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Definitions

• the described invention relates generally to methods of generating, isolating, and using functionally modified adult mononuclear cells.
• the described invention also relates to methods of generating, isolating, and using insulin producing cells derived from adult peripheral blood.
• Embryonic stem cells are stem cells derived from an embryo that are pluripotent, i.e., they are able to differentiate in vitro into endodermal, mesodermal and ectodermal cell types.
• Embryonic stem (ES) cells are attractive because of their high potential for self-renewal and their pluripotent differentiation capability, but ethical concerns have limited their availability and practical usefulness.
• adult stem cells include, but not limited to, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells, and skin stem cells.
• Bone marrow consists of a variety of precursor and mature cell types, including hematopoietic cells (the precursors of mature blood cells) and stromal cells (the precursors of a broad spectrum of connective tissue cells), both of which appear to be capable of differentiating into other cell types.
• hematopoietic cells the precursors of mature blood cells
• stromal cells the precursors of a broad spectrum of connective tissue cells
• CD34+ cells represent approximately 1% of bone marrow derived nucleated cells.
• Hematopoietic stem cells also known as the colony-forming unit of the myeloid and lymphoid cells (CFU-M,L), or CD34+ cells
• CD34 antigen also is expressed by immature endothelial cell precursors; mature endothelial cells do not express CD34+. Peichev, M. et al., Blood 95: 952-58 (2000).
• CD34+ cells derived from adult bone marrow give rise to a majority of the granulocyte/macrophage progenitor cells (CFU-GM), some colony-forming units-mixed (CFU-Mix) and a minor population of primitive erythroid progenitor cells (burst forming units, erythrocytes or BFU-E).
• CFU-GM granulocyte/macrophage progenitor cells
• CFU-Mix colony-forming units-mixed
• BFU-E primitive erythroid progenitor cells
• HSCs While there is no single cell surface marker exclusively expressed by hematopoietic stem cells, it generally has been accepted that human HSCs have the following antigenic profile: CD 34+, CD59+, Thy1+(CD90), CD38low/-, C-kit- low and, lin-.
• CD45 is also a common marker of HSCs, except platelets and red blood cells, which are CD45-. HSCs can generate a variety of cell types, including erythrocytes, neutrophils, basophils, eosinophils, platelets, mast cells, monocytes, tissue macrophages, osteoclasts, T lymphocytes, and B lymphocytes.
• hematopoietic stem cells The regulation of hematopoietic stem cells is a complex process involving self-renewal, survival and proliferation, lineage commitment and differentiation and is coordinated by diverse mechanisms including intrinsic cellular programming and external stimuli, such as adhesive interactions with the micro-environmental stroma and the actions of cytokines.
• Cytokines can be made by several cell types, but they are collected and concentrated by the extracellular matrix of the stromal (mesenchymal) cells at the sites of hematopoiesis. For example,
• GM-CSF granulocyte-macrophage colony-stimulating factor
• IL-3 multilineage growth factor
• HSCs reside in the bone marrow but can be forced into the blood, a process termed mobilization.
• Stem cell mobilization is a process whereby stem cells are stimulated out of the bone marrow into the bloodstream, so they are available for collection from the peripheral blood for future reinfusion.
• Current mobilization strategies used in the clinic mainly G-CSF cytokine, are well tolerated but often produce suboptimal number of collected HSCs.
• HSCs in the bone marrow niche generate energy mainly via anaerobic metabolism and have low levels of ROS, which promotes their self-renewal.
• Mobilization and homing are mirror processes depending on an interplay between chemokines, chemokine receptors, intracellular signaling, adhesion molecules and proteases. Homing to the bone marrow is necessary to optimize cell engraftment. The interaction between SDF-1/CXCL12 and its receptor CXCR4 is critical to retain HSCs within the bone marrow. (Suarez-Alvarez, B. et al,
• HSC hematopoietic stem cells
• hepatocytes can be turned into pancreatic beta cells and vice versa.
• Yi, F. et al "Rejuvenating liver and pancreas through cell transdifferentiation,” Cell Res. 2012; 22(4): 616-619).
• adult somatic cells can be reprogrammed to enter an embryonic stem cell-like state by being forced to express a set of transcription factors, for example, Oct-3/4 (or Pou5f1 , the Octamer transcription factor-3/4), the Sox family of transcription factors (e.g., Sox-1 , Sox-2, Sox-3, and Sox- 15), the Klf family
• a set of transcription factors for example, Oct-3/4 (or Pou5f1 , the Octamer transcription factor-3/4)
• Sox family of transcription factors e.g., Sox-1 , Sox-2, Sox-3, and Sox- 15
• Klf family of transcription factors e.g., Sox-1 , Sox-2, Sox-3, and Sox- 15
• transcription factors Klf-1 , Klf-2, Klf-4, and Klf-5
• Myc family of transcription factors e.g., c-Myc, N-Myc, and L-Myc.
• iPSCs human inducible Pluripotent Stem cells
• iPSCs are cells reprogrammed to express transcription factors that express stem cell markers and are capable of generating cells characteristic of all three germ layers (i.e., ectoderm, mesoderm, and endoderm).
• pluripotency a cell that reprogrammed to pluripotency (Id.).
• the minimal factors necessary to reprogram a cell depend on the endogenous "sternness" of the starting cell; for example, neural stem cells can be reprogrammed using Oct4 alone since they express high levels of the other factors (Id. Citing Kim, JB, et al, "Oct4-induced pluripotency in adult neural stem cells, " Cell 2009; 136: 411-19).
• Induced pluripotent stem cell reprogramming consists of three phases:
• the initiation phase is characterized by somatic genes being switched off by methylation, an increase in cell proliferation, a metabolic switch from oxidative phosphorylation to glycolysis, reactivation of teleomerase activity and a
• mesenchymal-to-epithelial transition (MET)(ld. Citing David, L, and Polo, JM, "Phases of reprogramming," Stem Cell Res. 2014; 12: 754-61), which involves the loss of mesenchymal characteristics, such as motility, and the acquisition of epithelial characteristics, such as cell polarity and expression of E-Cadherin (Id. Citing Redmer, T et al, "E-cadherin is crucial for embryonic stemcell pluripotency and can replace OCT4 during somatic cell reprogramming," EMBO Rep. 2011 ; 12: 720-26).
• Sox2 suppresses expression of Snail, an EMT inducer (Id.
• TGF ⁇ inhibitors promote Nanog expression, and mitogen-activated protein kinase (MAPK) signalling, activated by TGF , further induces the expression of mesodermal genes.
• MAPK mitogen-activated protein kinase
• Bone morphogenetic protein (BMP) signaling also plays an important role in the initiation stage of mouse iPS cell reprogramming by promoting MET via upregulation of epithelial genes, such as E-cadherin, occludin and epithelial cell adhesion molecule (Id. Citing Samavarchi-Tehrani, P. et al, "Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition In the initiation of somatic cell reprogramming," Cell Stem Cell 2010; 7: 64-77). However, constitutive BMP activation prevents human somatic cell reprogramming.
• epithelial genes such as E-cadherin, occludin and epithelial cell adhesion molecule
• Fibroblast growth factor (FGF) signaling has also been implicated at the initiation stage (Id. Citing Jiao, J. et al, "Promoting reprogramming by FGF2 reveals that the extracellular matrix is a barrier for reprogramming fibroblasts to
• FGF2 promotes the early stages of reprogramming through accelerating cell proliferation, facilitating MET and eliminating extracellular collagens.
• the minority of cells that undergo successful reprogramming also exhibit resistance to apoptosis and senescence by transgene expression (Id. Citing Papp, B, "Reprogramming to pluripotency: stepwise resetting of the epigenetic landscape,” Cell Res. 2011 ; 21 : 486-501).
• the initiation phase is also characterized by a metabolic switch from oxidative phosphorylation to glycolysis (Id. Citing Panopoulos, AD et al, 'The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming," Cell Res. 2012; 22: 168-77), which involves PI3K/AKT signaling (Id. Citing Zhu, S. et al, "Reprogramming of human primary somatic cells by OCT4 and chemical compounds," Cell Stem Cell 2010; 7: 651-55; Chen, M. et al,
• Wnt signaling also enhances the maturation phase of mouse somatic cell reprogramming, whereby exogenous stimulation of the pathway using Wnt3a after induction of reprogramming enhances formation of Nanog positive colonies (Id. Citing Ho, R, et al, "Stage- specific regulation of reprogramming to induced pluripotent stem cells by Wnt signaling and T cell factor proteins," Cell Rep. 2013; 3: 2113-26).
• the stabilization phase is characterized by transgene independence
• Platelets thrombocytes
• MK megakaryocytes
• Platelet adhesion Following vascular injury and the attendant endothelial damage, platelet adhesion, initiating the process of primary hemostasis, is mediated through receptor ligand interactions in a step-wise fashion.
• Extracellular von Willebrand factor (VWF)-platelet glycoprotein (GP) lb binding mediates initial platelet recruitment to the injured area.
• VWF von Willebrand factor
• GPVI Platelet GPVI interacts with fibrillary collagen and platelet ⁇ 1 integrin interacts with laminin, collagen and fibronectin, allowing for firm adhesion of platelets to the exposed extracellular matrix.
• Platelet activation Following platelet adhesion, a series of downstream signaling events results in an increase in intracellular calcium and subsequent platelet activation marked by exposure of negatively-charged phosphatidylserine (PS) on the platelet membrane surface, allowing for the assembly of coagulation factors; platelet alpha and delta granule secretion, resulting in the release of ADP, calcium, serotonin, VWF, coagulation factors V and VIII, and fibrinogen; platelet membrane GPIIb/llla integrin conversion to a high affinity state for VWF and fibrinogen binding; thromboxane A2 generation through arachidonic acid
• PS negatively-charged phosphatidylserine
• Platelet aggregation A key step for the development of a stable platelet aggregate is the conversion of the GPIIb/llla receptor into its high affinity
• Platelet markers [00311 Markers that appear on the platelet surface before activation. Platelet surface markers, which appear on the platelet surface before activation, include CD41 (GP llb/llla), CD42a (GPIX), CD42b (GPIb), and CD61 (avb3, vitronectin receptor).
• CD41 GP llb/llla
• CD42a GPIX
• CD42b GPIb
• CD61 avb3, vitronectin receptor
• CD41 is a heterodimeric integral membrane protein that undergoes post-translational modifications that result in two polypeptide chains linked by a disulfide bond. CD41 is expressed on platelets and megakaryocytes and on early embryonic hematopoietic stem cells. A CD41/CD61 complex formed by the integrin alpha chain associated with a beta 3 chain (CD61) Integrin ⁇ ⁇ 3 is a receptor for fibronectin, fibrinogen, von Willebrand factor, vitronectin and
• the GPIIb/llla receptor (integrin ⁇ ⁇ 3) is one of the most abundant cell surface receptors ( «80 000 per platelet) [Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller BS, Jordan RE. Analysis of GPIIb/llla receptor number by quantitation of 7E3 binding to human platelets. Blood. 1996;88:907-914], which represents about 15% of total surface protein. [Jennings, LK, Phillips, DR, "Purification of glycoproteins Mb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein lib-Ill complex. J Biol Chem.
• CD42a-d complex is a receptor for von Willebrand factor and thrombin.
• CD42a is also called platelet glycoprotein GPIX, GP9a.
• CD42b is also called platelet GPIb alpha, or glycoprotein 1b-alpha.
• markers which appear on the platelet surface during activation include activated llb/llla, CD62P (P-selectin), CD31 (PECAM) and CD63.
• P-selectin mediates the initial adhesion of activated platelets to monocytes and neutrophils via the P-selectin glycoprotein ligand-1 (PSGL-1) counterreceptor on the leukocyte surface.
• PSGL-1 P-selectin glycoprotein ligand-1
• Soluble CD40 ligand (sCD40L, CD154) is a plasma marker of in vivo platelet activation. Id. Release of sCD40L by activated platelets is the predominant source of plasma sCD40L; the mechanism of sCD40L release is proteolysis of platelet surface CD40L. Id. Accurate measurement of in vivo circulating sCD40L requires assay in plasma rather than serum. Id.
• Lysosomal Activated Membrane Protein is a cell surface glycoprotein that is known to complex with integrins. It may function as a blood platelet activation marker.
• Platelet surface P-selectin is a component of the a-granule membrane of resting platelets that is only expressed on the platelet surface membrane after a granule secretion.
• MK Megakaryocytes
• HSCs hematopoietic stem cells
• CMPs common myeloid progenitors
• thrombopoiesis Progressive commitment of CMPs to the megakaryocyte lineage is principally regulated by thrombopoietin (TPO).
• TPO thrombopoietin
• CFU-MK colony forming units-megakaryocyte
• the maturation of a megakaryocyte involves an increase in expression of the cell surface markers GPIIb/llla (also known as CD41 or allb/ ⁇ integrin receptor) and GPIb/GPIX/GPV receptors, and a substantial increase in cell mass, which results in cytosolic accumulation of a granules, dense bodies, and platelet-associated proteins like von Willebrand factor (vWF) and platelet factor-4.
• GPIIb/llla also known as CD41 or allb/ ⁇ integrin receptor
• GPIb/GPIX/GPV receptors GPIIb/GPIX/GPV receptors
• HSCs from peripheral blood (PB), bone marrow (BM) and CB are also capable of producing megakaryocytes and functional platelets.
• PB peripheral blood
• BM bone marrow
• CB CB
• CFU-MK colony-forming unit-megakaryocyte
• BFU-MK burst-forming unit-megakaryocyte
• BFU-MK and CFU-MK are thought to represent the primitive and mature progenitors restricted to the lineage, respectively. Id. Like their erythroid counterparts, the cytokine requirements for CFU-MK are simple; thrombopoietin stimulates the growth of 75% of all CFU-MK, with interleukin (IL)-3 being required along with thrombopoietin for the remainder. IL-3 or steel factor (SF) is required along with thrombopoietin for more complex, larger MK colony formation from primitive progenitor cells.
• IL interleukin
• SF steel factor
• Megakaryocytes also arise in clonal colonies containing cells of one or more additional hematopoietic lineages.
• the most primitive in vitro colony-forming cell is termed a colony-forming unit-granuloycte-erythrocyte-monocyte-megakaryocte (CFU-GEMM), mixed progenitor colony (CFU-Mix), or common myeloid progenitor (CMP; Id. Citing Akashi K, Traver D, Miyamoto T, Weissman IL. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature.
• a derivative of the CMP is the mixed MK/erythroid progenitor cell (MEP; Id. Citing Nakorn TN, Miyamoto T, Weissman IL. Characterization of mouse clonogenic megakaryocyte progenitors. Proc Natl Acad Sci U S A. 2003;100:205- 210).
