Flow cytometer analyses of the established erythroid progenitor cell lines.

<p>(A) Representative results of HiDEP cells. (B) Representative results of HUDEP cells and HUDEP-1 expressing TAL1, HUDEP-1-TAL1. (A, B) GPA, glycophorin A. CD71, transferrin receptor. c-KIT, the receptor of SCF. CD36, a marker of immature erythroid cells.</p

Dependency of the established erythroid progenitor cell lines on externally supplied culture factors.

<p>(A) The survival and proliferation of HiDEP-1 cells are dependent on DOX (HPV16-E6/E7) and EPO and partially dependent on DEX. (B) The survival and proliferation of HUDEP-1 cells are dependent on DOX (HPV16-E6/E7) and SCF and partially dependent on EPO. (A, B) DOX, doxycycline; expression of HPV16-E6/E7 is induced by DOX. SCF, stem cell factor. EPO, erythropoietin. DEX, dexamethasone. ALL, cells were cultured in the presence of DOX, SCF, EPO and DEX. –DOX, –SCF, –EPO, –DEX, cells were cultured after deprivation of DOX, SCF, EPO and DEX, respectively. Dependencies of other cell lines on externally supplied culture factors are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059890#pone.0059890.s006" target="_blank">Table S1</a>.</p

Cell pellets of the established erythroid progenitor cell lines.

<p>(A) HiDEP-1 cells before the induction of differentiation. (B) HUDEP-1 cells before and after the induction of differentiation. HUDEP-1 cells were cultured in erythroid differentiation medium on OP9 feeder cells to maintain cell viability during the differentiation process. All other cell lines also showed red cell pellets after the induction of differentiation.</p

An outline of the culture schedule used to establish immortalized cell lines from iPS cells.

<p>Human iPS cells (4x10⁴) were cultured in the presence of feeder cells with cytokines in a 100 mm dish. Attached cells indicate cells attached to the dish or to the feeder cells. Detached cells indicate cells detached from the dish or from the feeder cells. VEGF, vascular endothelial growth factor. IGF-II, insulin-like growth factor-II. SCF, stem cell factor. EPO, erythropoietin. DEX, dexamethasone.</p>a<p>OP9 feeder cells were used again for a further period.</p>b<p>Attached cells were cultured again on OP9 feeder cells.</p>c<p>All detached cells collected from the dish were cultured again in fresh medium.</p>d<p>Used OP9 cells were discarded together with any attached cells and a fresh batch of OP9 cells were used for the next culture step.</p>e<p>All detached cells collected from the dish were infected with lentivirus expressing HPV16-E6/E7, and were then cultured again.</p>f<p>The medium was changed twice per week.</p>g<p>Detached cells from the dish were either all re-cultured or some re-cultured and the remainder stored for further analyses.</p>h<p>From this time point, the detached cells could proliferate without feeder cells.</p>i<p>Very few attached cells were detected.</p>j<p>At approximately day 131, the requirement for essential factors for proliferation was evaluated.</p

Confirmation of enucleated cells.

<p>As a representative, the cells induced from HiDEP-1 cells were subjected to the analyses. (A) Supravital staining. Arrows indicate reticulocytes. (B) Immunohistostaining with glycophorin A antibody (pink) 12 days after the induction of differentiation. Nuclei are stained with SYTO16 (green). An arrow shows an enucleated cell. (C) Benzidine staining 10 days after the induction of differentiation (brown, left panel). After benzidine staining, cell nuclei were labeled with DAPI to distinguish nucleated and enucleated cells (blue stained nuclei in right panel). Arrows or arrowheads show enucleated and enucleating cells, respectively. (A–C) Scale bars indicate 10 µm.</p

Schematic outline of the procedures for establishing immortalized human erythroid progenitor cell lines from iPS cells and from hematopoietic stem/progenitor cells in umbilical cord blood.

<p>Schematic outline of the procedures for establishing immortalized human erythroid progenitor cell lines from iPS cells and from hematopoietic stem/progenitor cells in umbilical cord blood.</p

Effect of enforced expression of a transcription factor, TAL1, on induction of hematopoietic cells from human iPS cells. HiPS, human iPS cells (HiPS-RIKEN-3A).

<p>HiPS-TAL1, HiPS cells expressing TAL1 (HiPS-RIKEN-3A-TAL1). The cells were analyzed after the induction of differentiation of hematopoietic cells for 15 days. (A) Fold increase of production of hematopoietic cells from HiPS-TAL1 cells compared to HiPS cells. (B) Flow cytometer analysis. CD71, transferrin receptor. Glycophorin A (GPA), an erythroid specific marker. Percentages of GPA-positive cells are indicated in the figure. (C) Morphology of the cells derived from HiPS-TAL1 cells. Scale bar indicates 50 µm. The comparison of HiPS-RIKEN-4A and HiPS-RIKEN-4A-TAL1 showed similar results.</p

Gene expression profiles of the established erythroid progenitor cell lines estimated by quantitative RT-PCR analysis.

<p>Analyzed genes are indicated. FL, results with cDNA derived from human fetal liver. Relative expression was evaluated compared to that of FL. CB, cord blood. CB 6, 10 and 16, results with cDNA derived from cultured erythroid cells, i.e., CD34-positive cells in CB were induced to differentiate into mature erythroid cells for 6, 10 and 16 days, respectively, using the previously reported method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059890#pone.0059890-Miharada1" target="_blank">[4]</a>. B and A, Before and 2 days after induction of differentiation of HiDEP and HUDEP cells.</p

Analyses of cell viability and cell size during the induction of differentiation of the established erythroid progenitor cell lines.

<p>Cell viability and cell size were estimated by an automated cell counter. (A) Results of HiDEP-1 cells. (B) Results of HUDEP-1 cells. (A, B) <10 µm (%), the percentages of cells with a diameter less than 10 µm. Among all HiDEP and HUDEP cell lines, HiDEP-1 cells most efficiently differentiated into more mature cells.</p

Morphological analyses during the induction of differentiation of the established erythroid progenitor cell lines.

<p>(A) HiDEP-1 cells before (Day 0) and 6, 10 and 12 days after the induction of differentiation (Day 6, 10 and 12). Scale bar in Day 0 indicates 50 µm and other scale bars indicate 10 µm. (B) HUDEP-1 cells before (Day 0) and 2, 6 and 9 days after the induction of differentiation (Day 2, 6 and 9). HUDEP-1 cells were cultured in erythroid differentiation medium on OP9 feeder cells to maintain cell viability during the differentiation process. All scale bars indicate 50 µm. (A, B) Black arrows and arrowheads show enucleated and enucleating cells, respectively. Blue arrows indicate cell debris. Among all HiDEP and HUDEP cell lines, HiDEP-1 cells most efficiently produced enucleated cells.</p