METHODS AND PRODUCTION OF NOVEL PLATELETS
The present disclosure provides methods for generating megakaryocytes and novel platelet variants from the same CD34+ progenitor stem cells, which comprises at least two stages: stage zero (0) comprising an expansion and maintenance stage of the CD34+ progenitor stem cells for a period ranging between 0 hours to 48 hours; and, stage one (I) comprising a differentiation phase wherein the differentiation phase comprises differentiating the CD34+ progenitor stem cells in step (i) for a period sufficient to generate substantially matured megakaryocytes. Novel platelet variants are produced by passaging the megakaryocytes, produced by the CD34+ progenitor stem cells, through a bioreactor or a fluidic device. Formulations comprising megakaryocytes and platelet variants derived from CD34+ progenitor stem cells and methods of their use are also disclosed.
1 - 47 . (canceled)
48 . A method for generating megakaryocytes from progenitor stem cells comprising at least two stages:
(i) performing a stage zero (0) comprising an expansion and maintenance stage of the progenitor stem cells; and,
(ii) performing a stage one (I) comprising a differentiation phase wherein the differentiation phase comprises differentiating the progenitor stem cells in step (i) for a period sufficient to generate matured megakaryocytes, wherein the matured megakaryocytes are positive for one or more of CD61, CD42a, and CD42b.
49 . The method of claim 48 , wherein the progenitor stem cells are CD34+ progenitor stem cells.
50 . The method of claim 48 , further comprising culturing the matured megakaryocytes in a bioreactor subject to one or more of shear stress, mechanical strain and pulsed electromagnetic field.
51 . The method of claim 48 , further comprising differentiating the matured megakaryocytes to pro-platelets or platelets.
52 . A composition comprising platelets produced by the method of claim 48 , wherein the platelets are used in treating a disease or a disorder in subject.
53 . The composition of claim 52 , wherein the disease or disorder is selected from one or more of an immunoinflammatory disorder, a metabolic disorder, a neoplastic disorder, an autoimmune disorder, viral or bacterial-induced disorder.
54 . The composition of claim 52 , further comprising a cytotoxic agent selected from one or more of an antibody, a nucleic acid, a protein or a polypeptide, or a drug or a prodrug and a combination thereof.
55 . A method for generating genetically engineered megakaryocytes comprising at least:
(i) genetically engineering progenitor stem cells for forming genetically engineered megakaryocytes;
(ii) performing a stage zero (0) comprising an expansion and maintenance stage of the genetically engineered progenitor stem cells, and
(iii) performing a stage one (I) comprising a differentiation phase wherein the differentiation phase comprises differentiating the genetically engineered progenitor stem cells in step (i) for a period sufficient to generate matured engineered megakaryocytes, wherein the matured engineered megakaryocytes are positive for one or more of CD61, CD42a, and CD42b.
56 . The method of claim 55 , wherein the progenitor stem cells are CD34+ progenitor stem cells.
57 . The method of claim 56 , wherein the genetically engineered progenitor stem cells express one or more exogenous nucleic acids encoding for one or more of a therapeutic protein(s) or a polypeptide(s), a receptor, or a fragment thereof, selected from one or more of a cell-surface receptor or transmembrane receptor, an ion channel-linked receptor, a G-protein-coupled receptor, an enzyme-linked receptor or an internal receptor and a combination thereof.
58 . The method of claim 57 , wherein the therapeutic protein(s) or the polypeptide(s) is selected from one or more of an antibody or a fragment thereof, a growth factor, a hormone, an antigen, a cytokine and a combination thereof.
59 . The method of claim 55 , further comprising one or more of differentiating the genetically engineered megakaryocytes to engineered pro-platelets or platelets and culturing the genetically engineered megakaryocytes in a bioreactor.
60 . A method for generating platelet variants from progenitor stem cells comprising at least:
(i) performing an expansion and maintenance stage of the progenitor stem cells comprising culturing the progenitor stem cells;
(ii) performing a differentiation stage wherein the differentiation stage comprises differentiating the progenitor stem cells in step (i) for a period sufficient to generate matured megakaryocytes, wherein the matured megakaryocytes are positive for one or more of CD61, CD42a, and CD42b; and
(iii) passaging said matured megakaryocytes through a bioreactor wherein the matured megakaryocytes generate platelet variants.
61 . The method of claim 60 , wherein the progenitor stem cells are CD34+ progenitor stem cells.
62 . The method of claim 61 , wherein the CD34+ progenitor stem cells comprise an exogenous nucleic acid encoding for a protein.
63 . The method of claim 62 , wherein the protein is expressed in the platelet variants.
64 . A method for generating platelet variants from progenitor stem cells for administration into a subject in need thereof comprising passaging megakaryocytes through a bioreactor, wherein the megakaryocytes are positive for one or more of CD61, CD42a, and CD42b.
65 . The method of claim 64 , wherein the progenitor stem cells are CD34+ progenitor stem cells.
66 . The method of claim 65 , wherein a bioreactor gradient in the bioreactor generates platelet variants and wherein the platelet variants do not exhibit uncontrolled growth or tumor formation in vivo.
67 . A method for generating genetically engineered platelet variants from progenitor stem cells for administration into a subject in need thereof comprising:
(i) genetically engineering the progenitor stem cells and differentiating to produce genetically engineered megakaryocytes, wherein the genetically engineered megakaryocytes are positive for one or more of CD61, CD42a, and CD42b; and,
(ii) passaging the genetically engineered megakaryocytes through a bioreactor subject to one or more of shear stress, mechanical strain and pulsed electromagnetic field.