THREE DIMENSIONAL CLUSTERS OF TRANSDIFFERENTIATED CELLS, COMPOSITIONS AND METHODS THEREOF
The present disclosure provides compositions and methods for providing a cell replacement therapy to treat various diseases, including pancreatic diseases and diabetes. Specifically, the disclosure provides three-dimensional (3D) cell clusters of transdifferentiated insulin producing cells attached to scaffolds, such as a polysaccharide matrix, in order to provide a cell replacement therapy.
1 . A composition comprising a three-dimensional (3D) cell cluster comprising transdifferentiated insulin producing cells (IPC) attached to a polysaccharide matrix.
2 . The composition of claim 1 , wherein said polysaccharide matrix comprises a sulfated polysaccharide matrix, or a mix of sulfated polysaccharides and polysaccharides.
3 . The composition of claim 1 , wherein said polysaccharide matrix comprises an alginate polysaccharide, alginate sulfate, hyaluronan sulfate, or a combination thereof.
4 - 22 . (canceled)
23 . A method of generating a three-dimensional (3D) cell cluster comprising transdifferentiated insulin producing cells (IPC) attached to a polysaccharide matrix, the method comprising:
(a) providing the polysaccharide matrix;
(b) providing a human tissue;
(c) processing said tissue to recover primary human cells;
(d) propagating and expanding the cells of step (c) to a predetermined number of cells;
(e) transdifferentiating the cells of step (d); and
(f) attaching at least a subset of said cells to said polysaccharide matrix; thereby generating a 3D cell cluster comprising transdifferentiated insulin producing cells attached to a polysaccharide matrix.
24 . The method of claim 23 , wherein at step (e) said transdifferentiating comprises infecting said cells with:
(a) a first adenoviral vector comprising a nucleic acid encoding a human PDX-1 polypeptide;
(b) a second adenoviral vector comprising a nucleic acid encoding a second human pancreatic transcription factor polypeptide; and
(c) a third adenoviral vector comprising a nucleic acid encoding a human MafA polypeptide.
25 . The method of claim 24 , wherein said second pancreatic transcription factor is selected from NeuroD1 and Pax4.
26 . The method of claim 24 , wherein the infections with said first adenoviral vector and said second adenoviral vector are concurrent.
27 . The method of claim 23 , wherein step (d), step (e), or a combination thereof are executed under non-adherent cell culture conditions.
28 . The method of claim 23 , wherein said polysaccharide matrix comprises a sulfated polysaccharide matrix, or a mix of sulfated polysaccharides and polysaccharides.
29 . The method of claim 23 , wherein said polysaccharide matrix comprises an alginate polysaccharide, alginate sulfate, hyaluronan sulfate, or a combination thereof.
30 - 31 . (canceled)
32 . The method of claim 28 , wherein said sulfated polysaccharide matrix comprises a at least one bioactive polypeptide associated with a sulfate group of said sulfated polysaccharide matrix.
33 . (canceled)
34 . The method of claim 32 , wherein said bioactive polypeptide comprises a positively-charged polypeptide, a heparin-binding polypeptide, or a combination of both.
35 . The method of claim 32 , wherein said bioactive polypeptide is selected from a group comprising antithrombin III (ATIII), thrombopoietin (TPO), serine protease inhibitor (SLP1), CI esterase inhibitor (C1-INH), Vaccinia virus complement control protein (VCP), a fibroblast growth factor (FGF), a FGF receptor, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), a platelet-derived growth factor (PDGF), PDGF-ββ, bone morphogenetic protein (BMP), epidermal growth factor (EGF), CXC chemokine ligand 4 (CXCL4), stromal cell-derived factor-1 (SDF-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), Regulated on Activation, Normal T Expressed and Secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory peptide-1 (MIP-1), lymphotactin, fractalkine, an annexin, apolipoprotein E (ApoE), immunodeficiency virus type-1 (HIV-1) coat protein gp120, cyclophilin A (CypA), Tat protein, viral coat glycoprotein gC, gB or gD of herpes simplex virus (HSV), an envelope protein of Dengue virus, circumsporozoite (CS) protein of Plasmodium falciparum , bacterial surface adhesion protein OpaA, 1-selectin, P-selectin, heparin-binding growth-associated molecule (HB-GAM), thrombospondin type I repeat (TSR), peptide myelin oligodendrocyte glycoprotein (MOG), amyloid P (AP), transforming growth factor (TGF)-β1, or any combination thereof.
36 - 37 . (canceled)
38 . The method of claim 32 , wherein said association comprises a non-covalent bond.
39 . The method of claim 24 , wherein said 3D cell cluster is encapsulated by an encapsulation agent comprising a material selected from a group comprising: alginate, cellulose sulphate, collagen, chitosan, gelatin, agarose, polyethylene glycol (PEG), poly-L-lysine (PLL), polysulphone (PSU), polyvinyl alcohol (PVA), polylactic acid (PLA), acrylates, and low molecular weight dextran sulphate (LMW-DS), any derivatives thereof, and any combination thereof.
40 - 41 . (canceled)
42 . The method of claim 23 , wherein said transdifferentiated cells comprise improved glucose regulated C-peptide secretion, improved glucose regulated insulin secretion, increased insulin content, or increased expression of GCG and NKX6.1, or any combination thereof, compared to transdifferentiated non-pancreatic beta insulin producing cells cultured as a 3D cell cluster without a polysaccharide matrix.
43 . The method of claim 23 , wherein the viability of said transdifferentiated mammalian non-pancreatic beta insulin producing cells is similar to that of transdifferentiated non-pancreatic beta insulin producing cells cultured as a monolayer cell culture.
44 . The method of claim 23 , wherein said transdifferentiated mammalian non-pancreatic beta insulin producing cells are adult cells selected from a group comprising: epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, liver cells, blood cells, stem or progenitor cells, embryonic heart muscle cells, liver stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem and progenitor cells, pancreatic cells other than pancreatic beta cells, acinar cells, alpha-cells, or any combination thereof.
45 - 47 . (canceled)
48 . A method for treating a pancreatic disease or disorder in a subject, the method comprising administering said 3D cell cluster comprising transdifferentiated insulin producing cells of claim 23 .
49 . (canceled)
50 . The method of claim 48 , wherein said pancreatic disease or disease comprises type I diabetes, type II diabetes, gestational diabetes, pancreatic cancer, hyperglycemia, pancreatitis, pancreatic pseudocysts, pancreatic trauma caused by injury, or a disease caused by pancreatectomy, or any combination thereof.