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Microbiology JoVE (Open Access) Citable · DOI

Isolation of Murine Embryonic Hemogenic Endothelial Cells

DOI: 10.3791/54150-v
What you'll learn
  • ✓ Isolate hemogenic endothelial cells from murine embryonic tissues using flow cytometry
  • ✓ Distinguish hemogenic EC, HSPC, and non-hemogenic EC by surface marker expression
  • ✓ Perform tissue dissection, enzymatic digestion, and fluorescent antibody labeling protocols
  • ✓ Interpret flow cytometric data to identify blood-forming endothelial populations
Protocol

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Difficulty
advanced
Total time
~3–4 hours per embryo (dissection through flow cytometry analysis)
Model organism
Mouse (embryonic E10.5–E11.5)
Biosafety
BSL-1

Steps

1
Dissect murine embryonic yolk sac or AGM

Harvest yolk sac or aorta-gonad-mesonephros tissue from dissected embryos under a stereomicroscope. Remove surrounding membranes and connective tissue to isolate target organ.

▶ 01:12
2
Digest primary YS/AGM tissue enzymatically

Incubate isolated tissue in enzymatic digestion buffer (e.g., collagenase/DNase) at 37 °C to dissociate cells into single-cell suspension.

▶ 03:29
3
Label cells with dye and fluorescent antibodies

Stain dissociated cells with nucleic acid dyes and fluorescently conjugated antibodies against hemogenic endothelial and HSPC surface markers (e.g., CD31, CD41, c-Kit).

▶ 04:37
4
Identify hemogenic EC and HSPC by flow cytometry

Acquire labeled cells on a flow cytometer and apply gating strategy to distinguish hemogenic endothelial cells, hematopoietic stem/progenitor cells, and non-hemogenic endothelial populations.

▶ 06:19
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