Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

Summary

This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

Abstract

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool. Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants.

Protocol

Dissection and isolation of early hematopoietic stem cells from murine yolk sac and aorta-gonad-mesonephros region Before starting, it is important to have all the reagents and tools necessary for the procedure: Scissors Forceps, size 4, and surgical grade bent-tipped forceps, size 5/45 (optional) 30 gauge needles with syringe 35mm  and 100mm Petri dishes PBS with 10% and 20% fetal bovine serum and lastly, Collagenase To begi…