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.
Differentiation of Embryonic Stem Cells
EB dissociation and infection with MSCV-HoxB4-IRES-GFP
Culture and plating of OP9 stroma
Collection, fractionation and transplantation
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
C57Bl/6 Rag-2/gamma-c deficient mice | Animal | Taconic Farm | ||
Plate shaker | Tool | VWR | 33998-360 | Orbital Shaker by Ikaworks |
Multi-channel pipettor | Tool | VWR | 15000-174 | ePet by BioHit |
ES trypsin | Reagent | Invitrogen | 15090-046 | 0.25% Trypsin in PBS |
Standard trypsin | Reagent | Invtrogen | 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 | H2126 | freeze in 500 uL aliquots and avoid repeated freezing and thawing. |
DNAse | Reagent | Sigma | D4527 | |
DMEM | Invitrogen | 10-017CV | ||
Protamine sulfate | Reagent | Sigma | P3369 | 8 ug/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 |