• MEP mixed MK/erythroid progenitor cell
• cytokines most responsible for development of these two lineages erythropoietin and thrombopoietin, the two most closely related proteins in the hematopoietic cytokine family (Id. Citing Lok S, Kaushansky K, Holly RD, et al.
• the transcription factors expressed by megakaryocyte progenitors that allow for their commitment to the lineage include GATA1 , and FOG29.
• GATA1 is an X- linked gene encoding a 50 kDa zinc finger DNA binding protein.
• GATA1 is an X- linked gene encoding a 50 kDa zinc finger DNA binding protein.
• GATA1-/- condition is embryonic lethal due to failure of erythropoiesis (Id. Citing Pevny L, Simon MC, Robertson E, et al.
• GATA1 acts in concert with friend of GATA (FOG29), another protein that affects transcription without binding to DNA,
• the ets family of transcription factors includes approximately 30 members that bind to a purine box sequence, and consists of proteins that interact in both positive and antagonistic ways.
• PU.1 initially termed Spi-1 based on its association with spleen focus-forming virus products, blocks erythroid
• Doubeikovski A, Uzan G, Doubeikovski Z, et al. Thrombopoietin-induced expression of the glycoprotein Mb gene involves the transcription factor PU. 1 /Spi-1 in UT7-Mpl cells. J Biol Chem. 1997;272:24300-24307). Moreover, the ets factor Fli-1 is essential for megakaryopoiesis (Id. Citing Athanasiou M, Clausen PA,
• Thrombopoiesis is the process of formation of thrombocytes (platelets). On a molecular level, thrombopoiesis is a highly coordinate process, with sophisticated reorganization of membrane and microtubules and precise distribution of granules and organelles. Platelets form by fragmentation of mature megakaryocyte membrane pseudopodial projections termed proplatelets (Kaushansky citing Patel SR, Hartwig JH, Italiano JE., Jr The biogenesis of platelets from megakaryocyte proplatelets. J Clin Invest. 2005;115:3348-3354), in a process that consumes nearly the entire cytoplasmic complement of membranes, organelles, granules, and soluble macromolecules.
• thrombopoietin is the primary regulator of thrombopoiesis, little is known about what determines the size of mature platelets or how the mechanism of platelet formation is affected by the transcription factor GATA1 , the glycoprotein Ib/IX complex, the Wiskott Aldrich syndrome protein (WASP), and platelet myosin, as defects in each of these genes leads to unusually large or small platelets.
• thrombocytopenias toward a molecular understanding of disorders of platelet production. Curr Opin Pediatr. 2004;16:15-22).
• thrombopoietin for the generation of fully mature megakaryocytes from which platelets arise, elimination of the cytokine during the final stages of platelet formation is not detrimental (Id. Citing Choi ES, Nichol JL, Hokom MM, Hornkohl AC, Hunt P. Platelets generated in vitro from pro platelet-displaying human megakaryocytes are functional. Blood. 1995;85:402-413).
• UC-HS hematopoietic stem cells
• mesenchymal stem cells which in turn can be found in umbilical cord blood (UC-MS) or in Wharton's jelly (UC-MM).
• Umbilical cord (UC) vessels and the surrounding mesenchyma derive from the embryonic and/or extraembryonic mesodermis.
• these tissues, as well as the primitive germ cells are differentiated from the proximal epiblast, at the time of formation of the primitive line of the embryo, containing MSC and even some cells with pluripotent potential.
• the UC matrix material is speculated to be derived from a primitive mesenchyma, which is in a transition state towards the adult bone marrow
• the blood from the placenta and the umbilical cord which contains all the normal elements of blood - red blood cells, white blood cells, platelets and plasma - is relatively easy to collect in usual blood donation bags, which contain anticoagulant substances. Mononuclear cells then are separated by density gradient
• Ficoll-Paque density gradient centrifugation In Ficoll-Paque density gradient centrifugation, anticoagulant-treated and diluted cord blood is layered on the Ficoll-Paque solution and centrifuged.
• the mononuclear cell fraction includes two stem cell populations: (1) hematopoietic stem cells (HSC), which express certain characteristic markers (CD34, CD133); and (2) mesenchymal stem cells (MSC) that are capable of adhering to a culture surface under certain conditions (e.g., modified McCoy medium and lining of vessels with Fetal Bovine Serum (FBS) or Fetal Calf Serum (FCS)).
• HSC hematopoietic stem cells
• MSC mesenchymal stem cells
• FBS Fetal Bovine Serum
• FCS Fetal Calf Serum
• Umbilical cord blood MSCs can produce cytokines, which facilitate grafting in the donor and in vitro HSC survival compared to bone marrow MSC. (Zhang, X et al., Biochem Biophys Res Commun, 2006, 351 : 853- 859).
• MSCs from the umbilical cord matrix are obtained by different culture methods depending on the source of cells, e.g., MSCs from the connective matrix, from subendothelial cells from the umbilical vein or even from whole umbilical cord explant. They are generally well cultured in DMEM medium, supplemented with various nutritional and growth factors; in certain cases prior treatment of vessels with hyaluronic acid has proved beneficial (Baban, B. et al., J Reprod Immunol, 2004, 61 : 67-77).
• HUCB Human umbilical cord blood
• hematopoietic progenitor cells as measured in standard clonogenic assays for burst-forming units and granulocyte- macrophage colony-forming units.
• Crocuttini, FM and Boyd AW "Hematopoietic and lymphoid progenitor cells in human umbilical cord blood," Devel. Immunol. 4: 1-11 (1994)
• Broxmeyer, HE et al "Human umbilical cord blood as a potential source of transplantable hemopoietic stem/progenitor cells, " Proc. Natl Acad. Sci. USA (1989) 86: 3828-3719).
• CFU-GEMM multipotential
• BFU-E erythroid
• CFU-GM granulocyte-macrophage progenitor cells
• cord blood progenitors number of colonies formed/number of cells plated
• the frequency of cord blood progenitors equals or exceeds that of marrow and greatly surpasses that of adult blood.
• Progenitor cells from HUCB can be maintained for several weeks in long-term liquid culture systems, suggesting their production from more primitive cells (Id. citing Salahuddin, SZ et al, "Long term suspension cultures of human cord blood myeloid cells," 1981 ; Blood 58: 931-38; Smith, S & Broxmeyer, HE; "The influence of oxygen tension on the long-term growth in vitro of hemopoietic progenitor cells from human cord blood," Brit. J. Haematol. (1986): 63: 29-34).
• CFC colony-forming cells
• Cord blood B cells Human umbilical cord blood has been shown to be enriched for pre-B and B cells compared to adult peripheral blood. Id. The mean frequency of pre-B cells has been shown to be 0.7% of total lymphocytes in cord blood compared to 0.2% in adult blood (Id. citing Okino, F, "Pre-B cells and B lymphocytes in human cord blood and adult peripheral blood," Acta Paediatr. Jpn (1987): 29: 195-201). The mean relative frequency of B lymphocytes in cord blood is also higher, being 11.4% of total lymphocytes compared to 5.4% in adult blood (Id). In terms of absolute numbers of preB cells, cord blood contains 10 times the number in adult blood. Id.
• the antigens CD1C, CD38, CD5 and CD23 are highly expressed on cord blood B cells, but are normally expressed on only a small percentage of circulating B cells in normal adults. Id. It has been suggested that whereas neonatal B cells are probably functionally naive, their inherent potential for stimulation, which approaches that of adult B cells, can be realized as long as sufficiently strong T-cell help is available. Id.
• CD2 a surface antigen of the human T- lymphocyte lineage that is expressed on all peripheral blood T cells
• CD3 T lymphocyte marker
• CD8 marker for T cells with suppressor and cytotoxic activity
• CD4+ cells helper/inducer T cells
• cord blood CD4+ cells are deficient in their ability to provide help for antibody production. Id.
• cord blood CD4+ cells express high levels of CD45RA and L-selectin (Leu-8) (Id. citing Clement, LT et al, "Novel immunoregulatory functions of phenotypically distinct subpopulations of CD4+ cells in the human neonate," J. Immunol. (1990): 145: 102-108)) and have low levels of CD45RO (citing Sanders et al 1988). Their cytokine profiles suggest that they are naive THp cells. The dominant immunoregulatory phenotype of cord blood CD4+ cells has been shown to be largely immunosuppressive, consistent with the preponderance of CD4+CD45RA+ (and CD38+) cells (Id.
• PWM pokeweed mitogen
• cord blood CD4+ + CD45RA+ cells acquired the ability to provide help for B cell differentiation. This functional maturation was accompanied by conversion to the CD4+CD45RA-CD45RO+ phenotype.
• NK cells Natural Killer (NK) cells
• NK cells Human natural killer (NK) cells can be subdivided into different populations based on the relative expression of the surface markers CD16 and CD56.
• CD16 and CD56 CD56bright natural killer (NK) cells: an important NK cell subset
• Cord blood NK cells are heterogeneous. Although cells bearing the NK marker CD57+ are negligible in cord blood (Cicuttini, FM and Boyd AW, "Hematopoietic and lymphoid progenitor cells in human umbilical cord blood," Devel. Immunol.
• the hematopoietic stem cell is the common ancestor of all blood cells. Hematopoietic stem cell maturation involves the diversification of the lymphoid and myeloid cell lineages, the two major branches of hematopoietic cells. (Kondo, M. "Lymphoid and myeloid lineage commitment in multipotent hematopoietic
• Lymphoid lineage cells include T, B, and natural killer (NK) cells.
• the myeloid lineage includes
• granulocytes neurotrophils, eosinophils and basophils
• monocytes monocytes
• macrophages macrophages
• mast cells which belong to the myeloid lineage
• lymphoid and myeloid lineages are separable at the progenitor level.
• Common lymphoid progenitors CLPs
• CLPs can differentiate into all types of lymphocytes without noticeable myeloid potential under physiological conditions (Kondo M, Scherer DC, Miyamoto T, King AG, Akashi K, Sugamura K, et al. Cell-fate
• CMPs common myeloid progenitors
• B-cell potential A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature. 2000 Mar 9;404(6774):193-7.
• DCs dendritic cells
• CMPs can proliferate and differentiate into megakaryocyte-erythrocyte
• GM progenitors granulocyte-monocyte progenitors, which further give rise to megakaryocytes, erythrocytes, granulocytes, monocytes and others.
• GM progenitors granulocyte-monocyte progenitors, which further give rise to megakaryocytes, erythrocytes, granulocytes, monocytes and others.
• MKPs The monopotent megakaryocyte lineage-committed progenitor (MKPs) has been isolated downstream of MEPs by CD9, a megakaryocyte-associated surface protein. MKPs have the phenotype CD9+IL-7Ra- Lin- Sca-1- c-Kit+ Thy1.1- and represent only 0.01% of the total bone-marrow cells. (Iwasaki H, Akashi K. Myeloid lineage commitment from the hematopoietic stem cell. Immunity. 2007;26:726-740). MKPs give rise exclusively to various sizes of megakaryocyte colonies. (Id. Citing T.N. Nakorn, T.
• MEPs represent the majority of day 8 CFU-S activity; MKPs do not have CFU-S activity, and generate only megakaryocytes in vitro. Id.
• bipotent MEPs Like other primitive hematopoietic cells, bipotent MEPs resemble small lymphocytes but can be distinguished by a specific pattern of cell surface protein display, IL-7Ra-/Lin-/c-Kit+/Sca-1-/CD34-/FcRYlo.
• IL-7Ra-/Lin-/c-Kit+/Sca-1-/CD34-/FcRYlo a specific pattern of cell surface protein display, IL-7Ra-/Lin-/c-Kit+/Sca-1-/CD34-/FcRYlo.
• CD71 transferrin receptor
• CD36 thrombospondin receptor
• CMPs common myeloid progenitors
• GFPs granulocyte/macrophage
• megakaryocyte/erythrocyte progenitors reside in the lineage-negative (lin— ) CD34+CD38+ fraction of adult bone marrow as well as in cord blood. They are distinguishable by the expression of the IL-3Ra chain, the receptor of an early-acting hematopoietic cytokine, and CD45RA, an isoform of a phosphotyrosine phosphatase involved in negative regulation of cytokine signaling.
• Manz, MG, et al “Prospective isolation of human clonogenic common myeloid progenitors," Proc. Natl Acad. Sci. U.S. 2002 99(18): 11872-11877).
• the IL-3RaloCD45RA+ cells were termed GMPs and the IL-3Ra-CD45RA- cells termed MEPs.
• GMPs IL-3RaloCD45RA+ cells
• MEPs IL-3RaloCD45RA- cells
• SCF, IL-11 , FL, Epo, and Tpo after 72 h of culture.
• IL- 3RaloCD45RA- cells gave rise to all types of colonies (but CFU-GEMM)
• GMPs exclusively gave rise to granulocyte/macrophage colonies
• MEPs gave rise to megakaryocyte/ erythrocyte colonies and four (1.6%) granulocyte and macrophage colonies. Therefore, the IL-3Ra'°CD45RA- cells, which represent the CMP
• Myeloid progenitor cells with similar lineage restrictions can be found in cord blood. Although the distinct surface-marker expression profile was similar to adult bone marrow, percentages of the myeloid progenitor populations were slightly different in cord blood: IL-3Ra'°CD45RA- CMPs account for about 0.4%, IL- 3Ra'°CD45RA+ GMPs for about 0.3%, and IL-3Ra-CD45RA+ MEPs for about 0.05% of the mononuclear cell fraction of umbilical cord blood. HSC-enriched lin-CD34+CD38- cells and CMPs formed all types of colonies with cloning efficiencies of 68 and 83%, respectively. GMPs formed exclusively
• HSCs have been shown to express the gene for von Willebrand's factor, a platelet-associated peptide once thought to be restricted to the
• megakaryocyte lineage (Smith, BW, and Murphy, GJ, "Stem cells, megakaryocytes, and platelets,” Curr. Opin. Hematol. 2014; 21 (5): 430-37). These cells produce greater transcript levels of C-mpl, and are primed for megakaryocyte lineage commitment (Id. Citing Sanjuan-Pla, A., et al, "Platelet-biased stem cells reside at the apex of haematopoietic stem-cell hierarchy, " Nature 2013; 502: 232-36).
• megakaryocytes within the endosteal bone marrow niche in which TPO promotes niche expansion (Id. Citing Olson, TS, et al, "Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radio-ablative conditioning," Blood 2013; 121 : 5238-49) and mature megakaryocytes release cytokines to promote HSC proliferation (Heazlewood, SY et al, "Megakaryocytes co- localise with hemopoietic stem cells and release cytokines that up-regulate stem cell proliferation," Stem Cell Res. 2013; 11 :782-92)).
