Journal: Stem Cell Reports

Article Title: Analysis of the Spatiotemporal Development of Hematopoietic Stem and Progenitor Cells in the Early Human Embryo

doi: 10.1016/j.stemcr.2019.03.003

Figure Lengend Snippet: Testing Ex Vivo Culture Systems for Expansion of Hematopoietic Progenitors (A) Human embryos were staged, dissected, and either dissociated to make aggregates or cultured as explants at the air-liquid interface. When aggregates were made, aliquots were taken for the CFU-C assay (typically 0.1 ee). Aggregates were either self-reaggregated or co-aggregated with OP9 stromal cells. All tissues were cultured for 5–7 days at the air-liquid interface with variable addition of cytokines/small molecules. After culture, the tissue was dissociated and injected into sub-lethally irradiated NSG mice. After culture of both aggregates and explants, aliquots were taken for the CFU-C assay (up to 0.1 ee). The images of dissected tissue depict the lateral view of an AGM (left), embryonic liver (top right), and yolk sac (bottom right). (B–D) Number and type of CFU-Cs before and after culture using different culture systems; explant culture for 7 days (B), self-reaggregates cultured for 5 days (C), and co-aggregates with OP9s cultured for 7 days (D). SCF, IL3, FLT3LG 50–100 ng/mL were added as indicated in (E). Numbers shown above the graph indicate the mean fold expansion of the experiments shown. For (B), n = 3 for CS16, and n = 2 for CS17, and numbers before culture are taken from experiments with different embryos (see Figure 1 ). For (C) and (D), all bars are single experiments showing before and after culture from the same embryo. See also and . (E) The mean fold increase after culture with different culture conditions corresponding to the charts above. ∗ Indicates where the data are not shown on the above graphs. Here, three experiments only were performed with CS17 embryos. Error bars show SEM. ee, embryo equivalent. CS, Carnegie stage.

Article Snippet: Experimental variables for the culture medium included addition of human SCF 100–300 ng/mL (catalog no. 300-07), IL3 100 ng/mL (catalog no. 200-03), FLT3LG 100 ng/mL (catalog no. AF-300-19) (all Peprotech), TGF-β inhibitor (SB 431542) 5 μM (Tocris, catalog no. 1614), ROCK inhibitor Y-27632 10 μM (Sigma-Aldrich, catalog no. SCM075).

Techniques: Ex Vivo, Cell Culture, Injection, Irradiation