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Blood, Vol. 106, Issue 5, 1601-1603, September 1, 2005
Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation
Blood Ng et al.
106: 1601
Supplemental materials for: Ng et al, Vol 106, Issue 5, 1601-1603
Files in this Data Supplement:
- Document 1: Methods (PDF, 102 KB) -
This details hESC differentiation protocol, antibody staining and flow cytometry, immunocytochemistry, gene expression analysis, and oligonucleotide primers.
- Figure S1. Cellular morphology of blood cells developed from spin EBs established from 2000 cells/well (JPG, 32.2 KB)
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In this experiment, over 90% of wells formed blood cells containing a mixture of hemoglobinized erythroid and myeloid cells. Cytocentrifuge preparations at day 19 stained with May-Grünwald-Giemsa show developing erythroblasts (A,B,D), nucleated yolk sac erythrocyte (C), and macrophages (C,D). The morphology of these cells was correlated with their immunophenotype by staining some wells for glycophorin A (Figure S2) and others for CD45 (Figure S3) on the cell surface to confirm assignment to erythroid or myeloid lineages. Visibly hemoglobinized cells and clumps of cells stained brightly for cell surface glycophorin A and larger, nonhemoglobinized blood cells were CD45+. In this experiment, most wells contained a mixture of erythroid and myeloid cells.
- Figure S2. Erythroid cells from spin EBs express cell surface glycophorin A (JPG, 99.5 KB)
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Bright-field (A,D,G), fluorescence (B,E,H), and overlaid images (C,F,I) of wells containing visibly hemoglobinized cells stained with isotype control antibodies (A-C) or anti-glycophorin A (B-I) detecting human erythroid cells. Bright cell surface staining is observed in clumps and some individual cells although other cells are clearly negative and thus not of erythoid lineage.
- Figure S3. Myeloid cells from spin EBs express cell surface CD45 (JPG, 151 KB)
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Bright-field (A,D,G,J), fluorescence (B,E,H,K) and overlaid images (C,F,I,L) of wells containing myeloid cells stained with isotype control antibodies (A-C) or anti-CD45 (B-I) detecting human leukocytes. Bright cell surface staining of myeloid cells is observed.
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