• Runxl Multiple transcription factors, including Runxl , Gatal , Fli1 and cMyb, form complex networks that regulate the differentiation of megakaryocytes both positively and negatively.
• Runxl interacts with additional megakaryocyte factors including Gatal and Fli1.
• Gatal and its cofactor, Friend of Gatal (Fog1) are critical in promoting megakaryocyte-erythroid differentiation, while at the same time inhibiting expression of Pu.1 and myeloid differentiation.
• Gatal and FIIM Binding sites for Gatal and FIIM can be found in the enhancers of many megakaryocyte-specific genes (Id. Citing Eisbacher, M. et al, "Protein-protein interaction between Fli-1 and GATA-1 mediates synergistic expression of megakaryocyte-specific genes through cooperative DNA binding," Mol. Cell Biol.
• Fli1 enhances the activity of Gatal at megakaryocyte promoters, and represses the activity of erythroid factors at erythroid promoters.
• Fli1 expression may act to restrict the MEP to the megakaryocyte lineage.
• expression of the proto-oncogene c-Myb in the MEM favors erythropoiesis, and c-Myb expression is down regulated during megakaryopoiesis (Metcalf, D et al, "Anamaloous megakaryocytopoiesis in mice with mutations in the c-Myb gene," Blood 2005; 105: 3480-87).
• CD61/CD41/CD42 positive and CD34 negative over 13 days in culture 97% and 93% of P2 and P3 eels, respectively, were phosphatidylserine (PS) positive, whereas 93% of P1 cells were PS negative.
• PS phosphatidylserine
• the PS negative (P1) cells showed many typical features of bone marrow megakaryocytes by electron microscopy, including large size, polypoid nucleus, mitochondria and immature granules, although the demarcation membrane system was poorly developed. Virtually all of the PS positive P2 cells were apoptotic, lacked granules, and had no discernable nuclei. It was found that P1 gives rise to both the P2 and P3 populations, whereas P2 gave rise to no other population.
• Growth factors are extracellular polypeptide molecules that bind to a cell- surface receptor triggering an intracellular signaling pathway, leading to proliferation, differentiation, or other cellular response. These pathways stimulate the
• Platelet a granules contain several different growth factors, including platelet- derived growth factors (PDGF-AA, PDGF-BB, BDGF-AB), transforming growth factor- ⁇ (TGF- ⁇ 1 and TGF- ⁇ 2), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), and insulin-like growth factor- 1 (IGF-1), which are actively secreted by platelets (Aghideh, AN et al, "Platelet growth factors suppress ex vivo expansion and enhance differentiation of umbilical cord blood CD133+ stem cells to megakaryocyte progenitor cells," Growth Factors 2010; 28(6): 409-16, citing Marti eau, I., et al, "Effects of calcium and thrombin on growth factor release from platelet concentrates: kinetics and regulation of endothelial cell proliferation," Biomaterials 2004; 25: 4489-4502). Megakaryocytes express and store platelet factor
• MDF megakaryocyte growth and development factor
• TPO thrombopoietin
• Thrombopoietin the major regulator of megakaryocyte development and platelet production and a potent stimulator of thrombopoiesis, is a ligand for the Mpl receptor.
• Muench, M. and Barcena, A. "Megakaryocyte Growth and Development Factor is a Potent Growth Factor for Primitive Hematopoietic Progenitors in the Human Fetus," Ped. Res. 2004; 55(6): 1050-56).
• TPO belongs to the four-helix bundle family of cytokines, which includes erythropoietin, G-CSF, growth hormone and leukemia inhibitory factor among others. (Geddis, A.E., "Megakaryyopoiesis,” Semin. Hematol. 2010; 47(3): 212- 219).
• the TPO receptor c-Mp/ was identified based on its homology to the oncogne v-Mpl, already known at the time as the transforming factor of the murine myeloproliferative leukemia virus (Id. Citing Vigon I, et al.
• TPO and c-Mpl are critical for megakaryocyte growth and development, and in mouse models where one or the other is absent, platelets and megakaryocytes are reduced to approximately 10% of normal values (Gurney AL, et al. Thrombocytopenia in c-mpl-deficient mice. Science.
• HSCs In addition to megakaryocyte cells, HSCs also express c-Mpl and depend on TPO signaling for their maintenance and expansion (Id. Citing Fox N, et al,
• Thrombopoietin expands hematopoietic stem cells after transplantation. J Clin Invest. 2002;110:389-394).
• the c-Mpl gene encodes a 635 amino acid protein consisting of a 25 amino acid signal peptide (1-25), a 465 amino acid extracellular domain (26-491), a 22 residue transmembrane domain (492-513) and an intracellular domain containing two conserved motifs termed box 1 (528-536) and box 2 (565-574).
• the extracellular domain is composed of two repeating modules; the membrane distal module appears to have an inhibitory effect on signaling, as its deletion results in constitutive activation of the receptor (Id. Citing Sabath DF, Kaushansky K, Broudy VC. Deletion of the extracellular membrane-distal cytokine receptor homology module of Mpl results in constitutive cell growth and loss of thrombopoietin binding. Blood.
• c-Mpl does not have intrinsic kinase activity, but instead associates with the cytoplasmic tyrosine kinase Janus kinase 2 (Jak2) through its box 1 domain (Id. Citing Drachman JG, Kaushansky K. Structure and function of the cytokine receptor superfamily. Curr Opin Hematol. 1995;2:22-28). Additional elements regulate receptor internalization and subsequent degradation following TPO binding. These include dileucine repeats located within box 2, Tyr591 and Tyr625 (Id. Citing Dahlen DD, et al., Internalization of the thrombopoietin receptor is regulated by 2 cytoplasmic motifs. Blood. 2003;102:102-108; Hitchcock IS, et al, YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation. Blood. 2008).
• TPO signaling depends on the activation of Jak2.
• Jak2 associates with box 1 of c-Mpl through its FERM (band 4.1/ezrin/radixin/moesin) domain.
• FERM band 4.1/ezrin/radixin/moesin
• Jak2 molecules associated with the receptor are brought close enough to each other to become activated through trans-autophosphorylation (Id. Citing Witthuhn BA, Jo FW, Silvennoinen O, Yi T, Tang B, Miura O, et al. JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell. 1993;74:227-236). Active Jak2 then phosphorylates itself on multiple residues and phosphorylates c-Mpl on at least Tyr625 and Tyr630 (Id. Citing Drachman JG, Kaushansky K.
• Jak2 Following the activation of Jak2, multiple signaling molecules are activated and mediate the cellular response to TPO. These include members of the signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK) and phosphoinositol-3 kinase (PI3K) pathways (Id. Citing Geddis AE, et al, Thrombopoietin: a pan-hematopoietic cytokine. Cytokine Growth Factor Rev. 2002;13:61-73). Jak2 directly phosphorylates STAT family members including STAT1 , 3, 5a and 5b (Id.
• STAT signal transducer and activator of transcription
• MAPK mitogen-activated protein kinase
• PI3K phosphoinositol-3 kinase pathway
• Bcl-xL (Id. Citing Kirito K, et al, Thrombopoietin regulates Bcl- xL gene expression through Stat5 and phosphatidyl! nositol 3- kinase activation pathways. J Biol Chem. 2002;277:8329-8337) and cyclin D1 (Id. Citing Magne S, et al, STAT5 and Oct-1 form a stable complex that modulates cyclin D1 expression. Mol Cell Biol. 2003;23:8934-8945).
• Jak2 also activates the small GTPase Ras and the MAPK cascade, culminating in the activation of extracellular signal-related kinase (ERK)1/2.
• ERK extracellular signal-related kinase
• Ras then activates Raf-1 , mitogen-induced extracellular kinase (MEK) and finally Erk 1/2 (Id. Citing Avruch J, et al. Ras activation of the Raf kinase: tyrosine kinase recruitment of the MAP kinase cascade. Recent Prog Horm Res. 2001 ;56:127-155). Although activation of MAPK is significantly reduced in the absence of c-Mpl Tyr625 and Tyr630, it is not eliminated (Id. Citing Luoh SM, et al. Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo. Mol Cell Biol.
• the PI3K pathway is also essential for megakaryopoiesis (Id. Citing Geddis AE, Fox NE, Kaushansky K. Phosphatidylinositol 3- kinase is necessary but not sufficient for thrombopoietin-induced proliferation in engineered Mpl-bearing cell lines as well as in primary megakaryocyte progenitors. J Biol Chem.
• PI3K is composed of a kinase (p110) and a regulatory subunit (p85).
• TPO induces formation of a complex between phosphorylated p85 and the adaptor Gab, although this complex has not been found to bind directly to c- Mpl (Id. Citing Miyakawa Y, et al, Thrombopoietin induces phosphoinositol 3-kinase activation through SHP2, Gab, and insulin receptor substrate proteins in BAF3 cells and primary murine megakaryocytes. J Biol Chem. 2001 ; 276: 2494-2502);
• PI3K may become activated indirectly through Ras (Id. Citing Kodaki T, et al, The activation of phosphatidylinositol 3-kinase by Ras. Curr Biol. 1994;4:798- 806). TPO-induced PI3K phosphorylates and activates the serine/threonine kinase Akt whose substrates include Forkhead, glycogen synthase kinase 3 beta (GSK-3P) and Bad (Id. Citing Geddis AE, Fox NE, Kaushansky K.
• Phosphatidylinositol 3- kinase is necessary but not sufficient for thrombopoietin-induced proliferation in engineered Mpl-bearing cell lines as well as in primary megakaryocyte progenitors. J Biol Chem. 2001 ;276:34473-34479; Nakao T, et al, PI3K Akt FOX03a pathway contributes to thrombopoietin-induced proliferation of primary megakaryocytes in vitro and in vivo via modulation of p27(Kip1) Cell Cycle.
• PI3K also activates mammalian target of rapamycin (mTOR), whose targets SK6 and 4E-BP1 increase proliferation and maturation of megakaryocyte progenitors (Id. Citing Raslova H, et al. Mammalian target of rapamycin (mTOR) regulates both
• PI3K is itself negatively regulated by phosphatase and tensin homolog (PTEN), a tumor suppressor that promotes quiescence in hematopoietic stem cells (HSC) (Id. Citing Zhang J, et al. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature. 2006;441 :518-522).
• PTEN regulates the activity of Akt and mTOR, its role in TPO signaling and megakaryopoiesis has not yet been defined.
• This family includes at least 8 members that can inhibit Jak signaling in a variety of ways, including binding to the activation loop of Jak and targeting it for degradation, acting as a pseudosubstrate for Jak, or binding to phosphorylated tyrosines within the cytokine receptor itself (Id. Citing Alexander WS, Hilton DJ. The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu Rev Immunol. 2004;22:503-529).
• Jak signaling in a variety of ways, including binding to the activation loop of Jak and targeting it for degradation, acting as a pseudosubstrate for Jak, or binding to phosphorylated tyrosines within the cytokine receptor itself (Id. Citing Alexander WS, Hilton DJ. The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu Rev Immunol. 2004;22:503-529).
• Jak2 has other binding partners that regulate its activity.
• Lnk is an adaptor protein that inhibits growth in HSCs, erythroid and megakaryocyte cells (Id. Citing Tong W, Lodish HF. Lnk inhibits Tpo-mpl signaling and Tpo-mediated megakaryocytopoiesis. J Exp Med. 2004;200:569-580; Seita J, et al. Lnk negatively regulates self-renewal of hematopoietic stem cells by modifying thrombopoietin- mediated signal transduction. Proc Natl Acad Sci U S A. 2007;104:2349-2354).
• Lnk binds to phosphorylated tyrosines within Jak2 through its SH2-domain (Id. Citing Bersenev A, et al, Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2. J Clin Invest. 2008;118:2832- 2844); however, the exact mechanism by which it inhibits TPO signaling is not understood.
• Jak2 may be phosphorylated within the FERM domain, inducing its dissociation from c-Mpl and thus providing another mechanism to 'turn off' signaling (Id. Citing Funakoshi-Tago M, et al, Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2. Embo J. 2006;25:4763-4772).
• Some extracellular signal proteins can act as both growth factors and mitogens, stimulating both cell growth and cell-cycle progression. This functional overlap is achieved in part by overlaps in the intracellular signaling pathways that control these two processes.
• the signaling protein Ras for example, is activated by both growth factors and mitogens. It can stimulate the PI3-kinase pathway to promote cell growth and the MAP-kinase pathway to trigger cell-cycle progression. Similarly, Myc stimulates both cell growth and cell-cycle progression.
• Extracellular factors that act as both growth factors and mitogens help ensure that cells maintain their appropriate size as they proliferate.
• TGF- ⁇ inhibits the proliferation of several cell types, either by blocking cell-cycle progression in G1 or by stimulating apoptosis. TGF- ⁇ binds to cell-surface receptors and initiates an intracellular signaling pathway that leads to changes in the activities of gene regulatory proteins called Smads. This results in complex changes in the transcription of genes encoding regulators of cell division and cell death.
• Bone morphogenetic protein (BMP), a TGF- ⁇ family member, helps trigger the apoptosis that removes the tissue between the developing digits in the mouse paw. Like TGF- ⁇ , BMP stimulates changes in the transcription of genes that regulate cell death.
• FGF Fibroblast Growth Factor
• FGF fibroblast growth factor
• FGF1 is also known as acidic FGF
• FGF2 is sometimes called basic FGF (bFGF)
• FGF7 sometimes goes by the name keratinocyte growth factor.
• FGFs can activate a set of receptor tyrosine kinases called the fibroblast growth factor receptors (FGFRs).
• FGFRs fibroblast growth factor receptors
• the portion of the protein that binds the paracrine factor is on the extracellular side, while a dormant tyrosine kinase (i.e., a protein that can phosphorylate another protein by splitting ATP) is on the intracellular side.
• a dormant tyrosine kinase i.e., a protein that can phosphorylate another protein by splitting ATP
• the FGF receptor binds an FGF (and only when it binds an FGF)
• the dormant kinase is activated, and
• FGFs are associated with several developmental functions, including angiogenesis (blood vessel formation), mesoderm formation, and axon extension. While FGFs often can substitute for one another, their expression patterns give them separate functions. FGF2 is especially important in angiogenesis, whereas FGF8 is involved in the development of the midbrain and limbs.
• angiogenic factors such as VEGF, IGF, PDGF, HGF, FGF, TGFm Angiopoeitin-1 , and stem cell factor (SCF) have been found to differ amongst bone-derived-, cartilage-derived-, and adipose-derived MSCs. (Peng et al., 2008, Stems Cells and Development, 17: 761-774).
