Generation of mature myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin
A method for efficient generation of neutrophils, eosinophils, macrophages, osteoclasts, dendritic cells an Langerhans cells from human embryonic stem cells is disclosed.
1. A method of producing myeloid lineage cells, comprising the steps of (a) obtaining a population of mammalian pluripotent stem cells, wherein the stem cells are derived from mammalian embryos or are induced pluripotent stem cells, (b) inducing hematopoietic differentiation to obtain lin − CD34 + CD43 + CD45 + CD38 − multipotent hematopoietic cells by co-culturing the mammalian pluripotent stem cells with stromal cells, (c) expanding and differentiating lin − CD34 + CD43 + CD45 + CD38 − cells from step (b) by culture in the presence of GM-CSF to obtain CD34 + CD43 + CD45 + CD38 + myeloid progenitors and
(d) exposing the CD34 + CD43 + CD45 + CD38 + myeloid progenitors resulting from step (c) to:
(i) G-CSF for 7-9 days, wherein a population of neutrophils is isolated;
(ii) IL-3 and IL-5 for 12-14 days wherein a population of eosinophils is isolated;
(iii) M-CSF and IL-1β for 1-8 days wherein a population of macrophages is isolated;
(iv) GM-CSF, lα,25-dihydroxyvitamin D3 for 5-7 days, then GM-CSF, lα,25-dihydroxyvitamin D3 and RANKL for 7-14 days, wherein populations of osteoclasts are isolated;
(v) GM-CSF and IL-4 for 7-9 days, TNF-α being added after day 3 of GM-CSF and IL-4 exposure, wherein a population of dendritic cells is isolated; or (vi) GM-CSF and TGF-β, TNF-α being added after day 3 of GM-CSF and TGF-β exposure, wherein a population of Langerhans cells is isolated.
2. The method of claim 1 , wherein the pluripotent stem cells of step (a) form embryoid bodies during step (b).
3. The method of claim 1 , wherein the stromal cells are selected from the group consisting of S17, AM20.1B4, UG26.1B6, FH-B-hTERT, primary stromal cells and OP9 mouse bone marrow stromal cells.
4. The method of claim 3 , wherein the stromal cells are OP9 mouse bone marrow stromal cells.
5. The method of claim 1 , wherein the cells of step (c) are cultured for 2-8 days.
6. The method of claim 1 , wherein step (d) comprises exposing the expanded cells to G-CSF for 7-9 days wherein a population of neutrophils is isolated.
7. The method of claim 6 , wherein G-CSF is present at 100±50 ng/mL.
8. The method of claim 1 , wherein step (d) comprises exposing the expanded cells to IL-3 and IL-5 for 12-14 days wherein a population of eosinophils is isolated.
9. The method of claim 8 , wherein IL-3 and IL-5 are present at 10±5 ng/mL respectively.
10. The method of claim 5 , wherein the cells of step (c) are cultured for 2 days, and step (d) comprises exposing the expanded cells to M-CSF and IL-1β for 1-8 days wherein a population of macrophages is isolated.
11. The method of claim 10 , wherein M-CSF is present at 20±10 ng/mL and IL-1β is present at 10±5 ng/mL.
12. The method of claim 5 , wherein the cells of step (c) are cultured for 8 days, and step (d) comprises exposing the expanded cells to M-CSF and IL-1β for 2-3 days wherein a population of macrophages is isolated.
13. The method of claim 12 , wherein M-CSF is present at 20±10 ng/mL and IL-1β is present at 10±5 ng/mL.
14. The method of claim 1 , wherein step (d) comprises exposing the expanded cells to GM-CSF and IL-4 for 7-9 days, TNF-α being added after day 3 of GM-CSF and IL-4 exposure, wherein a population of dendritic cells is isolated.
15. The method of claim 14 , wherein GM-CSF is present at 20±10 ng/mL, IL-4 is present at 20±10 ng/mL and TNF-a is present at 2.5±1 ng/mL.
16. The method of claim 1 , wherein step (d) comprises exposing the expanded cells to GM-CSF and TGF-β, TNF-α being added after day 3 of GM-CSF and TGF-β exposure, wherein a population of Langerhans cells may be isolated.
17. The method of claim 16 , wherein GM-CSF is present at 20±2 ng/mL, TGF-β is present at 5±2.5 ng/mL and TNF-a is present at 1±0.5 ng/mL.
18. method of claim 1 , wherein step (d) comprises exposing the expanded cells to GM-CSF and lα,25-dihydroxyvitamin D3 for 5-7 days , then GM-CSF, lα,25-dihydroxyvitamin D3 and RANKL for 7-14 days, wherein a population of osteoclasts is isolated.
19. The method of claim 18 , wherein GM-CSF is present at 100±50 ng/mL for the first 5-7 days and 50±25 ng/mL for the last 7-14 days, lα,25-dihydroxyvitamin D3 is present at 200+100 nmol and RANKL is present at 10±5 ng/mL.
20. The method of claim 18 , wherein 1α,25-dihydroxyvitamin D3 is substituted with vitamin D3 like compounds selected from the group consisting of calcitriol, calcidiol, 19-nor-(20S)- lα,25-dihydroxyvitamin D3, and 22-Oxacalcitriol.
21. The method of claim 1 , wherein 1α,25-dihydroxyvitamin D3 is added to step(c), and step (d) comprises exposing the expanded cells to GM-CSF, lα,25-dihydroxyvitamin D3 for 5-7 days, then GM-CSF, 1α,25-dihydroxyvitamin D3 and RANKL for 7-14 days, wherein populations of osteoclasts are isolated.
22. The method of claim 21 , wherein lα,25-dihydroxyvitamin D3 is substituted with vitamin D3 like compounds selected from the group consisting of calcitriol, calcidiol, 19-nor-(20S)- 1α,25-dihydroxyvitamin D3, and 22-Oxacalcitriol.
23. The method of claim 1 , wherein the mammal is a human.
24. A method of producing myeloid progenitors, comprising the steps of (a) obtaining a population of mammalian pluripotent stem cells, wherein the stem cells are derived from mammalian embryos or are induced pluripotent stem cells (b) inducing hematopoietic differentiation to obtain lin − CD34 + CD43 + CD45 + CD38 − multipotent hematopoietic cells by co-culturing the mammalian pluripotent stem cells with stromal cells, and (c) expanding and differentiating lin − CD34 + CD43 + CD45 + CD38 − cells from step (b) by culture in the presence of GM-CSF, whereby CD34 + CD43 + CD45 + CD38 + myeloid progenitors are produced.