Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Summary

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Abstract

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny. Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability. Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types. The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol. HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders. However, many patients that might benefit from HSC therapy lack access to suitable donors. ESC could provide an alternative source of HSC for these patients. The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC. In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation. EBs are then dissociated and infected with retroviral HoxB4. Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days. During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells. These cells can then be used to rescue and reconstitute lethally irradiated mice.

Protocol

Differentiation of Embryonic Stem Cells

1. Collect a near confluent flask of embryonic stem cells (ESC) via treatment with ESC trypsin.
2. Resuspend cells in 5 mL of Differentiation media and transfer to a non-gelatin coated T25. Incubate at 37°C for 45 minutes. Retrieve supernatant and collect cells via centrifugation.
   
   Note: Mouse embryonic fibroblasts (MEFs) from the ESC cultures attach readily to the non-gelatin coated T25 and thus are depleted from the culture during this plating step. If you are not culturing your ESC on MEFs, you can skip the pre-plate.
   
   
3. Resuspend MEF-depleted ESC in Differentiation media ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57Bl/6 Rag-2/gamma-c deficient mice	Animal	Taconic Farm
Plate shaker	Tool	VWR international	33998-360	Orbital Shaker by Ikaworks
Multi-channel pipettor	Tool	VWR international	15000-174	ePet by BioHit
ES trypsin	Reagent	Invitrogen	15090-046	0.25% Trypsin in PBS
Standard trypsin	Reagent	Invitrogen	25300-062	0.05% Trypsin-EDTA
PBS	Buffer	Invitrogen	20012-027
Enzyme-free dissociation buffer	Buffer	Invitrogen	13151-014
Dissociation enzyme mix	Buffer	Invitrogen	17104-019	500 mg Collagenase IV
Hyaluronidase	Reagent	Sigma-Aldrich	H2126	freeze in 500 μL aliquots and avoid repeated freezing and thawing.
DNAse	Reagent	Sigma-Aldrich	D4527
DMEM		Invitrogen	10-017CV
Protamine sulfate	Reagent	Sigma-Aldrich	P3369	8 μg/mL
EB differentiation media	medium			Please view recipe on attached Protocol.doc
10% IMDM	medium			Please view recipe on attached Protocol.doc
10% IMDM+cytokines	medium			Please view recipe on attached Protocol.doc
20% anti-MEM				Please view recipe on attached Protocol.doc