• IGF-1 Insulin-like Growth Factor
• IGF- 1 a hormone similar in molecular structure to insulin, has growth- promoting effects on almost every cell in the body, especially skeletal muscle, cartilage, bone, liver, kidney, nerves, skin, hematopoietic cells, and lungs. It plays an important role in childhood growth and continues to have anabolic effects in adults. IGF-1 is produced primarily by the liver as an endocrine hormone as well as in target tissues in a paracrine/autocrine fashion. Production is stimulated by growth hormone (GH) and can be retarded by undernutrition, growth hormone insensitivity, lack of growth hormone receptors, or failures of the downstream signaling
• GH growth hormone
• IGF1 R Insulin-like growth factor 1 receptor
• IGF-1 R a receptor tyrosine kinase
• IGF-1 is one of the most potent natural activators of the AKT signaling pathway, a stimulator of cell growth and proliferation, and a potent inhibitor of programmed cell death.
• IGF-1 is a primary mediator of the effects of growth hormone (GH). Growth hormone is made in the pituitary gland, released into the blood stream, and then stimulates the liver to produce IGF-1. IGF-1 then stimulates systemic body growth. In addition to its insulin-like effects, IGF-1 also can regulate cell growth and development, especially in nerve cells, as well as cellular DNA synthesis.
• GH growth hormone
• TGF- ⁇ Transforming Growth Factor beta
• TGF- ⁇ superfamily There are over 30 structurally related members of the TGF- ⁇ superfamily, and they regulate some of the most important interactions in development.
• the proteins encoded by TGF- ⁇ superfamily genes are processed such that the carboxy- terminal region contains the mature peptide. These peptides are dimerized into homodimers (with themselves) or heterodimers (with other TGF- ⁇ peptides) and are secreted from the cell.
• the TGF- ⁇ superfamily includes the TGF- ⁇ family, the activin family, the bone morphogenetic proteins (BMPs), the Vg-1 family, and other proteins, including glial-derived neurotrophic factor (GDNF, necessary for kidney and enteric neuron differentiation) and MüIIerian inhibitory factor, which is involved in mammalian sex determination.
• TGF- ⁇ family members TGF- ⁇ 1 , 2, 3, and 5 are important in regulating the formation of the extracellular matrix between cells and for regulating cell division (both positively and negatively).
• TGF- ⁇ 1 increases the amount of extracellular matrix epithelial cells make both by stimulating collagen and fibronectin synthesis and by inhibiting matrix degradation.
• TGF- ⁇ s may be critical in controlling where and when epithelia can branch to form the ducts of kidneys, lungs, and salivary glands.
• TGF- ⁇ 1 was by far the most potent enhancer of mRNA expression of bone marrow stromal TPO, a commitment of lineage specificity.
• TheTPO in turn, induced TGB- ⁇ receptors I and II on megakaryoblasts at the midmegakaryopoietic stage.
• TGF- ⁇ 1 was able to arrest the maturation of megakaryocyte colony forming units (CFU-Meg) in a dose-dependent manner. This effect was relatively specific when compard with its effect on burst-forming unit-erythroid (BFU-E) or CFU-GM.
• BFU-E burst-forming unit-erythroid
• CFU-GM burst-forming unit-erythroid
• Activin A and BMP 2 induce cell commitment and differentiation toward erythropoiesis, even in the absence of erythropoietin (EPO). Their biological activities are antagonized by binding with follistatin or FLRG (follistatin-related gene), 2 secreted glycoproteins expressed by human bone marrow and regulated by TGF- ⁇ and activin A ((Jeanpierre, S. et al, "BMP4 regulation of human
• FLRG member of the follistatin family, a new player in hematopoiesis. Mol Cell Endocrinol 2004;225:109-118).
• FLRG and follistatin are involved in the regulation of human hematopoietic cell dhesiveness in immature hematopoietic progenitors and stem cells through direct interactions between the type I motifs of fibronectin and follistatin domains.
• BMPs Bone Morphogenetic Proteins
• BMPs can be programmed cell death, cell migration, and differentiation.
• the BMPs include proteins such as Nodal (responsible for left-right axis formation) and BMP4
• BMP2, BMP4 and BMP7 regulate the proliferation, maintenance (Jeanpierre, S. et al, "BMP4 regulation of human megakaryocyte differentiation is involved in thrombopoietin signaling," Blood 2008; 112: 3154-63) citing Hutton, JF, et al," Bone morphogenetic protein 4 contributes to the maintenance of primitive cord blood hematopoietic progenitors in an ex vivo stroma-non contact co-culture system," Stem Cell Dev. 2006; 15: 805-13), clonogenicity, and repopulating capacity of CD34+CD38- primitive hematopoietic populations (Id. Citing Bhatia, M.
• BMP4 cooperates with SCF to modulate the primitive hematopoietic stem cell compartment in the absence of any other cytokine. Id.
• BMP4 has the same capacity as TPO to induce early and late MK markers, and similar terminal differentiation properties, such as polyploidization, secretion of PF4, and platelet production.
• BMP4 an element of a key signaling pathway involved in the regulation of the hematopoietic "niche” (Id. Citing Zhang, et al, “Identification of the haematopoietic stem cell niche and control of the niche size,” Nature 2003; 425: 836-41), which is mainly produced by the bone marrow stroma (Id. Citing Martinovic, S.
• PEAR-1 platelet endothelial aggregation receptor- 1
• RAD001 an mTOR inhibitor
• Wnt3a has been implicated as a repressor of human megakaryocyte progenitor expansion in an in-vitro iPSC derivation system that causes production of CD41/CD235 dual positive progenitors (Id. Citing Paluru, P. et al.'The negative impact of Wnt signaling on megakaryocyte and primitive erythroid progenitors derived from hyman embryonic stem cells," Stem Cell Res. 2014; 12: 441-51).
• a role for the aryl hydrocarbon receptor (AHR) in the regulation of iPSC-based in vitro megakaryopoiesis Id. Citing Smith, BW et al, 'The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation," Blood. 2013; 122: 376-85) has been described.
• Platelets have a well-described physiological role in hemostasis and coagulation, but recently, they have also been shown to participate in immunity, tissue repair and development (Elzey BD, et al., The emerging role of platelets in adaptive immunity. Cell Immunol. 2005; 238:1-9; Jenne CN et al., Platelets: bridging hemostasis, inflammation, and immunity. Int J Lab Hematol. 2013;35:254-61 ;
• Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling. Blood. 2010;116:661-70).
• Platelet-derived extracellular vesicles can provide the molecules necessary to orchestrate these diverse functions. Platelets can generate microvesicles or microparticles (MPs), which are derived from the plasma membrane, and exosomes (EXOs), which are derived from endosomal pathways (Aatonen MT et al., Isolation and characterization of platelet- derived extracellular vesicles, J. Extracellular Vesicles, vol. 3 (2014) 24692).
• MPs microvesicles or microparticles
• EXOs exosomes
• Platelet plasma membrane derived microparticles are generally known to be 100 to 1000 nm in size. Platelets are also known to produce exosomes, which are 40 to 100 nm in size, from multivesicular bodies. In contrast to the heterogenous PMPs, exosomes in general form a more homogenous population, both by size and molecular content, but in platelets, the normally distinct formation processes of the two are jumbled because of a-granules.
• Multivesicular bodies the source of exosomes, are also considered to be pre stages of a-granules, which may then liberate exosomes on fusion with the plasma membrane, and several a- granule-derived molecules are also present on PMPs.
• microparticles, and platelet-derived exosomes include, without limitation, the following: Growth factors such as VEGF, bFGF, PDGF, TGF-beta1 ; Immune response factors such as CD40L(CD154); Chemokines/cytokines such as
• CX3CR1 and beta-thromboglobulin
• Complement proteins such as CD55, CD59, C5b-9, C1q, C3B, C1-INH, Factor H
• Apoptosis markers such as Caspace-3, Caspace-9, FasR(CD95); Coagulation factors such as Fva, FVIII, TFPI, TF, PAR-1 , FXIIIA
• Active Enzymes such as PDI, 12-LO, NADPH oxidase, iNOS2, Heparnase
• Adhesion proteins such as alpha-lib/beta3 (CD41/CD61), GPIb (CD42b), GPIX (CD42a), P-selectin (CD62P), PECAM-1 (CD31), GPIIIb (CD36), CD49, CD29, CD47, CD9, JAM-A, vWF, fibrinogen, thrombospondin, vitronectin
• Bioactive lipids such as PS, AA, L
• the common exosomal marker, CD63 is not only enriched in the platelet-derived exosomes but is also present on PMPs and, vice versa, many common PMP proteins are detected on subsets of exosomes.
• PMVs Platelet-derived microvesicles
• MVs microvesicles
• PMVs functionally variable MVs tailored for the purpose.
• the effect of PMVs can be either direct, that is, mediated by the PMV itself, such as acting as a catalytic surface, or indirect, that is, mediated by the recipient cells, which change their phenotype on PMV fusion.
• the presence of a molecule is not a guarantee for its function, as demonstrated by the unexpected anti-inflammatory response induced by CD40L(CD154)-containing PMPs (Aatonen et al., Seminars in Thrombosis and Hemostasis Vol. 38, No. 1 (2012)).
• the ultimate effect of PMVs is likely to depend on the cellular milieu (both temporally and spatially), which may explain, for example, the apparently
• PMVs can transfer fully operational surface receptors (CXC4R, CD41 ) onto the recipient cells. Receptor transfer by PMVs may confound the origin of cells: PMP-mediated transfer of CD31 and von Willebrand factor into monocytes falsely implied a presence of endothelial progenitor cells. PMVs also contain and transfer active enzymes, for example protein disulfide isomerase for platelet aggregation, inducible nitric oxide synthase II and nicotinamide adenine dinucleotide phosphate oxidase during endothelial dysfunction, and 12-lipo-oxygenase in lipoxin A4 production from mast cells.
• active enzymes for example protein disulfide isomerase for platelet aggregation, inducible nitric oxide synthase II and nicotinamide adenine dinucleotide phosphate oxidase during endothelial dysfunction, and 12-lipo-oxygenase in
• Platelets harbor RNA molecules which are translated into proteins in an activation-dependent manner, for example CD41 , CD61 , and IL-1 ⁇ 33 which are all members of the PMP proteome. It has been suggested that agonist-dependent changes in the platelet translatome may underlie the molecular, or even the functional, differences of PMP species (Aatonen et al., Seminars in Thrombosis and Hemostasis Vol. 38, No. 1 (2012)).
• PB- SC peripheral blood-stem cells
• the described invention provides umbilical cord blood derived plateletlike cells that can be used to generate induced pluripotent stem cells from adult mononuclear cells without the safety concerns involved in the generation of induced pluripotent stem cells by viral- or drug-induced transduction that may be stable when transferred to the patient.
• the described invention provides a method of functionally reprograming adult cells to insulin-producing cells comprising: (a) isolating a population of peripheral blood mononuclear cells (PBMCs) from a human subject; (b) isolating a platelet rich fraction comprising platelet-like cells from umbilical cord blood or peripheral blood; (c) contacting the population of PBMCs of step (a) with the platelet rich fraction of step (b) in vitro, wherein the contacting is effective to reprogram the PBMCs to an immature cell type that expresses one or more embryonic biomarkers; and (d) expanding the immature cell type in vitro under culture conditions effective to generate an insulin-producing cell population, wherein the insulin-producing cell population expresses human beta-cell specific transcription factors and is functionally equivalent to human pancreatic beta-cells.
• PBMCs peripheral blood mononuclear cells
• the adult PBMCs of step (a) are isolated from a Ficoll-Paque gradient fraction.
• the platelet rich fraction comprising platelet-like cells is isolated from a Ficoll-Paque gradient fraction.
• the platelet rich fraction comprising platelet-like cells comprises one or more of whole cells, microparticles, exosomes, lysed cells, and alpha granules.
• the platelet-rich fraction comprising platelet-like cells comprising one or more of whole cells, microparticles, exosomes, lysed cells, and alpha granules contains transcription factors, growth factors, or both.
• the whole cells comprise one or more of hematopoietic stem cells, hematopoietic progenitor cells, common lymphoid progenitors, common myeloid progenitors, megakaryocyte-erythrocyte progenitors; granulocyte-monocyte progenitors, megakaryocyte lineage-committed progenitors, megakaryocytes, and platelet-like cells.
• the immature cell type that expresses one or more embryonic biomarkers from step (c) comprises one or more of OCT3/4, NANOG, NKX6.1 , MAFA, Sur1 , Kir6.2, PD-L1 , CD270, Galectin 9, TGF- ⁇ 1 , AIRE, CCR3, CXCR4, and CCL2.
• the described invention provides a method for treating a recipient subject suffering from a disease characterized by hyperglycemia comprising: (a) isolating a population of peripheral blood
• PBMCs mononuclear cells
• the donor and the recipient subject are
• hyperglycemia disease is an autoimmune disease.
• the disease is diabetes.
• the autoimmune disease is type 1 diabetes.
• the donor is allogeneic to the recipient subject.
• the described invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a cell product containing a therapeutic amount of an insulin-producing cell population, wherein the insulin-producing cell population expresses human beta-cell specific transcription factors and is
• the pharmaceutical composition produced by a process comprising: (a) isolating a population of peripheral blood mononuclear cells (PBMCs) from a human donor; (b) isolating a platelet rich fraction comprising platelet-like cells from umbilical cord blood or peripheral blood of the donor; (c) contacting the population of PBMCs of step (a) with the platelet rich fraction of step (b) in vitro, wherein the contacting is effective to reprogram the PBMCs to an immature cell type that expresses one or more embryonic biomarkers; (d) expanding the immature cell type in vitro under culture conditions effective to generate a cell product containing a therapeutically effective amount of an insulin-producing cell population, wherein the insulin-producing cell population expresses human beta-cell specific transcription factors and is
• step (e) formulating the cell product with a pharmaceutically acceptable carrier to form the pharmaceutical composition, wherein the cell product containing the therapeutically effective amount of the insulin-producing cell population from step (d) is effective to reduce symptoms of the hyperglycemia disease; and the immature cell type that expresses one or more embryonic biomarkers of step (c) comprises a population of cells positive for one or more of OCT3/4, NANOG, NKX6.1 , MAFA, Sur1 , Kir6.2, PD-L1 , CD270, Galectin 9, TGF- ⁇ 1 , AIRE, CCR3, CXCR4, and CCL2; and negative for CXCL10, CCR4, CCR5, CCR7, CXCR1 , CXCR2, CXCR3, CD62L, and CXCL1.
• the described invention provides a population of functionally reprogrammed adult cells that present one or more of OCT3/4, NANOG, NKX6.1 , MAFA, Sur1 , Kir6.2, PD-L1 , CD270, Galectin 9, TGF- ⁇ 1 , AIRE, CCR3, CXCR4, and CCL2 that are negative for CXCL10, CCR4, CCR5, CCR7, CXCR1 , CXCR2, CXCR3, CD62L, and CXCL1.
