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Figure 1 Schematic representation of the generation of HPC lines from LH2-transduced ES cells. SF, Steel factor; epo, erythropoietin.
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 | Figure 2 Analysis of LH2 expression by in situ hybridizations of day 6 EBs using an antisense (A–C) and sense control (D) probes for the LH2 gene. EBs were generated from control ES cells (A), polyclonal MSCV-LH2 ES cells (B), and MSCV-LH2 subline #7 ES cells (C and D). Scale bar indicates 100  m.
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Figure 3 LH2-expressing EBs give rise to unique HPC colonies. Two types of colonies derived from control EB cells replated in Steel factor and erythropoietin (A). The small tight colonies consist of primitive erythroid (eryp) cells and the large irregular shaped colony consists of definitive erythroid (eryd) cells. (B) and (C) show the cell morphology by May-Grünwald Giemsa staining of cells within eryd colonies and eryp colonies, respectively. A distinct type of colony termed HPC colony formed when LH2-expressing EBs were replated in Steel factor and erythropoietin (D and F). (D) shows a HPC colony (right) which is 12 days old and is compared to an eryd colony (left), and (F) shows a 14 days old HPC colony. Note that (D) is a higher magnification as compared with (F). May-Grünwald Giemsa staining show the immature 'blast-like' morphology of cells within the HPC colony (E) and of an HPC line that has been expanded in liquid culture in the presence of Steel factor (G). May-Grünwald Giemsa staining of cells showing mast cell morphology derived from wild-type EBs after two weeks of culture in liquid culture in the presence of Steel factor (H). Scale bars: (A and D) 0.25 mm, (F) 0.5 mm, (B, C and E) 50 m, (G and H) 20 m.
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 | Figure 4 Steel factor dependence for proliferation and maintenance of established HPC lines. Growth curves of an HPC line cultured in the indicated factors (A). After six days of culture the HPCs cultured in Steel factor (B), Tpo (C), and IL-3 (D) were stained by May-Grünwald Giemsa stain. Scale bars indicate 20 m.
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Figure 5 Multilineage colonies generated from HPCs in precursor assays containing Steel factor + factor mix (IL-3/Tpo/GM-CSF/G-CSF/M-CSF/Epo) (A and B). May-Grünwald Giemsa staining of a representative multilineage colony is shown in (C). May-Grünwald Giemsa staining of the cells within representative bi-lineage colony (D), single-lineage (E) colony, and the mast cells generated when cells within multilineage colonies were replated in secondary progenitor assays containing Steel factor + factor mix (F). Relationship between the number of HPCs plated and the total number of colonies that develop in two independent experiments carried out at a three months interval (G). ery, anucleated definitive erythroid cell; neut, neutrophilic granulocyte; mac, macrophage; and meg, megakaryocyte. Scale bars: (A and B) 0.5 mm, (C) 50 m, (D, E and F) 20 m.
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 | Figure 6 Analysis of MSCV-derived transcripts in different cell populations by Northern blot and RT–PCR. (A) LH2 expression was analysed by Northern blot analysis of total RNA (10 g) from two independent sublines (#3 and #7) of LH2-transduced ES cells, the EBs after in vitro differentiation, and one established HPC line derived from each ES cell subline (LH2). Sizes indicate locations of the 28 and 18S rRNA. The same blot was stripped and rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe followed by a second stripping and rehybridization to a Neo probe (Neo).(B) RT–PCR analysis was performed on cDNA generated from eryd colonies derived from control EB cells (eryd), HPC colonies derived from EBs generated from LH2-transduced ES cells (HPC col.), and HPCs that were continuously cultured in liquid culture (HPC LQ). LH2 expression analyses on the differentiated progeny of HPCs was performed on either multilineage colonies (HPC mix) or mast cells (HPC mast). Negative controls are PCR carried out on a mock RNA preparation (neg.), and on poly(A)+ RNA preparation of continuously cultured HPCs where no cDNA synthesis have been carried out [HPC LQ poly(A)–RT].
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Figure 7 Northern blot analysis of hematopoietic progenitor/stem cell associated transcription factors. Total RNA (10 g) from two independent HPC lines and the ES cell sublines they were generated from was loaded onto gels and subsequently blotted and hybridized to probes of the indicated genes. Equal loading and integrity of the RNA was confirmed by hybridization to a GAPDH probe. Sizes indicate locations of the 28 and 18S rRNA.
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 | Figure 8 FACS analysis of a representative HPC line cultured in Steel factor. Histograms displaying cell surface marker expression on the HPCs. Percentage of positive cells is indicated in each histogram. Stippled line histograms represent unstained cells and solid line histograms represent cells labelled with antibodies specific for the indicated cell surface markers.
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Figure 9 FACS analysis of an HPC line induced to differentiate for 10 days in Steel factor and factor mix (A). See legend to Figure 8 for further details. Analysis of ploidy by propidium iodide staining of HPCs cultured in Steel factor (SF) and thrombopoietin (Tpo) for 6 days (B).
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