In vitro production of red blood cells with sortaggable proteins
Methods for the in vitro production of enucleated red blood cells and the enucleated red blood cells thus prepared are provided. Such enucleated red blood cells may express a sortaggable surface protein, which allows for surface modification in the presence of a sortase. Also described herein are surface modified enucleated red blood cells, e.g., conjugated with an agent of interest such as a peptide, a detectable label, or a chemotherapeutic agent, and uses thereof in delivering the agent to a subject.
1. A method for producing human enucleated red blood cells, the method comprising:
(i) providing a population of human CD34+ progenitor cells;
(ii) expanding the population of human CD34+ progenitor cells in a first medium for 1-6 days, wherein the first medium comprises serum-free culture medium, Flt-3 ligand, stem cell factor (SCF), interleukin 3 (IL-3), and interleukin 6 (IL-6);
(iii) differentiating the expanded human CD34+ progenitor cells from step (ii) in a second medium for 4-7 days, wherein the second medium comprises erythroid differentiation culture medium, 5-20 μg/mL insulin, dexamethasone, 1-10 ng/mL IL-3, 10-500 ng/mL SCF, holo human transferrin, and erythropoietin (EPO); wherein the second medium is free of Flt-3 ligand, IL-6, or a combination thereof;
(iv) differentiating the cells from step (iii) in a third medium for 3-5 days, wherein the third medium comprises erythroid differentiation culture medium, 5-20 μg/mL insulin, 10-100 ng/mL SCF, holo human transferrin, and EPO; wherein the third medium is free of Flt-3 ligand, IL-6, dexamethasone, IL-3, or a combination thereof;
(v) differentiating the cells from step (iv) in a fourth medium for 4-12 days to produce human enucleated red blood cells, wherein the fourth medium comprises erythroid differentiation culture medium, 5-20 μg/mL insulin, holo human transferrin, and EPO; wherein the fourth medium is free of Flt-3 ligand, IL-6, dexamethasone, β-estradiol, IL-3, SCF or a combination thereof; and
(vi) collecting the human enucleated red blood cells from step (v), wherein the total time period for steps (ii)-(v) ranges from 11-25 days, and wherein the human enucleated red blood cells comprise cells with similar hemoglobin content and cell size to normal human red blood cells and synchronized expression of cell surface differentiation markers during steps (ii)-(v).
2. The method of claim 1 , wherein the human CD34 + progenitor cells are human CD34 + peripheral blood cells.
3. The method of claim 1 , wherein in step (ii), the population of human CD34 + progenitor cells is cultured in the first medium for 4 days.
4. The method of claim 1 , wherein in step (ii), the population of human CD34 + progenitor cells is cultured in the first medium for 5 days.
5. The method of claim 1 , wherein in step (ii), the population of human CD34 + progenitor cells is cultured in the first medium for 6 days.
6. The method of claim 1 , wherein in step (iii), the expanded human CD34 + progenitor cells are differentiated in the second medium for 5 days.
7. The method of claim 1 , wherein in step (iii), the expanded human CD34 + progenitor cells are differentiated in the second medium for 6 days.
8. The method of claim 1 , wherein in step (iii), the expanded human CD34 + progenitor cells are differentiated in the second medium for 7 days.
9. The method of claim 1 , wherein in step (iv), the cells are differentiated in the third medium for 3 days.
10. The method of claim 1 , wherein in step (iv), the cells are differentiated in the third medium for 4 days.
11. The method of claim 1 , wherein in step (iv), the cells are differentiated in the third medium for 5 days.
12. The method of claim 1 , wherein in step (v), the cells are differentiated in the fourth medium for 4 days.
13. The method of claim 1 , wherein in step (v), the cells are differentiated in the fourth medium for 5 days.
14. The method of claim 1 , wherein in step (v), the cells are differentiated in the fourth medium for 6 days.
15. The method of claim 1 , wherein step (v) is performed in a culture container having a surface coating of an extracellular matrix component.
16. The method of claim 1 , wherein the erythroid differentiation culture medium in one or more of the first, second, third, and fourth media is Iscove Modified Dulbecco Media (IMDM).
17. The method of claim 1 , wherein the second medium comprises 100 nM to 5 μM dexamethasone.
18. The method of claim 1 , wherein the third medium comprises 250-1500 μg/mL holo human transferrin.
19. The method of claim 1 , wherein the fourth medium comprises 250-1500 μg/mL holo human transferrin.
20. The method of claim 1 , wherein the human CD34 + progenitor cells are genetically modified to express a sortaggable surface fusion protein comprising a red blood cell membrane protein and a peptide.
21. The method of claim 20 , wherein the sortaggable surface fusion protein comprises a type I red blood cell transmembrane protein fused to an acceptor peptide at the N-terminus of the type I red blood cell transmembrane protein.
22. The method of claim 21 , wherein the type I red blood cell transmembrane protein is glycophorin A.
23. The method of claim 21 , wherein the acceptor peptide is an oligoglycine.
24. The method of claim 20 , wherein the sortaggable surface fusion protein comprises a type II red blood cell transmembrane protein fused to a peptide comprising a sortase recognition motif at the C-terminus of the type II red blood cell transmembrane protein.
25. The method of claim 24 , wherein the sortase recognition motif is recognizable by a sortase A.
26. The method of claim 24 , wherein the sortase recognition motif is LPXTG (SEQ ID NO: 1), wherein X is any amino acid residue.
27. The method of claim 24 , wherein the type II red blood cell transmembrane protein is Kell or CD71.
28. The method of claim 20 , wherein the red blood cell membrane protein is a type III red blood cell transmembrane protein.
29. The method of claim 28 , wherein the type III red blood cell transmembrane protein is glucose transporter 1 (GLUT1), Aquaporin 1, or Band 3.
30. The method of claim 17 , wherein the second medium further comprises β-estradiol.