• OCT3/4 NANOG
• NKX6.1 MAFA
• Sur1 Sur1
• Kir6.2 PD-L1
• CD270 Galectin 9
• TGF- ⁇ 1 fibroblast growth factor-associated fibroblasts
• AIRE Galectin 9
• CCR3, CXCR4, and CCL2 that are negative for CXCL10, CCR4, CCR5, CCR7, CXCR1 , CXCR2, CXCR3, CD62L, and CXCL1.
• the functionally reprogrammed cells are capable of producing insulin.
• the described invention provides use of a pharmaceutical composition
• a pharmaceutical composition comprising a cell product containing a therapeutic amount of an insulin-producing cell population, wherein the insulin-producing cell population expresses human beta-cell specific transcription factors and is
• PBMCs peripheral blood mononuclear cells
• PBMCs peripheral blood mononuclear cells
• platelet rich fraction comprising platelet-like cells from umbilical cord blood or peripheral blood of the donor
• the contacting is effective to reprogram the PBMCs to an immature cell type that expresses one or more embryonic biomarkers
• the hyperglycemia results from an autoimmune disease and the therapeutic amount is effective to ameliorate symptoms of the autoimmune disease.
• the disease is type 1 diabetes.
• Figure 1 shows data of platelet-like human cord blood cells expressing human ES cell markers.
• Platelet-like cells were purified from human cord blood (A - C) and from adult peripheral blood units (D - F).
• A Analysis of purified platelet-like cells by flow cytometry. The gated platelet-like cells in dot plot (top left panel, blue) were analyzed by using markers CD41 , CD42, and ES marker OCT3/4.
• B Flow cytometry after double staining with CD41 and ES marker SOX2. The isotype-matched IgGs served as controls for flow cytometry.
• C Western blot show the expression of ES markers in cord blood platelet-like cells.
• D Flow cytometry of OCT3/4 and Sox2 expression in adult blood platelet-like cells.
• FIG. 2 depicts flow cytometry data showing the interaction of platelet-like cells with other immune cells in cord blood.
• CBMCs Cord Blood Mononuclear Cells
• B The distribution of the gated CD42+ platelet-like cells in dot plot of CBMCs. The histogram (left panel) showed the gated CD42+ platelet like-cells (T), with broad distribution (green color) among the different regions of four populations (right panel).
• C The distribution of platelet-like cells on other immune cells.
• CBMCs were applied for flow cytometry.
• D Quantify the double positive cells with platelet markers and lineage markers. Representative data were from three experiments.
• E CBMCs were treated with EDTA/trypsin for 5 minutes at room temperature, washed with PBS at 1000 rpm for 5 minutes. CBMCs without treatment with
• EDTA/trypsin served as control for flow cytometry (left panel). Representative data were from three experiments. Expression of CD41 on granulocytes was markedly declined after the treatment with EDTA/trypsin. However, there are still significant numbers of CD41+ platelet-like cells that adhere to the monocytes. (F) Dot plot shows the gated CD14+CD41+ cells (blue).
• FIG. 3 shows data representing the interaction of platelets with monocytes/macrophages (Mo/M). Platelets were purified from adult blood units of healthy donors (New York Blood Bank).
• A Flow cytometry show the percentage of CD14+CD41 b+ and CD14+CD42a+cells were increased after permeabilization. Isotype-matched IgGs served as controls. Representative data were from three experiments. Transmission electronic microscopy shows the interaction of platelets with ⁇ .
• B The ultrastructure of platelets (top panel) and ⁇ .
• C The interaction of pseudopods between ⁇ and platelets. A pseudopod from ⁇ (yellow arrow) extended into platelets.
• Figure 5 shows data of expression of pancreatic islet ⁇ cell-related markers in platelets.
• A Real time PCR analysis.
• B Western blotting for islet ⁇ cell-specific transcription factors.
• Figure 6 depicts data showing expression of immune modulation-related markers in platelet-like cells.
• Platelet-like cells were purified from human cord blood (A - D) and adult peripheral blood units (E - 1).
• A Flow cytometry shows the expression of co-inhibitory surface markers CD270 and Galectin 9 on cord blood platelet-like cells.
• B Intra-cellular staining show the expression of TGF- ⁇ 1 in cord blood platelet-like cells.
• C Western blotting shows the expression of AIRE in seven cord blood preparations.
• D Double staining show the expression of AIRE in CD41+ cord blood platelet like-cells. Isotype- matched IgGs served as controls.
• E Flow cytometry shows the expression of co-inhibitory surface markers PO-L1 , CD270, and Galectin 9 on adult blood platelets.
• F Intra-cellular staining shows the expression of TGF- ⁇ 1 in adult blood platelets.
• G Western blotting show the expression of AIRE in nine adult blood preparations.
• H Double staining shows the expression of AIRE in CD41+ adult blood platelets. Isotype-matched IgGs served as controls.
• I Flow cytometry show the expressions of chemokine receptors and ligands at varied levels on adult blood platelets.
• alpha cell or "a-cell” are used interchangeably herein to refer to a type of cell in the pancreas that makes and releases the hormone glucagon when blood glucose level falls too low. Glucagon stimulates the liver to release glucose into the blood for energy.
• Beta cells or " ⁇ -cells” as used herein refers to a pancreatic cell that makes insulin.
• bi potent refers to a cell that can differentiate into two cell lineages.
• CD3 is a protein complex composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD35 chain, and two CD3£ chains, which associate with the T cell receptor (TCR) and the ⁇ -chain to generate an activation signal in T lymphocytes. Together, the TCR, the ⁇ -chain and CD3 molecules comprise the TCR complex.
• the intracellular tails of CD3 molecules contain a conserved motif known as the immunoreceptor tyrosine-based activation motif (ITAM), which is essential for the signaling capacity of the TCR.
• ITAM immunoreceptor tyrosine-based activation motif
• the CD3 chain can bind ZAP70 (zeta associated protein), a kinase involved in the signaling cascade of the T cell.
• Integrins are receptors that mediate attachment between a cell and the tissues surrounding it and are involved in cell-cell and cell-matrix interactions. In mammals, 18 a and 8 ⁇ subunits have been characterized. Both a and ⁇ subunits contain two separate tails, both of which penetrate the plasma membrane and possess small cytoplasmic domains.
• Integrin ⁇ IGAM
• CD11 b CD11 b
• macrophage- 1 antigen Mac-1
• complement receptor 3 (CR3) is a protein subunit of the heterodimeric integrin ⁇ 2 molecule.
• the second chain of ⁇ 2 is the common integrin ⁇ 2 subunit (CD18).
• ⁇ 2 is expressed on the surface of many leukocytes including
• ⁇ 2 mediates inflammation by regulating leukocyte adhesion and migration. Further, ⁇ 2 is thought to have a role in phagocytosis, cell-mediated cytotoxicity, chemotaxis and cellular activation, as well as being involved in the complement system due to its capacity to bind inactivated complement component 3b (iC3b).
• the ITGAM subunit of integrin ⁇ 2 is involved directly in causing the adhesion and spreading of cells, but cannot mediate cellular migration without the presence of the ⁇ 2 (CD18) subunit.
• CD14 is a cell surface protein expressed mainly by macrophages and, to a lesser extent, neutrophil granulocytes.
• CD14+ cells are monocytes that can differentiate into a host of different cells; for example, differentiation to dendritic cells is promoted by cytokines such as GM-CSF and IL-4.
• CD14 acts as a co-receptor (along with toll-like receptor (TLR) 4 and lymphocyte antigen 96 (MD-2)) for the detection of bacterial lipopolysaccharide (LPS).
• TLR toll-like receptor
• MD-2 lymphocyte antigen 96
• CD14 only can bind LPS in the presence of lipopolysaccharide binding protein (LBP).
• CD15 (3-fucosyl-N-acetyl-lactosamine; stage specific embryonic antigen 1 (SSEA-1)) is a carbohydrate adhesion molecule that can be expressed on glycoproteins, glycolipids and proteoglycans. CD15 commonly is found on SSEA-1.
• CD16 is an Fc receptor (FcYRIIIa and FCYRIIID) found on the surface of natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes and macrophages. Fc receptors bind to the Fc portion of IgG antibodies.
• CD19 is a human protein expressed on follicular dendritic cells and B cells. This cell surface molecule assembles with the antigen receptor of B
• lymphocytes in order to decrease the threshold for antigen receptor-dependent stimulation. It generally is believed that, upon activation, the cytoplasmic tail of CD19 becomes phosphorylated, which allows binding by Src-family kinases and recruitment of phosphoinositide 3 (PI-3) kinases.
• PI-3 phosphoinositide 3
• CD20 is a non-glycosylated phosphoprotein expressed on the surface of all mature B-cells. Studies suggest that CD20 plays a role in the development and differentiation of B-cells into plasma cells. CD20 is encoded by a member of the membrane-spanning 4A gene family (MS4A). Members of this protein family are characterized by common structural features and display unique expression patterns among hematopoietic cells and nonlymphoid tissues.
• MS4A membrane-spanning 4A gene family
• CD31 platelet/endothelial cell adhesion molecule; PECAM1
• PECAM1 normally is found on endothelial cells, platelets, macrophages and Kupffer cells, granulocytes, T cells, natural killer cells, lymphocytes, megakaryocytes, osteoclasts and
• CD31 has a key role in tissue regeneration and in safely removing neutrophils from the body. Upon contact, the CD31 molecules of macrophages and neutrophils are used to communicate the health status of the neutrophil to the macrophage.
• CD34 is a monomeric cell surface glycoprotein normally found on hematopoietic cells, endothelial progenitor cells, endothelial cells of blood vessels, and mast cells. The CD34 protein is a member of a family of single-pass
• transmembrane sialomucin proteins and functions as a cell-cell adhesion factor.
• CD34 also may mediate the attachment of stem cells to bone marrow extracellular matrix or directly to stromal cells.
• CD44 the "hyaluronan receptor”
• hyaluronan receptor a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration, is used to identify specific types of mesenchymal cells.
• CD45 protein tyrosine phosphatase, receptor type, C; PTPRC
• CD45 is a protein tyrosine phosphatase (PTP) with an extracellular domain, a single transmembrane segment, and two tandem intracytoplasmic catalytic domains, and thus belongs to receptor type PTP.
• PTP protein tyrosine phosphatase
• CD45 also suppresses JAK kinases, and thus functions as a regulator of cytokine receptor signaling.
• the CD45 family consists of multiple members that are all products of a single complex gene.
• Various known isoforms of CD45 include: CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, and CD45R (ABC).
• Different isoforms may be found on different cells. For example, CD45RA is found on naive T cells and CD45RO is found on memory T cells.
• CD56 neural cell adhesion molecule
• NCAM neural cell adhesion molecule
• CD59 refers to a glycosylphosphatidylinositol (GPI)-linked membrane glycoprotein which protects human cells from complement-mediated lysis.
• GPI glycosylphosphatidylinositol
• the CD66 antigen family identifies a neutrophil-specific epitope within the hematopoietic system that is expressed by members of the carcinoembryonic antigen family of adhesion molecules, which belong within the immunoglobulin gene superfamily.
• the extracellular portions of all CD66 (a-f) molecules possess a N- terminal V-set IgSF domain which, lacks the canonical inter-b -sheet disulfide of the CD-2 family.
• CD66a is heavily glycosylated type 1 glycoprotein with more than 60% of the mass contributed by N-linked glycans, which bear sialylated Lex (sLe x, CD15s) structures. In CD66a they are spaced further apart, VxYxxLx21 IxYxxV, and resemble motifs which bind tyrosine phosphatases such as SHIP-1 and-2.
• CD66a is expressed on granulocytes and epithelial cells.
• CD66a-d Products of 4 of the 7 functional carcinoembryonic antigen (CEA) family genes, CD66a-d, are known to be expressed on hematopoietic cells.
• the expression of these molecules on hematopoietic cells is generally restricted to the myeloid lineage. These molecules are present at low levels on resting mature granulocytes but expression increases rapidly following activation with inflammatory agonists, probably as a result of exocytosis from storage granules.
• CD66a is detected on some macrophages in tissue sections and has been reported on T cells and a subpopulation of activated NK cells.
• CD66b ((CGM1 ); CD67, CGM6, NCA-95) is a
• glycosylphosphatidylinositol (GPI)-linked protein that is a member of the
• CD66b expressed on granulocytes, generally is believed to be involved in regulating adhesion and activation of human eosinophils.
• CD90 or Thy-1 is a 25-37 kDa heavily N-glycosylated
• CD90 glycophosphatidylinositol (GPI) anchored conserved cell surface protein with a single V-like immunoglobulin domain, originally discovered as a thymocyte antigen. It belongs to the immunoglobulin gene superfamily. The complex carbohydrate side chains vary in composition between tissues and species.
• CD90 is expressed on hematopoietic stem cells and neurons. CD90 is highly expressed in connective tissue, on various fibroblast and stromal cell lines and is expressed on all thymocytes and peripheral T cells in mice. In humans, CD90 is expressed only on a small number of fetal thymocytes, 10%-40% of blood CD34+ cells in bone marrow, and ⁇ 1% of CD3+CD4+ lymphocytes in peripheral circulation. CD90 also is expressed in the human lymph node HEV endothelium but not on other endothelia and lastly, is expressed on a limited number of lymphoblastoid and leukemic cell lines.
• CD105 is a homodimeric integral membrane glycoprotein composed of disulfide-linked subunits of 90-95 kDa. In humans, it is expressed at high levels on vascular endothelial cells and on syncytiotrophoblast of term placenta. During human heart development, it is expressed at high levels on endocardial cushion tissue mesenchyme during heart septation and valve formation;
• Endoglin is an accessory protein of multiple kinase receptor complexes of the TGF- ⁇ superfamily.
• the TGF- ⁇ 1 superfamily of structurally related peptides includes the TGF- ⁇ isoforms, ⁇ 1 , ⁇ 2, ⁇ 3, and ⁇ 5, the activins and the bone morphogenetic proteins (BMPs).
• TGF-p-like factors are a multifunctional set of conserved growth and differentiation factors that control biological processes such as embryogenesis, organogenesis, morphogenesis of tissues like bone and cartilage, vasculogenesis, wound repair and angiogenesis, hematopoiesis, and immune regulation.
• Signaling by ligands of the TGF- ⁇ superfamily is mediated by a high affinity, ligand-induced, heteromeric complex consisting of related ServThr kinase receptors divided into two subfamilies, type I and type II.
• the type II receptor transphosphorylates and activates the type I receptor in a Gly/Ser-rich region.
