Compositions and methods for producing megakaryocytes
Methods for producing megakaryocytic progenitors (preMKs) and megakaryocytes (MKs) from stem cells are provided. The present disclosure further provides compositions comprising preMKs and MKs and their lysates, and also methods of use of preMKs, MKs, their lysates and compositions thereof.
1. A method for megakaryocyte production comprising:
culturing dissociated pluripotent stem cells in a matrix-independent culture and under continuous agitation such that the pluripotent stem cells form self-aggregating pluripotent cell spheroids;
differentiating, under continuous agitation, the self-aggregating pluripotent cell spheroids in a first culture medium into hemogenic endothelial cell spheroids; and
differentiating, under continuous agitation, the hemogenic endothelial cell spheroids in a second culture medium to produce megakaryocytic progenitors, causing the hemogenic endothelial cell spheroids to release the megakaryocytic progenitors into suspension while maintaining the hemogenic endothelial cell spheroids for subsequent production and release of the megakaryocytic progenitors.
2. The method of claim 1 wherein the first culture medium comprises one or more of Bone morphogenic protein 4 (BMP4), Basic fibroblast growth factor (bFGF), and Vascular endothelial growth factor (VEGF).
3. The method of claim 1 wherein the second culture medium comprises one or more of Stem cell factor (SCF), Thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3-L), Interleukin-3 (IL-3), Interleukin-6 (IL-6) and Heparin.
4. The method of claim 1 wherein the pluripotent stem cells are human induced pluripotent stem cells.
5. The method of claim 1 further comprising the step of seeding the megakaryocytic progenitors onto a non-adherent surface in a culture medium before differentiating the megakaryocytic progenitors into megakaryocytes.
6. The method of claim 1 further comprising the step of differentiating the megakaryocytic progenitors in a third culture medium into megakaryocytes.
7. The method of claim 6 wherein the third culture medium comprises one or more of Stem cell factor (SCF), Thrombopoietin (TPO), Interleukin-6 (IL-6), Interleukin-9 (IL-9) and Heparin.
8. A method for megakaryocyte production comprising:
differentiating, in a matrix-independent culture, pluripotent stem cells in a first culture medium into hemogenic endothelial cells; and
differentiating the hemogenic endothelial cells in a second culture medium into megakaryocytic progenitors,
wherein each of the differentiating the pluripotent stem cells and the differentiating the hemogenic endothelial cells is carried out under continuous agitation to enable the pluripotent stem cells to self-aggregate and differentiate into hemogenic endothelial cell spheroids and the hemogenic endothelial cells in the hemogenic endothelial cell spheroids to differentiate into megakaryocyte progenitors, causing the hemogenic endothelial cell spheroids to release the megakaryocytic progenitors into suspension while maintaining the hemogenic endothelial cell spheroids for subsequent production and release of additional megakaryocytic progenitors.
9. The method of claim 8 wherein the first culture medium comprises one or more of Bone morphogenic protein 4 (BMP4), Basic fibroblast growth factor (bFGF), and Vascular endothelial growth factor (VEGF) and the second culture medium comprises one or more of Stem cell factor (SCF), Thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3-L), Interleukin-e (IL-3), Interleukin-6 (IL-6) and Heparin.
10. The method of claim 8 wherein the pluripotent stem cells are human induced pluripotent stem cells.
11. The method of claim 8 further comprising the step of differentiating the megakaryocytic progenitors in a third culture medium into megakaryocytes, the third culture medium comprising one or more of Stem cell factor (SCF), Thrombopoietin (TPO), Interleukin-6 (IL-6), Interleukin-9 (IL-9) and Heparin.