Figure 1.
Blood cell induction from pluripotent stem cells starts with commitment into primitive streak.
a. A schema of stepwise haematopoietic differentiation of human ES/iPS cells. b. Gene expression analysis of colonies at the beginning of differentiation (day 0) and the end of step 1 (day 4) with/without 40 ng/mL BMP4. Data from KhES-1 are shown as representative. c. Phase contrast microscopies and immunofluorescence staining of colonies during initial differentiation. Data from KhES-1 are shown as representative. d. Relative expression of T at day 4 of differentiation with different combinations of BMP4 and its inhibitor Noggin. Where shown, bars represent standard deviation of the mean of three independent experiments; Scale bars, 500 µm. Data from KhES-1 and 253G4 strains are shown as representative. e. Flow cytometric analysis of differentiating cells on day 4, indicating the down-regulation of immature cell markers and up-regulation of differentiated progenitor markers. Data from KhES-1 are shown as representative.
Figure 2.
Characterization of cells during initial differentiation with lineage-specific marker expression.
a. Expression analysis of lineage-specific marker genes at the beginning of differentiation (day 0) and the end of step 2 (day 6). Bars represent standard deviation of the mean of three independent experiments. Data from KhES-1 are shown as representative. b. The development of progenitors on day 6 positive for lateral mesoderm markers but negative for paraxial mesoderm and haematopoietic cell markers. Leftmost column shows the gating strategy for eliminating dead cells and debris. Data from KhES-1 are shown as representative. c. Efficacy of inducing KDR+CD34+ or − mesodermal progenitors from each two lines of human ES cells and iPS cells. Bars represent standard deviation of the mean of three independent experiments.
Figure 3.
Human ES/iPS cell-derived haematopoiesis in a monolayer culture free from animal serum or stromal cells.
a. Sequential phase contrast pictures showing haematopoietic development. Scale bars, 500 µm (left two panels) and 100 µm (right two panels). Data from KhES-1 are shown as representative. b. Floating cells harvested on day 30 showing various lineages of haematopoietic cells; MPO-positive myeloid lineage cells (leftmost panels), pan-human Hb-positive erythroid lineage cells (centre panels), and CD41-positive megakaryocytes (rightmost panels). Scale bars, 100 µm. Data from KhES-1 are shown as representative. c. Expression of lineage-specific antigens on floating cells harvested on day 30; Myeloid lineages (CD13 and CD45), erythroid lineages (CD235a), T cells (CD3), and B cells (CD19). Data from KhES-1 are shown as representative. d. Numbers and fraction of blood cells induced from each two lines of human ES cells and iPS cells. Bars represent standard deviation of the mean of three independent experiments.
Figure 4.
Functional blood cells derived from human ES/iPS cells.
a. Number of migrated cells that permeated through the transwell membrane with or without fMLP. Values were normalised to the number of counting beads, and the control values were arbitrarily set to the condition without fMLP. Data from KhES-1 are shown as representative. b. Assay for phagocytosis-induced respiratory burst activity using chemiluminescent microspheres (luminol-binding microspheres). Abbreviation: RLU, relative light units. Data from KhES-1 are shown as representative. c. Oxygen dissociation curves of erythroid cells derived from human ES/iPS cells (harvested on day32 of differentiation), human cord blood, and adult peripheral blood. Where shown, bars represent standard deviation of the mean of three independent experiments.
Figure 5.
Hematopoietic stem/progenitor cells in culture.
a. Sequential FCM analysis of cells harvested on indicated days showing the existence of CD34+CD45+ haematopoietic progenitor cells in culture. Data from KhES-1 are shown as representative. b. Various colony types on MTC-containing medium clonally emerged from single haematopoietic progenitor cells. Data from KhES-1 are shown as representative. c. Numbers of each colony type derived from different days of culture. Bars represent standard deviation of the mean of three independent experiments. Data from KhES-1 and 253G4 strains are shown as representative.
Figure 6.
Haematopoietic differentiation from KDR+CD34+ mesodermal progenitors.
a. Schema of the protocol for measuring haematopoietic activities of depicted fractions on day 6. b. Each sorted fraction-derived haematopoiesis on day 25 detected by fluorescent microscopy and FCM analysis. Data from KhES-1 are shown as representative. c. Ratio of CD45+ cells in GFP+ fraction on day 25 showing the strongest haematopoietic activity of fraction C followed by fraction B. d. Single KDR+CD34+CD45− cell-derived haematopoietic colonies (HC), VE-cadherin+ endothelial colonies (EC), and HC+EC colonies generated on OP9 cell layers. Data from KhES-1 are shown as representative. e. Number of wells that showed HC, EC, and EC+HC development.