or presence (+Dox) of dox. Factor mix is Tpo, IL-3, IL-6, Flt3L, epo. C. Number of definitive hematopoietic progenitor cells responding to the indicated growth factor combinations in the absence (−Dox) or presence (+Dox) of dox. * p<0,002, ** p<0,01, *** p<0,005.</p

Efficiency in generating DoxHPC lines from individual colonies in clonal assays of EB cells

*<p>(Number of DoxHPC lines generated/Number of colonies picked)</p><p>ND, Not done.</p

Lhx2 expression in intact EBs transiently induce expansion of definitive hematopoietic progenitor cells in the absence of exogenously added growth factors.

<p>A. Gene expression analysis by real-time PCR on day 6 EB generated from the iLhx2 ES cells comparing control EBs (−Dox) to those where Lhx2 expression was turned on at day 4 of differentiation (+Dox). B. Frequency of definitive CFCs responding to SCF/IL-6/epo in clonal assays of day 6 EBs in the presence (+Dox) or absence (−Dox) of dox, if dox was added at day 3 or 4 of differentiation, or if dox was present between day 3 and 5 of differentiation. Control is day 6 EBs generated without dox. C. Frequency of definitive CFCs responding to SCF/IL-6/epo in clonal assays of day 6, 7 and 8 EBs in the presence (−Dox) of absence (+Dox) of dox if dox was added or not at day 4 of differentiation. Hence, the following combinations were analysed: −− no dox added to the ES cells differentiation or to clonal assays of EB cells, −+ dox was added to clonal assays of day 6, 7 and 8 EB cells. +− dox was added to day 4 EB and no dox was added to clonal assays of the day 6, 7 and 8 EB cells. ++ dox was added to day 4 EBs and to the clonal assays of the day 6, 7 and 8 EB cells. Asterisks indicate where the difference in CFC between clonal assays performed in the absence or presence of dox is statistically significant, *p<0,005, **p<0,05.</p

Analyses of the effect of Lhx2 expression at different stages of ES cell differentiation.

<p>A. Frequency of definitive CFCs in EB cells generated from the iLhx2 ES cells at the indicated days of differentiation starting at day 2,5 analysed in clonal assays in the absence (−Dox) or presence (+Dox) of dox. B. Frequency of CFC-Blast in day 3,25 EBs analysed in clonal assays with SCF and VEGF in the absence (−Dox) or presence (+Dox) of dox. C. Frequency of EryP precursor cells in day 6 EBs if dox is added during the indicated time points of ES cell differentiation and omitted in the clonal assays, compared to when no dox is added (Control) or if dox is added to clonal assays of control EBs (Control +Dox). The latter two control experiments are equivalent to the experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002025#pone-0002025-g002" target="_blank">Figure 2A</a> using the iLhx2 ES cells. D. Relative number EB cells recovered at day 6 of differentiation if dox is added to the iLhx2 ES cell line at the indicated time points during differentiation compared to when EBs are generated in the absence of dox (Control) which is arbitrarily set as 1. * p<0,005 compared to control. ** p<0,01 compared to control. E. Frequency of formation of secondary EBs in clonal assays of day 6 EBs if dox is added at the indicated time points during differentiation. Control EBs are generated without dox. F. Frequency of definitive CFCs in day 6 EBs when dox is added at indicated days of differentiation. Control EBs are generated without dox. G. Relative expression of the <i>Brachyury</i> gene during differentiation of the iLhx2 ES cell line revealing the progression of the gastrulation process in this particular ES cell line during differentiated <i>in vitro</i>.</p

Lhx2 expression in clonal assays inhibits proliferation of EryP precursor cells and increase the frequency of definitive CFCs. A.

<p>Frequency of EryP precursor cells in day 6 EB cells generated from the iLhx2 or iLhx2-GFP ES cell lines if clonal assays were performed in the absence (−Dox) or presence (+Dox) of dox B. Relative frequency of definitive CFCs in day 6 EB cells generated from the iLhx2 or iLhx2-GFP ES cell lines if the clonal assays were performed in the absence (−Dox) or presence (+Dox) of dox. C. Relative expression of Lhx2 analysed by real-time PCR comparing iLhx2 and iLhx2-GFP ES cells cultured in the presence of dox for 2 days. D. Relative Lhx2 expression analysed by real-time PCR in iLhx2 ES cells cultured for 2 days in the presence of dox at the indicated concentrations. Maximal Lhx2 expression is reproducibly achieved in the presence of 2 µg/ml of dox which is arbitrarily set as 1. E. Frequency of EryP precursor cells in clonal assays of day 6 EBs performed in the presence of dox at the indicated concentrations. F. Frequency of definitive CFCs in clonal assays of day 6 EBs performed in the presence of dox at the indicated concentrations. *p<0,01 compared to no dox addition (0 ng/ml). **p<0,02 compared to no dox addition.</p

An overview of the effects of Lhx2 expression during ES cell differentiation <i>in vitro</i>.

<p>1) Lhx2 expression and simultaneous activation of the c-kit receptor tyrosine kinase and gp130 signal transducer via IL-6 directly induce self-renewal of a distinct multipotential definitive hematopoietic progenitor cell (Def. HPC) present in the EB (1a) leading to the generation of HSC-like cell lines (DoxHPC lines) strictly dependent on Lhx2 expression and SCF/IL-6 for continuous self-renewal <i>in vitro</i> (1b). 2) Lhx2 expression inhibits proliferation of committed primitive erythroid (EryP) precursor cells (2a) and definitive erythroid (EryD) precursor cells (2b). 3) Lhx2 expression interferes with the initial step in ES cell differentiation whereas it does not interfere with the gastrulation process. Mast, mast cells. Mac, macrophages. Neut, neutrophilic granulocyte. Meg, megakaryocytes.</p

Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible expression-2

<p><b>Copyright information:</b></p><p>Taken from "Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible expression"</p><p>BMC Genomics 2006;7():75-75.</p><p>Published online 6 Apr 2006</p><p>PMCID:PMC1459142.</p><p>Copyright © 2006 Richter et al; licensee BioMed Central Ltd.</p> off (upper panel) and 10 genes with up-regulated expression when expression is turned off (lower panel). Relative expression of each gene is set as 1 when is expressed (Day 0) and is compared to the expression at day 3 after dox withdrawal