Hematopoietic colony-forming cells derived from human embryonic stem cells - PubMed (original) (raw)

Hematopoietic colony-forming cells derived from human embryonic stem cells

D S Kaufman et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Human embryonic stem (ES) cells are undifferentiated, pluripotent cells that can be maintained indefinitely in culture. Here we demonstrate that human ES cells differentiate to hematopoietic precursor cells when cocultured with the murine bone marrow cell line S17 or the yolk sac endothelial cell line C166. This hematopoietic differentiation requires fetal bovine serum, but no other exogenous cytokines. ES cell-derived hematopoietic precursor cells express the cell surface antigen CD34 and the hematopoietic transcription factors TAL-1, LMO-2, and GATA-2. When cultured on semisolid media with hematopoietic growth factors, these hematopoietic precursor cells form characteristic myeloid, erythroid, and megakaryocyte colonies. Selection for CD34(+) cells derived from human ES cells enriches for hematopoietic colony-forming cells, similar to CD34 selection of primary hematopoietic tissue (bone marrow, umbilical cord blood). More terminally differentiated hematopoietic cells derived from human ES cells under these conditions also express normal surface antigens: glycophorin A on erythroid cells, CD15 on myeloid cells, and CD41 on megakaryocytes. The in vitro differentiation of human ES cells provides an opportunity to better understand human hematopoiesis and could lead to a novel source of cells for transfusion and transplantation therapies.

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Figures

Figure 1

Figure 1

Flow cytometric analysis of undifferentiated human ES (H1) cells and differentiated H1 cells. (A) Undifferentiated H1 cells analyzed by single-color flow cytometry. Appropriate isotype control antibody is demonstrated by line and indicated antibody by filled plot. SSEA-1 and SSEA-4 were unconjugated antibodies and a secondary FITC-conjugated goat anti-mouse (GAM) antibody was used. All other antibodies were directly conjugated to flurochrome. (B) H1 cells allowed to differentiate on S17 cells (H1/S17 cells) analyzed by two-color flow cytometry. Percentages of positive cells are indicated in each quadrant.

Figure 2

Figure 2

Methylcellulose hematopoietic colony-forming assays. (A) Production of CFCs from human ES cells. H1/S17 cells, H1.1/S17, H1/C166 cells, and differentiated H1/MEF cells were harvested after 14–20 days of culture, placed in methylcellulose-based media supplemented with hematopoietic growth factors, and scored for total hematopoietic colonies after 14 days. Results are mean ± SE of seven trials with H1/S17, four trials with H1.1/S17, four trials with H1/C166, and three trials with H1/MEF. Data are presented as colonies per 105 cells harvested from the differentiated H1 cultures. (B) Time course of H1/S17 cell differentiation into hematopoietic CFCs. H1 cells cocultured with S17 cells for the indicated number of days before colony assay. (C) Percent of erythroid (burst-forming unit-erythroid) and myeloid (CFU-GM, CFU-M, and CFU-G) colonies derived from H1/S17 cells harvested at day indicated. (D) CD34+ H1/S17 cells are enriched for CFCs. Unsorted H1/S17 cells and H1/S17 cells sorted for CD34+ cells and CD34− cells by magnetic column were placed in hematopoietic colony assay. These results are mean ± SE of three separate trials.

Figure 3

Figure 3

Photographs of hematopoietic colonies and cells derived from H1/S17 cells. H1 cells allowed to differentiate on S17 cells for ≈17 days, harvested, and allowed to form colonies in semisolid media for 14 days before scoring colony phenotypes. (A) CFU-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). Colony of mixed erythroid and myeloid cells. (B) Burst-forming unit-erythroid. Large unstained, red (hemoglobin) colony. (C) CFU-GM, unstained myeloid colony. (D) CFU-M, unstained myeloid colony, less dense than CFU-GM colony. (E) CFU-Mk. Colony of cells stained with platelet/megakaryocyte-specific antibody against CD41 (GPIIb/IIIa) with alkaline phosphatase-conjugated secondary antibody and Fast Red/naphthol reagent to provide red stain. (F) Cytospin of CFU-GM cells demonstrating granulocytes with esterase-positive red granules. (Scale bars: A_–_D, 100 μm; E, 40 μm; F, 20 μM.)

Figure 4

Figure 4

Flow cytometric analysis of hematopoietic cells derived from methylcellulose colony assay of H1/S17 cells. Cells were washed free of methylcellulose before incubation with indicated antibodies and analyzed by two-color (Upper) or one-color (Lower) flow cytometry. (Upper) Isotype controls are shown (Left), and percent positive cells in each quadrant is indicated. (Lower) Isotype control is demonstrated by line, and indicated antibody is demonstrated by filled plot. Percent positive cells are shown by labeled marker.

Figure 5

Figure 5

Hematopoietic gene expression by RT-PCR of H1/S17 cells. (A) H1 cells were allowed to differentiate on either S17 cells for 17 days (lanes 1 and 2) or to differentiate on MEF cells for 17 days (lanes 3 and 4), or harvested after culture on MEFs for 6 days, before evidence of differentiation (undifferentiated H1 cells, lanes 5 and 6) and subjected to RT-PCR analysis. Irradiated S17 cells were examined to demonstrate positive bands in the H1/S17 samples were not from these feeder cells (lanes 7 and 8). The erythroleukemia cell line K562 was used as a positive control (lanes 9 and 10). Oligonucleotide primers specific for genes of interest are shown. Each sample was done with RT added (+, lanes 1, 3, 5, 7, and 9) and without RT added (−, lanes 2, 4, 6, 8, and 10) to demonstrate positive bands are not caused by genomic DNA. (B) Time course of hematopoietic gene expression. H1 cells were allowed to differentiate on S17 cells for the number of days indicated prior isolation of RNA for RT-PCR analysis. Day 0 (d. 0) indicates undifferentiated H1 cells. Controls of PCR done on samples without RT added did not have any positive bands (data not shown).

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