Methods for preparing mature erythrocytes from conditionally immortalized hematopoietic stem cells
View Patent ↗Provided herein are methods for preparing differentiated cells, and the cells prepared by such methods.
1. A method of preparing a plurality of anucleated cells comprising: culturing conditionally immortalized hematopoietic stem cells in the presence of an agent for inducing differentiation to erythrocytes; wherein the agent consists of IL-3 and EPO, and wherein the agent induces the differentiation of conditionally immortalized hematopoietic stem cells to mature erythrocytes thereby producing a plurality of anucleated cells;
wherein the conditionally immortalized hematopoietic stem cells were generated by:
a. providing a plurality of hematopoietic stem cells with an exogenous MYC molecule; and
b. providing the hematopoietic stem cells with an exogenous Bcl-2 molecule, an exogenous Bcl-X molecule, or a combination thereof;
and wherein the activity of the exogenous MYC molecule was suppressed before differentiation.
2. The method of claim 1 , wherein the exogenous MYC molecule is c-Myc, l-Myc, n-Myc, s-Myc, or a combination thereof.
3. The method of claim 1 , wherein the exogenous MYC molecule is c-Myc.
4. The method of claim 1 , wherein the generation of the conditionally immortalized hematopoietic stem cells further comprised culturing the stem cells with IL-3, IL-6, stem cell factor, thrombopoietin, Flt3 ligand, or a combination thereof.
5. The method of claim 1 , wherein the exogenous MYC molecule is provided by transfecting the hematopoietic stem cells with a first vector comprising:
a nucleic acid sequence encoding a MYC molecule; wherein the exogenous Bcl-2 molecule, the exogenous Bcl-X molecule, or combination thereof is provided by transfecting the stem cells with a second vector comprising: a nucleic acid sequence encoding a Bcl-2 molecule, a Bcl-X molecule, or a combination thereof.
6. The method of claim 5 , wherein the first vector further comprises the hormone-binding domain of the human estrogen receptor.
7. The method of claim 6 , further comprising contacting the plurality of hematopoietic stem cells with: estradiol (E2), 4-hydroxytamoxifen (4-OHT), or both.
8. The method of claim 7 , wherein the activity of the exogenous MYC molecule was suppressed by no longer contacting the plurality of hematopoietic stem cells with: estradiol (E2), 4-hydroxytamoxifen (4-OHT), or both.
9. The method of claim 5 , further comprising inducing translocation of the exogenous MYC molecule to a nucleus.
10. The method of claim 5 , wherein the first vector, the second vector, or both the first vector and the second vector is Murine Stem Cell Virus (MSCV), or a derivative thereof.
11. The method of claim 5 , wherein the first vector, the second vector, or both the first vector and the second vector is MSCV-(IRES)-GFP, or a derivative thereof.
12. The method of claim 5 , wherein the first vector, the second vector, or both the first vector and the second vector is MSCV-(IRES)-GFP, and further comprises woodchuck hepatitis B virus RNA regulatory element (WRE).
13. The method of claim 5 , wherein the first vector further comprises the hormone-binding domain of the human glucocorticoid receptor.
14. The method of claim 13 , further comprising contacting the plurality of hematopoietic stem cells with mifepristone.
15. The method of claim 13 , wherein the activity of the exogenous MYC molecule was suppressed by no longer contacting the plurality of hematopoietic stem cells with mifepristone.