Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression

doi:10.1242/dev.00149

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doi: 10.1242/10.1242/dev.00149

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Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression

Yun Shin Chung¹^,*, Wen Jie Zhang¹^,*, Elizabeth Arentson¹, Paul D. Kingsley², James Palis² and Kyunghee Choi¹^,

^¹ Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA
^² Department of Pediatrics and Center for Human Genetics and Molecular Pediatric Disease, University of Rochester Medical Center, Rochester, NY, USA

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Fig. 1. Generation of human CD4 (CD4) knock-in ES cells. (A) Targeting strategy used to insert CD4 gene into the Scl locus is shown. The mouse Scl locus, targeting construct, and the targeted allele are shown. The black boxes and numbers below indicate the exons. The ATG codon starts within exon IV. (B) Southern blot analysis of the targeted allele. DNA was digested with enzymes indicated and run on an agarose gel. Top left, genomic DNA was digested with EcoRI and probed with genomic DNA as indicated below. Both targeted and wild-type alleles generated a 5.2 kb DNA band. Top right: genomic DNA was digested with EcoRI and probed with exon 6 probe as indicated below. The wild-type allele (upper band) and the targeted allele (lower band) are shown. Bottom: genomic DNA was digested with MunI and probed with human CD4 gene. Only the targeted allele gave 7.3 and 3 kb DNA bands. The gel was run for 48-72 hours for good separation of DNA. The enzymes used are as follows: X, XhoI; R, EcoRI; N, NotI; B, BamHI; H, HindIII; M, MunI.

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Fig. 2. (A-I) In situ hybridization of Scl. CD4⁺ and CD4^- cells were sorted from day 5 EBs and subjected to in situ hybridization. (A-D) CD4⁺ cells; (E-H) CD4^- cells; (I) E8.5 yolk sac. bi, blood islands; e, endoderm. (J) Kinetic analyses of FLK1 and SCL expression during EB development. In vitro differentiated ES cells (from day 2.75 to day 8) from wild-type R1 and three independent knock-in clones (1-4, 1-19, and 1-68) were subjected to FACS analyses for FLK1 and CD4 expression. Numbers in a given box indicate the percentage of cells that are FLK1⁺CD4^-, FLK1⁺CD4⁺ or FLK1^-CD4⁺.

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Fig. 3. In vitro progression of hematopoietic and endothelial cell lineages. (A) FLK1⁺CD4^- and FLK1^-CD4^- cells were FACS-sorted from day 2.5 EBs and further cultured in vitro. The cells were then analyzed for FLK1 and CD4 expression for up to 20 hours. The cells at 0 hour indicate the starting cell population after sorting. (B) FLK1⁺CD4⁺ and FLK1⁺CD4^- cells were FACS-sorted from day 4 EBs and further subjected to in vitro culture as in A. The cells were analyzed for FLK1 and CD4 expression for up to 20 hours. The cells at 0 hours show the sorting purity.

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Fig. 4. FLK1⁺CD4⁺ cells from D2.75 EBs are enriched for the hemangioblasts. (A) FLK1⁺CD4⁺, FLK1⁺CD4^- and FLK1^-CD4^- cells were FACS-sorted and subjected to blast colony replating (6x10⁴ cells/ml). The resulting blast colonies were counted 4 days later. Secondary EBs were also counted and shown. Error bars indicate standard deviations from triplicate plates. (B) Gene expression analysis. RNA from FLK1⁺CD4⁺, FLK1⁺CD4^- and FLK1^-CD4^- cells were amplified and probed with Flk1, Scl, Gata1, Gata2, Lmo2 and L32. 1, unsorted 2.75 EBs; 2, FLK1⁺CD4⁺; 3, FLK1⁺CD4^-; 4, FLK1^-CD4^-; 5, water — a negative control for RT-PCR.

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Fig. 5. Hematopoietic and endothelial cell analysis. Day 6 EB cells were subjected to three-color analyses for FLK1, CD4 and hematopoietic or endothelial cell markers. Cells gated on VE-cadherin⁺, CD31⁺, CD34⁺, Ter-119⁺ or CD45⁺ were analyzed for FLK1 and CD4 expression.

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Fig. 6. Hematopoietic and endothelial cell lineage development. (A) Day 6 EB cells were sorted for FLK1⁺CD4^-, FLK1⁺CD4⁺, FLK1^-CD4⁺ and FLK1^-CD4^- and subjected to hematopoietic replating (5x10⁴ cells/ml). The hematopoietic colonies were counted after 5-7 days. Ery, erythroid; Mac, macrophage; Ery+Mac, bi-potential erythroid and macrophage colony. (B) FLK1⁺CD4^-, FLK1⁺CD4⁺, FLK1^-CD4⁺ and FLK1^-CD4^- cells sorted from day 6 EBs were cultured on type IV collagen coated plates for 4 days in the presence of VEGF (50 ng/ml). Four days later, the adherent cells were stained with PECAM1 (CD31) antibodies (right) or just treated with secondary antibody alone (left). FLK1^-CD4⁺ cells did not generate any adherent cells (not shown). (C) Day 8 EBs were sorted for FLK1^-CD4^-, FLK1⁺CD4^- and FLK1^-CD4⁺ cells and subjected to hematopoietic replating (5x10⁴ cells/ml). The colonies were counted 5-7 days later.

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Fig. 7. Schematic diagram of the hematopoietic and endothelial cell lineage development within EBs.