• the type I receptor in turn phosphorylates and transduces signals to a novel family of recently identified downstream targets, termed Smads.
• Endoglin binds transforming growth factor (TGF) TGF- ⁇ 1 and - ⁇ 3 by associating with the TGF- ⁇ type II receptor, interacts with activin-A, interacts with bone morphogenic protein (BMP)-7 via activin type II receptors, ActRII and ActRIIB, and binds BMP-2 by interacting with the ligand binding type I receptors ALK3 and ALK6.
• TGF transforming growth factor
• BMP-7 bone morphogenic protein
• CD166 antigen a 556 amino acid glycoprotein belonging to the immunoglobulin gene superfamily, is encoded by the activated leukocyte-cell adhesion molecule (ALCAM) gene in humans. It contains a secretory signal sequence, an extracellular domain which contains 3 I g -I ike C2-type domains, 2 Ig- like V-type domains and 9 potential N-linked glycosylation sites, a hydrophobic transmembrane spanning domain and a 32 amino acid cytoplasmic domain with no known motifs. The N-terminal Ig domain is the binding site for both homophilic and CD166-CD6 interactions.
• CD166 is anchored to the actin cytoskeleton via the cytoplasmic domain but the receptors involved in this interaction are unknown.
• the soluble CD166 is produced by proteolytic cleavage of extracellular domains or by alternative splicing. It is expressed on mesenchymal stem cells and progenitor cells and on cortical thymic epithelial cells and medullary thymic epithelial cells, neurons, activated T cells, B cells, monocytes, fibroblasts, endothelium, epithelium, primitive subsets of hematopoietic cells including pluripotent stem cells, blastocysts and endometrium.
• CD270 also known as TR2
• Herpesvirus entry mediator A HVEMA
• Tumor necrosis factor receptor superfamily member 14, TNFRSF14, Tumor necrosis factor receptor like 2
• HVEM Herpesvirus entry mediator A
• Tumor necrosis factor receptor superfamily member 14, TNFRSF14, Tumor necrosis factor receptor like 2
• HVEM is widely expressed in blood vessels, brain, heart, kidney, liver, lung, prostate, spleen, thymus and other organs. Resting T cells and naive and memory B cells express high levels of HVEM as well. In humans, HVEM is not expressed in germinal center B cells. Immature dendritic cells express high levels of HVEM that is downregulated upon maturation.
• CXCR-4 refers to a G-protein-linked chemokine receptor.
• cell is used herein to refer to the structural and functional unit of living organisms and is the smallest unit of an organism classified as living.
• chemokine refers to a class of chemotactic cytokines that signal leukocytes to move in a specific direction.
• chemotaxis or “chemotactic” refer to the directed motion of a motile cell or part along a chemical concentration gradient towards environmental conditions it deems attractive and/or away from surroundings it finds repellent.
• clonogenicity and its other grammatical forms as used herein refers to the property of a single stem cell to produce a colony of cells through self-renewal.
• component refers to a constituent part, element or ingredient.
• connecting peptide or "C-peptide” as used herein refers to a short 31 -ami no-acid polypeptide that connects insulin's A-chain to its B-chain in the proinsulin molecule. It is used as a marker in autoimmune diseases like diabetes. Increased levels are an indication for insulin release as they are released at equimolar quantities and a better outcome for a patient. A very low C-peptide confirms type 1 diabetes and insulin dependence and is associated with high glucose variability, lack of glucose homeostasis and increased complications with poor outcome. Measurement of C-peptide levels is clinically validated by
• contact and its various grammatical forms as used herein refers to a state or condition of touching or of immediate or local proximity.
• Contacting a composition to a target destination may occur by any means of administration known to the skilled artisan.
• CB-SCs cord blood-derived stem cells
• CBMCs cord blood mononuclear cells
• cytokine refers to small soluble protein substances secreted by cells which have a variety of effects on other cells. Cytokines mediate many important physiological functions including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Generally, cytokines act locally.
• type I cytokines which encompass many of the interleukins, as well as several hematopoietic growth factors
• type II cytokines including the interferons and interleukin-10
• TNF tumor necrosis factor
• IL-1 immunoglobulin super-family members
• chemokines a family of molecules that play a critical role in a wide variety of immune and inflammatory functions.
• the same cytokine can have different effects on a cell depending on the state of the cell. Cytokines often regulate the expression of, and trigger cascades of other cytokines.
• Nonlimiting examples of cytokines include e.g., IL-1.alpha., IL-.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11 , IL-12/IL-23 P40, IL13, IL-17, IL-18, TGF-beta., IFN-gamma., GM-CSF,
• the term "derived from” as used herein encompasses any method for receiving, obtaining, or modifying something from a source of origin.
• the platelet -like cells from the platelet-like fraction may be derived from cord blood, meaning the platelet-like cells, directly or indirectly, came from the cord blood.
• whole platelet-like cells, lysed platelet-like cells, components of platelet-like cells including exosomes, microparticles, nucleic acids, growth factors, etc. may be derived from cord blood, meaning each of those components, directly or indirectly, came from the cord blood.
• lysed platelet-like cells may be derived from whole platelet-like cells, meaning that the lysed platelet-like cells, directly or indirectly, came from whole platelet-like cells.
• detectable marker encompasses both selectable markers and assay markers.
• selectable markers refers to a variety of gene products to which cells transformed with an expression construct can be selected or screened, including drug-resistance markers, antigenic markers useful in
• adherence markers such as receptors for adherence ligands allowing selective adherence, and the like.
• detectable response refers to any signal or response that may be detected in an assay, which may be performed with or without a detection reagent.
• Detectable responses include, but are not limited to, radioactive decay and energy (e.g., fluorescent, ultraviolet, infrared, visible) emission, absorption, polarization, fluorescence, phosphorescence, transmission, reflection or resonance transfer.
• Detectable responses also include chromatographic mobility, turbidity, electrophoretic mobility, mass spectrum, ultraviolet spectrum, infrared spectrum, nuclear magnetic resonance spectrum and x-ray diffraction.
• a detectable response may be the result of an assay to measure one or more properties of a biologic material, such as melting point, density, conductivity, surface acoustic waves, catalytic activity or elemental composition.
• a "detection reagent” is any molecule that generates a detectable response indicative of the presence or absence of a substance of interest. Detection reagents include any of a variety of molecules, such as antibodies, nucleic acid sequences and enzymes. To facilitate detection, a detection reagent may comprise a marker.
• differentiated label generally refers to a stain, dye, marker, or antibody used to characterize or contrast structures, components or proteins of a single cell or organism.
• differentiation refers to the process of development with an increase in the level of organization or complexity of a cell or tissue, accompanied with a more specialized function.
• differentiation inducer refers to a compound that is a direct, or indirect, causative agent of the process of cell differentiation. A “differentiation inducer” while sufficient to cause differentiation is not essential to differentiation.
• enrich refers to increasing the proportion of a desired substance, for example, to increase the relative frequency of a subtype of cell compared to its natural frequency in a cell population.
• Positive selection, negative selection, or both are generally considered necessary to any enrichment scheme.
• Selection methods include, without limitation, magnetic separation and FACS. Regardless of the specific technology used for enrichment, the specific markers used in the selection process are critical, since developmental stages and activation-specific responses can change a cell's antigenic profile.
• negative magnetic selection can be accomplished by mixing a cell population marked by antibodies with a suspension of paramagnetic particles that bind to the antibody tag. Application of a magnetic field will then separate bead- cell aggregates from the unmarked cells, which can be collected by aspiration.
• positive magnetic selection can be accomplished by labeling the cells of interest with antibody-labeled -magnetic particles directed to known cell markers; generally, small particles (e.g., 50 nm) are used to minimize potential effects of the antibody-particle complexes on the biology of the selected cells.
• FACS separation allows the simultaneous application of positive and negative selection for a variety of surface markers.
• positive and negative selection by FACS can be modified by substituting magnetic selections using the same combinations of antibodies.
• unconjugated primary antibodies are used in combination with magnetic beads conjugated to secondary immunoglobulins for negative selection; for positive selection, an avidin-biotin system using biotinylated antibodies followed by avidin-conjugated microbeads can be used.
• negative selection can be used before FACS sorting to reduce cellularity of the sample.
• samples subjected to positive selection can subsequently be processed to isolate specific cellular subsets by FACS. Pre-enrichment of a target population before FACS can have a significant impact on the final purity of the isolated cell populations.
• factor refers to nonliving components that have a chemical or physical effect.
• a "paracrine factor” is a diffusible signaling molecule that is secreted from one cell type that acts on another cell type in a tissue.
• a "transcription factor” is a protein that binds to specific DNA sequences and thereby controls the transfer of genetic information from DNA to mRNA.
• fragment refers to a small part, derived from, cut off, or broken from a larger unit which retains the desired biological activity of the larger unit.
• flow cytometry refers to a tool for
• interrogating the phenotype and characteristics of cells It senses cells or particles as they move in a liquid stream through a laser (light amplification by stimulated emission of radiation)/light beam past a sensing area. The relative light-scattering and color-discriminated fluorescence of the microscopic particles is measured. Flow Analysis and differentiation of the cells is based on size, granularity, and whether the cells is carrying fluorescent molecules in the form of either antibodies or dyes. As the cell passes through the laser beam, light is scattered in all directions, and the light scattered in the forward direction at low angles (0.5-10°) from the axis is proportional to the square of the radius of a sphere and so to the size of the cell or particle.
• Light may enter the cell; thus, the 90 ° light (right-angled, side) scatter may be labled with fluorochrome-linked antibodies or stained with fluorescent membrane, cytoplasmic, or nuclear dyes.
• the differentiation of cell types, the presence of membrane receptors and antigens, membrane potential, pH, enzyme activity, and DNA content may be facilitated.
• Flow cytometers are multiparameter, recording several measurements on each cell; therefore, it is possible to identify a
• Fluorescence- activated cell sorting which allows isolation of distinct cell populations too similar in physical characteristics to be separated by size or density, uses
• fluorescent tags to detect surface proteins that are differentially expressed, allowing fine distinctions to be made among physically homogeneous populations of cells.
• growth factor refers to extracellular polypeptide molecules that bind to a cell-surface receptor triggering an intracellular signaling pathway, leading to proliferation, differentiation, or other cellular response. Growth factors include, but are not limited to, cytokines and hormones.
• hematopoietic stem cell refers to a cell isolated from the blood or from the bone marrow that can renew itself, differentiate to a variety of specialized cells, mobilize out of the bone marrow into the circulating blood, and undergo programmed cell death (apoptosis).
• hematopoietic stem cells derived from human subjects express at least one type of cell surface marker, including, but not limited to, CD34, CD38, HLA-DR, c-kit, CD59, Sca-1 , Thy-1 , and/or CXCR-4, or a combination thereof.
• isolated refers to the separation of cells from a population through one or more isolation methods such as, but not limited to, mechanical separation or selective culturing.
• An "isolated" population of cells does not have to be pure.
• Other cell types may be present. According to some embodiments, and isolated population of a particular cell type refers to greater than 10% pure, greater than 20% pure, greater than 30% pure, greater than 40% pure, greater than 50% pure, greater than 60% pure, greater than 70% pure, greater than 80% pure, greater than 90% pure, or greater than 95% pure.
• labeling refers to a process of distinguishing a compound, structure, protein, peptide, antibody, cell or cell component by introducing a traceable constituent.
• traceable constituents include, but are not limited to, a fluorescent antibody, a fluorophore, a dye or a fluorescent dye, a stain or a fluorescent stain, a marker, a fluorescent marker, a chemical stain, a differential stain, a differential label, and a radioisotope.
• labile refers to subject to increased degradation.
• marker or “cell surface marker” are used interchangeably herein to refer to an antigenic determinant or epitope found on the surface of a specific type of cell.
• Cell surface markers can facilitate the characterization of a cell type, its identification, and eventually its isolation.
• Cell sorting techniques are based on cellular biomarkers where a cell surface marker(s) may be used for either positive selection or negative selection, i.e., for inclusion or exclusion, from a cell population.
• matrix refers to a surrounding substance within which something is contained or embedded.
• mechanical agitation refers to a process whereby tissue is physically shaken or churned via mechanical means.
• mechanical means include, but are not limited to, a mixer or other mechanical device.
• MSCs meenchymal stem cells
• MSCs secrete many biologically important molecules, including interleukins 6, 7, 8, 11 , 12, 14, and 15, M-CSF, Flt-3 ligand, SCF, LIF, bFGF, VEGF, PIGF and MCP1 (Majumdar, et al., J. Cell Physiol. 176: 57-66 (1998), Kinnaird et al, Circulation 109: 1543-49 (2004)). In 2004, it was reported that no single marker that definitively identifies MSCs in vivo had yet been identified, due to the lack of consensus from diverse documentations of the MSC phenotype. Baksh, et al., J. Cell. Mol. Med. 8(3): 301-16, 305 (2004).
• MSCs lack typical hematopoietic antigens, namely CD14, CD34, and CD45. (Id.; citing Pittenger, M.F. et al., Science 284: 143-47 (1999)).
• multilineage differentiation capability refers to the property of a single stem cell to generate different types of mature progenies.
• multipotent refers to a cell, such as mesenchymal stem cells and several other adult stem cells, which can differentiate into multiple cell lineages, but not all the lineages derived from the three germ layers.
• nonexpanded refers to a cell population that has not been grown in culture (in vitro) to increase the number of cells in the cell population.
• PDGF Platinum Derived Growth Factor
• PDGF Activation of PDGF receptors leads to stimulation of cell growth, but also to changes in cell shape and motility; PDGF induces reorganization of the actin filament system and stimulates chemotaxis, i.e., a directed cell movement toward a gradient of PDGF. In vivo, PDGF plays a role in embryonic development and during wound healing.
• platelet rich fraction of blood or “platelet rich blood fraction” or “platelet rich fraction” as used herein refers to a fraction of human or animal blood obtained via a fractionation method that separates one or more components of blood wherein platelets account for at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the total number of cells in the fraction.
• the platelet rich fraction of blood may comprise other components of blood, including, without limitation, serum, mononuclear cells including progenitor cells, granulocytes, erythrocytes,
• the platelet rich fraction of blood may be further processed to isolate or remove one or more components of blood that are present in the platelet rich fraction.
• a platelet rich fraction of blood may be obtained via Ficoll-Paque gradient separation.
• the Ficoll-Paque separated fractions of blood can comprise (from top to bottom), the plasma fraction, the mononuclear cell fraction, the Ficoll-Paque media fraction, and the granulocyte/erythrocyte fraction.
• the Ficoll-Paque separated platelet rich fraction of blood comprises one or more of the plasma fraction and the mononuclear cell fraction.
• cord blood platelet-like cells comprises cord blood platelet-like cells.
