Compositions for Drug Delivery and Methods of Use Thereof
Methods for producing megakaryoctyres and platelets derived from inducible pluripotent stem cells and comprising a therapeutic agent are provided. The present disclosure further provides methods and compositions for loading a platelet or a megakaryocyte with a therapeutic agent and for genetically modifying a platelet or a megakaryocyte to express an agent.
1 .- 62 . (canceled)
63 . A method for preparing a lysate composition, the method comprising:
differentiating induced pluripotent stem cells (iPSCs) into megakaryocytes; and
lysing the megakaryocytes so as to obtain the lysate composition comprising one or more of Interleukin 1-beta, Interleukin 16, Interleukin 12P40, TNF-beta, BCA-1, IP-30, Fractalkine, GCkine, MCP-4, MIP-1alpha, MIP-1beta, SDF-1alpha, SDF-1beta, or a combination thereof.
64 . The method of claim 63 , wherein the megakaryocytes are CD42b + , CD61 + , and DNA + .
65 . The method of claim 63 , wherein the step of differentiating the iPSCs into megakaryocytes comprises:
expanding induced pluripotent cells in a matrix independent culture using single cell passaging; and
dissociating the expanded pluripotent cells into a single cell suspension; and
differentiating the dissociated pluripotent cells in a first culture medium into hemogenic endothelial cells.
66 . The method of claim 65 , 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).
67 . The method of claim 65 , wherein the first culture medium comprises BMP4 for a first time period, and for a second time period bFGF and VEGF are added to the first culture medium.
68 . The method of claim 67 , wherein for the first time period, the first culture medium comprises BMP4 without bFGF and VEGF.
69 . The method of claim 65 , wherein the step of differentiating the iPSCs into megakaryocytes comprises differentiating the hemogenic endothelial cells in a second culture medium into megakaryocytic progenitors.
70 . The method of claim 69 , 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.
71 . The method of claim 69 , wherein the step of differentiating the iPSCs into megakaryocytes comprises differentiating the megakaryocytic progenitors in a third culture medium into megakaryocytes.
72 . The method of claim 71 , 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.
73 . The method of claim 69 , wherein one or more of the dissociated pluripotent cells, the hemogenic endothelial cells or the megakaryocytic progenitors are differentiated under continuous agitation in a matrix independent culture.
74 . The method of claim 65 , wherein the dissociated pluripotent cells are cultured so as to form self-aggregating pluripotent cell spheroids in a matrix independent culture.
75 . The method of claim 74 , wherein the self-aggregating pluripotent cell spheroids are differentiated so as to form hemogenic endothelial cell spheroids.
76 . The method of claim 75 , wherein the hemogenic endothelial cell spheroids are differentiated so as 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.
77 . The method of claim 63 , wherein the lysate composition comprises Interleukin 1-beta, Interleukin 16, Interleukin 12P40, TNF-beta, BCA-1, IP-30, Fractalkine, GCkine, MCP-4, MIP-1alpha, MIP-1beta, SDF-1alpha, SDF-1beta.
78 . A method for preparing a lysate composition, the method comprising:
differentiating induced pluripotent stem cells (iPSCs) into megakaryocyte progenitors; and
lysing the megakaryocyte progenitors so as to obtain the lysate composition comprising one or more of Interleukin 1-beta, Interleukin 16, Interleukin 12P40, TNF-beta, BCA-1, IP-30, Fractalkine, GCkine, MCP-4, MIP-1alpha, MIP-1beta, SDF-1alpha, SDF-1beta, or a combination thereof.
79 . The method of claim 78 , wherein differentiating iPSCs into megakaryocyte progenitors comprises:
differentiating the iPSCs in a first culture medium into hemogenic endothelial cells, the first culture medium comprising BMP4 for a first time period and bFGF and VEGF for a second time period after the first time period; and
differentiating the hemogenic endothelial cells in a second culture medium into megakaryocytic progenitors.
80 . The method of claim 78 , wherein the lysate composition comprises Interleukin 1-beta, Interleukin 16, Interleukin 12P40, TNF-beta, BCA-1, IP-30, Fractalkine, GCkine, MCP-4, MIP-1alpha, MIP-1beta, SDF-1alpha, SDF-1beta.
81 . A composition comprising:
a megakaryocyte lysate, wherein the megakaryocyte lysate is produced from lysing of megakaryocytes that have been differentiated from iPSCs, and wherein the megakaryocyte lysate comprises at least one factor produced during the differentiation of the iPSCs into the megakaryocytes, the at least one factor selected from Interleukin 1-beta, Interleukin 16, Interleukin 12P40, TNF-beta, BCA-1, IP-30, Fractalkine, GCkine, MCP-4, MIP-1alpha, MIP-1beta, SDF-1alpha, SDF-1beta, or a combination thereof.
82 . The composition of claim 81 , wherein the megakaryocyte lysate comprises Interleukin 1-beta, Interleukin 16, Interleukin 12P40, TNF-beta, BCA-1, IP-30, Fractalkine, GCkine, MCP-4, MIP-1alpha, MIP-1beta, SDF-1alpha, and SDF-1beta.