• platelet-like cell refers to a cell in the platelet-rich fraction of a Ficoll-Paque gradient capable of platelet function (e.g., adhesion, activation, aggregation), or that comprises one or more platelet markers, platelet growth factors, or platelet nucleic acids.
• a platelet-like cell includes cell precursors and one or more exosomes or
• pluripotent refers to the ability to develop into all the cells of the three embryonic germ layers, forming the body organs, nervous system, skin, muscle and skeleton. Examples include the inner cell mass of the eblastocyst, embryonic stem cells, and reprogrammed cells, such as iPS cells.
• progenitor cell refers to an early descendant of a stem cell that can only differentiate, but can no longer renew itself. Progenitor cells mature into precursor cells that mature into mature phenotypes. Hematopoietic progenitor cells are referred to as colony-forming units (CFU) or colony-forming cells (CFC). The specific lineage of a progenitor cell is indicated by a suffix, such as, but not limited to, CFU-E (erythrocytic), CFU-F (fibroblastic), CFU-GM
• CMPs common myeloid progenitors
• MEPs megakaryocyte/erythrocyte progenitors
• MegE megakaryocyte-erythrocyte progenitors
• MKPs megakaryocyte lineage- committed progenitors
• purification refers to the process of isolating or freeing from foreign, extraneous, or objectionable elements.
• self-renewal refers to the capacity for extensive proliferation and generation of stem cells with the same properties as the parent cell.
• stem cells refers to undifferentiated cells having high proliferative potential with self-renewal and clonogenic properties capable of multi- lineage differentiation. Stem cells are distinguished from other cell types by two characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.
• totipotent refers to a stem cell that can form the embryo and the trophoblast of the placenta.
• TGFp transforming growth factor beta
• TGFp transforming growth factor beta
• TGF ⁇ superfamily ligands bind to a type II receptor, which recruits and phosphorylates a type I receptor.
• the type I receptor then phosphorylates receptor- regulated SMADs (R-SMADs) which can now bind the coSMAD SMAD4.
• R- SMAD/coSMAD complexes accumulate in the nucleus where they act as
• a method for reprogramming adult mononuclear cells comprises:
• the contacting is effective to reprogram cells, which can be identified by biomarkers.
• the mononcuclear fraction of cells suitable for reprogramming contains adult mononuclear cells.
• the adult mononuclear cells are isolated from peripheral blood, bone marrow, liver, spleen, pancreas, kidney, brain, spinal cord, thyroid, lung, stomach intestines, or any other body part that can be affected by autoimmune diseases.
• the collecting of the UC blood cells or adult peripheral blood cells step is performed by Ficoll-Paque gradient.
• the platelet rich fraction of cord blood or adult peripheral blood is obtained from a fraction of the Ficoll-Paque gradient.
• the contacting of the adult mononuclear cells with the platelet rich fraction of cord blood is effective to transfer one or more components of the platelet rich fraction of cord blood to the adult mononuclear cells.
• the contacting of the isolated mononuclear cells with the platelet rich fraction is in the presence of 2mM CaCI 2 at 37o C for between 10 minutes and 2 hours (Baj-Krzyworzeka et al., Platelet derived microparticles stimulate proliferation, survival, adhesion, and chemotaxis of hematopoietic cells, Exp. Hematology 30 (2002) 450-459).
• the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 10 minutes.
• the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 20 minutes.
• the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 30 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 40 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 50 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 60 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 70 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 80 minutes.
• the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 90 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 100 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 110 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the platelet rich fraction is for at least 120 minutes.
• the contacting occurs in a cell suspension at 37o C without agitation.
• the platelet rich fraction comprising platelet-like cells is activated prior to contacting.
• the contacting occurs in a cell suspension at 37o C without agitation for a time that is sufficient for phagocytosis of platelets; e.g. at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes.
• the contacting of the isolated mononuclear cells is with a lysed platelet rich fraction.
• the platelet rich fraction of blood is lysed with a whole cell lysis buffer. According to some embodiments, the lysed platelet rich fraction of blood is cleared by centrifugation.
• the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 10 minutes.
• the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 20 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 30 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 40 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 50 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 60 minutes.
• the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 70 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 80 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 90 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 100 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 110 minutes. According to some embodiments, the contacting of the isolated mononuclear cells with the lysed platelet rich fraction is for at least 120 minutes.
• the isolated mononuclear cells are washed in buffer and then cultured to expand the total number of reprogrammed cells.
• the adult mononuclear cells are peripheral blood mononuclear cells (PBMCs).
• the method comprises contacting isolated adult PBMCs in vitro, with a platelet rich fraction of cord blood.
• the PBMCs and a platelet rich fraction of cord blood are derived from genetically distinct individuals.
• the PBMCs and platelet rich fraction of cord blood are derived from the same individual.
• the method comprises contacting the isolated adult peripheral blood mononuclear cells (PBMCs) in vitro, with an enriched population of platelet-like cells derived from cord blood.
• PBMCs peripheral blood mononuclear cells
• the PBMCs are treated with trypsin/EDTA prior to the contacting to remove mature or adult platelets from the surface of the PBMCs.
• the method comprises contacting in the adult peripheral blood mononuclear cells (PBMCs) in vitro with a lysate of a platelet rich fraction of blood.
• PBMCs peripheral blood mononuclear cells
• the peripheral blood mononuclear cells are contacted with a whole cell lysate derived from the platelet rich fraction of blood.
• the lysate of the platelet rich fraction of blood comprises alpha granule contents.
• the contacting comprises fusing the PBMCs with one or more of the components of the lysate of the platelet rich fraction of blood.
• the method comprises contacting the adult peripheral blood mononuclear cells (PBMCs) with one or more of microparticles and exosomes derived from the platelet-rich fraction.
• the microparticles and exosomes are derived from a platelet rich fraction of cord blood.
• the microparticles and exosomes comprise one or more of embryonic stem cell like mRNA and protein.
• the microparticles and/or exosomes comprise one or more of growth factors such as VEGF, bFGF, PDGF, TGF-beta1 ; Immune response factors such as CD40L(CD154); Chemokines/cytokines such as
• Rantes CCL5, CCL23, CXCL7, CXCR4, PF-4(CXCL4), TNF-RI-II, IL-1 beta, CX3CR1 , and beta-thromboglobulin;
• Complement proteins such as CD55, CD59, C5b-9, C1q, C3B, C1-INH, Factor H;
• Apoptosis markers such as Caspace-3, Caspace-9, FasR(CD95); Coagulation factors such as Fva, FVIII, TFPI, TF, PAR-1 , FXIIIA; Active Enzymes such as PDI, 12-LO, NADPH oxidase, iNOS2, Heparnase;
• Adhesion proteins such as alpha-lib/beta3 (CD41/CD61), GPIb (CD42b), GPIX (CD42a), P-selectin (CD62P), PECAM-1 (CD31), GPIIIb (CD36), CD49, CD29, CD
• the method comprises contacting the adult peripheral blood mononuclear cells (PBMCs) with alpha granules of the platelet-like cells.
• PBMCs peripheral blood mononuclear cells
• the alpha granules are acquired from a platelet rich fraction of cord blood.
• the platelet rich fraction of human blood comprises platelets, platelet like cells or other components associated with stem cells.
• the platelet rich fraction of human blood comprises cell signaling molecules associated with signaling pathways underlying induced pluripotent stem cell reprogramming.
• the platelet rich fraction of human blood comprises platelet-like cells comprising embryonic stem cell markers.
• the platelet rich fraction of human blood comprises the transcription factors OCT3/4 and SOX2 (See Fig. 1 A and 1 B).
• the platelet rich fraction of human blood comprises one or more of the proteins OCT3/4, SOX2, NANOG, CRIPTO, GATA-4, and C-myc (See Fig. 1C).
• the platelet fraction of human blood comprises one or more of proteins OCT3/4, SOX2, NANOG, and C-myc (See Fig. 1 D, 1 E, and 1 F).
• the PBMCs comprise one or more of the markers CD14, CD66, CD4, CD8, CD19, CD56, CD41 b, and CD42a.
• the method comprises contacting peripheral blood mononuclear cells (PBMCs) with a platelet rich fraction of blood to reprogram monocytes and macrophages into a more stem-like state.
• PBMCs are contacted with a platelet rich fraction of blood to reprogram monocytes and macrophages into peripheral blood insulin producing cells (PB-IPC).
• PB-IPC peripheral blood insulin producing cells
• the method comprises contacting the PBMCs with a platelet rich fraction of blood wherein one or more of transcription factors and nucleic acids are transferred from the platelet rich fraction to the mononuclear cells.
• platelet-like cells from the platelet rich fraction fuse with PBMCs, thereby transferring the transcription factors and nucleic acids from the platelet to the PBMCs.
• microparticles from the platelet rich fraction fuse with mononuclear cells, thereby transferring one or more of transcription factors and nucleic acids to the PBMCs.
• exosomes from the platelet rich fraction fuse with PBMCs, thereby transferring one or more of transcription factors or nucleic acids.
• PBMCs are reprogramed by contacting with a platelet rich fraction of blood to display one or more of tetraspanin CD9, leukocyte common antigen CD45, and stem cell factor receptor CD117.
• the method comprises contacting peripheral blood insulin-producing cells (PB-IPCs) with the platelet-rich cell fraction.
• PB-IPCs peripheral blood insulin-producing cells
• the isolated PB-IPCs are derived by culturing PBMCs within a vessel with a hydrophobic surface.
• the PB-IPCs may be obtained by providing a sample of adult human peripheral blood; removing red cells from the sample to obtain mononuclear cells; culturing the mononuclear cells on a hydrophobic surface with a net positive charge and obtaining a cell population which is attached to the surface (Zhou Y. et al., US Pat. No. 8,835,163, the entirety of which is herein incorporated by reference).
• the method comprises contacting PB-IPCs isolated from PBMCs with a platelet rich fraction of cord blood.
• the method comprises contacting PB-IPCs isolated from PBMCs with one or more of microparticles and exosomes acquired from a platelet rich fraction of cord blood. According to some embodiments, the method comprises contacting the PB-IPCs isolated from PBMCs with one or more of microparticles and exosomes acquired from adult peripheral blood.
• the method comprises contacting peripheral blood insulin producing cells (PB-IPCs) with a platelet rich fraction of blood to enhance potential for insulin production.
• PB-IPCs display embryonic stem cell-associated transcription factors including OCT- 4 and NANOG, along with the hematopoetic markers CD9, CD45, and CD117.
• the PB-IPCs lack expression of hematopoetic stem cell marker CD34 as well as lymphocyte and monocyte/macrophage markers. According to some embodiments, the PB-IPCs demonstrate characteristics of islet beta-cell progenitors including the expression of beta-cell specific insulin gene transcription factors and prohormone convertases, production of insulin, and formation of insulin granules. According to some embodiments, PB-IPCs have the ability to reduce hyperglycemia and migrate into pancreatic islets after
• the instant invention discloses a method of enhancing the insulin-producing characteristics of PB-IPCs, and improving the capacity to reduce hyperglycemia and migrate into pancreatic islets by contacting PB-IPCs from adult blood with a platelet rich fraction of cord blood or platelet rich fraction of adult blood.
• the PB-IPCs are obtained directly from whole cord blood or whole adult blood.
• the PB-IPCs are isolated from whole blood by culturing mononuclear cells on a hydrophobic tissue culture surface.
• the PB-IPCs are contacted with a platelet rich fraction of blood wherein one or more of transcription factors and nucleic acids are transferred from the platelet rich fraction to the PB-IPCs.
• plateletlike cells from the platelet rich fraction fuse with PB-IPCs, thereby transferring the transcription factor and nucleic acids from the platelet-like cell to the PB-IPC.
• microparticles from the platelet rich fraction fuse with PB-IPCs, thereby transferring one or more of transcription factors or nucleic acids to the PB-IPCs.
• exosomes from the platelet rich fraction fuse with PB-IPCs, thereby transferring one or more of transcription factors or nucleic acids.
• the PB-IPCs contacted with platelet rich fraction have an enhanced ability to migrate into pancreatic islets and become functional producers of insulin.
• the PB-IPCs contacted with platelet rich fraction differentiate into beta-cells.
• the contacting of PBMCs with the platelet rich fraction of human blood produces fibroblast-like macrophages.
• the contacting of PBMCs with the platelet rich fraction of blood produces a population of cells comprising a proportion of cells for particular markers of undifferentiated cells and/or differentiated cells.
• markers of undifferentiated cells such as Oct4, neuroprogenitor markers such as nestin and Ngn-3, and markers of mature neuron markers such as beta-tubulin and TPH2 can be assessed by quantitative RT-PCR.
• production and localization of markers of undifferentiated cells can be assessed by immunocytochemistry.
• Markers of undifferentiated and differentiated cells are assayed by any of various methods such as antibody-based detection techniques using an antibody specific for a particular marker. Antibody-based techniques include
• assays include assays for detection of mRNAs encoding a particular marker. Such assays include polymerase chain reaction, blot hybridization (also known as Northern blots) and in situ hybridization. Details of these and other such assays are described herein and in standard references including J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd ed., 2001 ; F. M.
• the contacting of PBMCs with a platelet rich fraction of blood results in a reprograming of one or more of the following cell types comprising the PBMCs: monocytes, macrophages, and lymphocytes.
• the contacting of PBMCs with a platelet rich fraction of blood results in the reprogramming of one or more cell types comprising the PBMCs into a functional insulin producing cell.
• the contacting of PBMCs with a platelet rich fraction of blood results in the reprogramming of one or more cell types comprising the PBMCs into a functional islet beta-cell.
• the contacting of PBMCs with a platelet rich fraction of blood results in a reprograming of one or more of the cell types comprising the PBMCs to minimize or eliminate an immune response against the reprogrammed cell when administered to a subject.
• the functionally modulated adult blood mononuclear cells display stem-like, hematopoietic, or differentiated phenotypic characteristics.
• the functionally modulated adult blood mononuclear cells display one or more of the following pancreatic markers: MafA, Nkx6.1 , Pdx-1 , Onecutl , NeuroDI , Nkx2.2, insulin, glucagon, pancreatic polypeptide, somatostatin, ghrelin, Sur-1 and Kir6.2.
• the functionally modulated adult blood is functionally modulated adult blood
• the mononuclear cells display one or more of the following embryonic/hematopoietic markers: tetra-spanin CD9, leukocyte common antigen CD45, stem cell factor receptor CD117.
• the functionally modulated adult blood mononuclear cells display small amounts of or the absence of one or more of the following: hematopoietic stem cell marker CD34, lymphocyte markers CD3 (T cells) and CD20 (B cells).
• the functionally modulated adult blood mononuclear cells are derived from a hematopoietic lineage, and not a mesenchymal lineage, from peripheral blood.
• the functionally modulated adult mononuclear cells display small amounts of, or the absence of, monocyte/macrophage specific antigens CD14 and CD11 b/Mac-1. According to some embodiments, the functionally modulated adult mononuclear cells display small amounts of, or the absence of, HLA-DR, CD40, and CD80. According to some embodiments, the functionally modulated adult mononuclear cells display embryonic stem cell related transcription factors Oct-4 and NANOG.
• the functionally modulated adult blood mononuclear cells display one or more of the following molecules: OCT3/4, NANOG, NKX6.1 , MAFA, Sur1 , Sur2, PDL-1 , CD270, Galectin 9.
• the functionally modulated adult blood cells display embryonic stem characteristics, including one or more of stem cell markers Oct-4, Nanog, and Sox-2, together with other embryonic stem (ES) cell-related genes, e.g., Zinc finger and SCAN domain containing 10 (ZNF206, also named ZSCAN10), Zic family member 3 heterotaxy 1 (ZIC3), Zic family member 2 (ZIC2), Growth associated protein 43 (GAP43), PR domain containing 14
• PRDM14 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1), Podocalyxin-like (PODXL), Polyhomeotic homolog 1 (PHC1), and Zinc finger protein 589 (ZNF589).
• PTPRZ1 Z polypeptide 1
• PODXL Podocalyxin-like
• PLC1 Polyhomeotic homolog 1
• ZNF589 Zinc finger protein 589
• the functionally modulated adult blood mononuclear cells are CD45+.
• the functionally modulated adult blood mononuclear cells are phenotypically distinct from lymphocytes, dendritic cells, macrophages and monocytes, in that they are negative for one or more of the following antigenic markers: CD3, CD20 (B- lymphocyte cell-surface antigen B1 , Accession No. M27394), CD11c (integrin, alpha
• CD11 b/Mac-1 complement component 3 receptor
• the functionally modulated adult blood mononuclear cells are phenotypically distinct from hematopoietic stem cells in that they are CD34 negative (Hematopoietic progenitor cell antigen CD34, Accession No. P28906) (Craig et al. 1994, British Journal of Haematology, 88:24-30; Lansdorp, P. A I. and Dragowaka, W. (1992) J. Exp. Med. 175:1501-1509; Sutherland, H. J., et al. (1989), Blood 74.1563-1570.)).
• CD34 negative Hematopoietic progenitor cell antigen CD34, Accession No. P28906
• the functionally modulated adult blood mononuclear cells are capable of differentiating into other cell types including, but not limited to, insulin producing cells.
• the functionally modulated adult blood mononuclear cells that are insulin-producing cells display glucagon-like peptide 1 (GLP-1) receptor.
• administration of a long acting agonist of GLP-1 , exendin-4 increases insulin production and cell differentiation of the functionally modulated adult blood
• the reprogrammed functionally modulated adult blood mononuclear cells can be expanded in culture. According to some embodiments, the expanded reprogrammed adult peripheral blood
• mononuclear cells comprise cells having the characteristics of pluripotent stem cells that may differentiate into functional pancreatic islet beta-cells.
• the reprogrammed functionally modulated adult blood mononuclear cells comprise one or more embryonic stem cell markers, human islet beta-cell specific transcription factors or both derived from the cord platelet-like cells. According to some embodiments, the reprogrammed functionally modulated adult blood mononuclear cells express immune tolerance- related markers.
• mononuclear cells comprises:
• the functionally modulated cord blood mononuclear cells display stem-like, hematopoietic, or differentiated phenotypic characteristics.
• the functionally modulated cord blood mononuclear cells display one or more of the following pancreatic markers: MafA, Nkx6.1 , Pdx-1 , Onecutl , NeuroDI , Nkx2.2, insulin, glucagon, pancreatic polypeptide, somatostatin, ghrelin, Sur-1 and Kir6.2.
• the functionally modulated cord blood is functionally modulated cord blood
• the functionally modulated cord blood mononuclear cells display one or more of the following embryonic/hematopoietic markers: tetra-spanin CD9, leukocyte common antigen CD45, stem cell factor receptor CD117.
• the functionally modulated cord blood mononuclear cells display small amounts of or the absence of one or more of the following: hematopoietic stem cell marker CD34, lymphocyte markers CD3 (T cells) and CD20 (B cells).
• the functionally modulated cord blood mononuclear cells are derived from a hematopoietic lineage, and not a mesenchymal lineage, from peripheral blood.
• the functionally modulated cord mononuclear cells display small amounts of, or the absence of, monocyte/macrophage specific antigens CD14 and CD11 b/Mac-1. According to some embodiments, the functionally modulated cord blood mononuclear cells display small amounts of, or the absence of, HLA-DR, CD40, and CD80. According to some embodiments, the functionally modulated cord blood mononuclear cells display embryonic stem cell related transcription factors Oct-4 and NANOG.
• the functionally modulated cord blood mononuclear cells display one or more of the following molecules: OCT3/4, NANOG, NKX6.1 , MAFA, Sur1 , Sur2, PDL-1 , CD270, Galectin 9.
• the functionally modulated cord blood cells display embryonic stem characteristics, including one or more of stem cell markers Oct-4, Nanog, and Sox-2, together with other embryonic stem (ES) cell-related genes, e.g., Zinc finger and SCAN domain containing 10 (ZNF206, also named ZSCAN10), Zic family member 3 heterotaxy 1 (ZIC3), Zic family member 2 (ZIC2), Growth associated protein 43 (GAP43), PR domain containing 14
• PRDM14 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1), Podocalyxin-like (PODXL), Polyhomeotic homolog 1 (PHC1), and Zinc finger protein 589 (ZNF589).
• PTPRZ1 Z polypeptide 1
• PODXL Podocalyxin-like
• PLC1 Polyhomeotic homolog 1
• ZNF589 Zinc finger protein 589
• the functionally modulated cord blood mononuclear cells are CD45+.
• the functionally modulated cord blood mononuclear cells are phenotypically distinct from lymphocytes, dendritic cells, macrophages and monocytes, in that they are negative for one or more of the following antigenic markers: CD3, CD20 (B-lymphocyte cell- surface antigen B1 , Accession No. M27394), CD11c (integrin, alpha X, Accession
• CD11 b/Mac-1 complement component 3 receptor 3 subunit
• the functionally modulated cord blood mononuclear cells are phenotypically distinct from hematopoietic stem cells in that they are CD34 negative (Hematopoietic progenitor cell antigen CD34,
• the platelet rich fraction of cord blood comprises platelets, platelet like cells or other components associated with stem cells.
• the platelet rich fraction of cord blood comprises cell signaling molecules associated with signaling pathways underlying induced pluripotent stem cell reprogramming.
• the platelet rich fraction of cord blood comprises platelet-like cells comprising embryonic stem cell markers.
• the platelet rich fraction of cord blood comprises the transcription factors OCT3/4 and SOX2 (See Fig. 1 A and 1 B).
• the platelet rich fraction of cord blood comprises one or more of the proteins OCT3/4, SOX2, NANOG, CRIPTO, GATA-4, and C-myc (See Fig. 1C).
• the platelet fraction of cord blood comprises one or more of proteins OCT3/4, SOX2, NANOG, and C-myc (See Fig. 1 D, 1 E, and l F).
• the cord blood mononuclear cells comprise one or more of the markers CD14, CD66, CD4, CD8, CD19, CD56, CD41 b, and CD42a.
• the described invention discloses a cell product comprising a pharmaceutical composition containing the functionally modulated adult blood mononuclear cells of the described invention.
• a method for treating a subject in need thereof comprises administering the cell product to a diabetic mammalian subject, wherein the cell product may be effective to increase a population of functional cells in the pancreas of the subject.
• the cell product may be effective to increase the population of functional ⁇ cells in the pancreas of the subject.
• the cell product may be effective to migrate to the pancreas of the subject following administration.
• the pharmaceutical composition containing the functionally modulated adult blood mononuclear cells may be formulated with an excipient, carrier or vehicle including, but not limited to, a solvent.
• excipient refers to carrier materials suitable for formulation and administration of the functionally modulate adult blood mononuclear cell product described herein.
• Carriers and vehicles useful herein include any such materials known in the art which are nontoxic and do not interact with other components.
• pharmaceutically acceptable carrier refers to any substantially-non-toxic carrier useable for formulation and administration of the composition of the described invention in which the functionally modulated adult blood mononuclear cell product of the described invention will remain stable and bioavailable.
• the pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the mammal being treated. It further should maintain the stability and bioavailability of an active agent.
• the pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition.
• the pharmaceutically acceptable carrier may be, without limitation, a binding agent (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodium starch glycolate, etc.), or a wetting agent (e.g., sodium lauryl sulfate, etc.).
• a binding agent e.g., pregelatinized maize starch, poly
• pharmaceutically acceptable carriers for the compositions of the described invention include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethyleelluloses, polyvinylpyrrolidones and the like.
• carrier solutions also can contain buffers, diluents and other suitable additives.
• buffer refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.
• buffers envisioned by the described invention include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), and normal/physiologic saline (0.9% NaCI).
• PBS Dulbecco's phosphate buffered saline
• D5W 5% dextrose in water
• normal/physiologic saline (0.9% NaCI).
• the infusion solution is isotonic to subject tissues.
• the infusion solution is hypertonic to subject tissues.
• Compositions of the described invention that are for parenteral administration may include pharmaceutically acceptable carriers such as sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in a liquid oil base.
• the functionally modulated adult blood mononuclear cell product of the described invention may be administered parenterally in the form of a sterile injectable aqueous or oleaginous suspension.
• parenteral or “parenterally” as used herein refers to introduction into the body by way of an injection (i.e., administration by injection), including, but not limited to, infusion techniques.
• the functionally modulated adult blood mononuclear cell product of the described invention may be a sterile solution or suspension in a nontoxic
• a solution generally is considered as a homogeneous mixture of two or more substances; it is frequently, though not necessarily, a liquid. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent.
• a suspension is a dispersion (mixture) in which a finely-divided species is combined with another species, with the former being so finely divided and mixed that it does not rapidly settle out. In everyday life, the most common suspensions are those of solids in liquid water.
• the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride (saline) solution. According to some embodiments, hypertonic solutions are employed.
• suitable vehicles consist of solutions, e.g., oily or aqueous solutions, as well as suspensions, emulsions, or implants.
• Aqueous suspensions may contain substances, which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
• Additional functionally modulated adult blood mononuclear cell product of the described invention readily may be prepared using technology, which is known in the art, such as described in Remington's Pharmaceutical Sciences, 18th or 19th editions, published by the Mack Publishing Company of Easton, Pa., which is incorporated herein by reference.
• “pharmaceutically effective amount” refer to the amount of the compositions of the invention that result in a therapeutic or beneficial effect following its administration to a subject.
• the effective amount of the composition may vary with the age and physical condition of the biological subject being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the timing of the infusion, the specific compound, composition or other active ingredient employed, the particular carrier utilized, and like factors.
• a skilled artisan may determine a pharmaceutically effective amount of the inventive compositions by determining the dose in a dosage unit (meaning unit of use) that elicits a given intensity of effect, hereinafter referred to as the "unit dose.”
• dose-intensity relationship refers to the manner in which the intensity of effect in an individual recipient relates to dose.
• the intensity of effect generally designated is 50% of maximum intensity.
• the corresponding dose is called the 50% effective dose or individual ED50.
• the use of the term “individual” distinguishes the ED50 based on the intensity of effect as used herein from the median effective dose, also abbreviated ED50, determined from frequency of response data in a population.
• Effectiveness refers to the property of the compositions of the described invention to achieve the desired response, and “maximum efficacy” refers to the maximum achievable effect.
• the amount of the chemotactic hematopoietic stem cell product in the pharmaceutical compositions of the described invention that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and may be determined by standard clinical techniques. (See, for example, Goodman and Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Joel G. Harman, Lee E.
• the functionally modulated adult blood mononuclear cell product of the described invention may be administered initially, and thereafter maintained by further administrations.
• the functionally modulated adult blood mononuclear cell product of the described invention may be administered initially, and thereafter maintained by further administrations.
• the functionally modulated adult blood mononuclear cell product of the described invention may be administered initially, and thereafter maintained by further administrations.
• the functionally modulated adult blood mononuclear cell product of the described invention may be administered initially, and thereafter maintained by further administrations.
• the functionally modulated adult blood mononuclear cell product of the described invention may be administered initially, and thereafter maintained by further administrations.
• the functionally modulated adult blood mononuclear cell product of the described invention may be administered initially, and thereafter maintained by further administrations.
• mononuclear cell product of the described invention may be administered by one method of injection, and thereafter further administered by the same or by different method.
• the functionally modulated adult blood mononuclear cell product of the described invention can be administered to a subject by direct injection to a desired site, systemically, or in combination with a pharmaceutically acceptable carrier.
• the growth and/or differentiation of the functionally modulated adult blood mononuclear cell product of the described invention, and the therapeutic effect of the functionally modulated adult blood mononuclear cell product of the described invention may be monitored.
• the functionally modulated adult blood mononuclear cell product of the described invention administered to treat diabetes may be monitored by testing blood glucose and/ or insulin levels in a subject.
• the immunological tolerance of the subject to the functionally modulated adult blood mononuclear cell product of the described invention after administration may be tested by various methods known in the art.
• anticoagulant-treated adult blood or umbilical cord blood is diluted in the range of 1 :2 to 1 :4 with PBS/EDTA to reduce aggregation of erythrocytes.
• the diluted blood is then layered above a Ficoll-Paque solution in a centrifuge tube, without mixing.
• the layered blood/Ficoll-Paque is centrifuged for 40 minutes at 400 x g between 18o and 20o C, without the use of the centrifuge brake. This results in the formation of blood fractions comprising, from top to bottom: a first fraction comprising blood plasma and platelet-like cells; a second fraction
• the fractions are further processed to isolate specific fraction components. Briefly, to further process mononuclear cells, the second fraction comprising mononuclear cells and platelet-like cells is carefully removed from the Ficoll-Paque gradient using a Pasteur pipet. The second fraction is then washed and centrifuged at 300 x g, 18o and 20o C, three times with PBS/EDTA, discarding the supernatant after each round.
• the fractions are further processed to isolate platelet-like cells.
• the first fraction comprising blood plasma and platelet-like cells is removed from the Ficoll-Paque gradient.
• Equal volume of HEP buffer with 1 ⁇ prostaglandin E1 (PGE1) is then added to the platelet rich fraction and mixed gently.
• the fraction is then centrifuged at 100 x g for 15-20 at room temperature with no brake to pellet any contaminating red or white blood cells.
• the supernatant is then transferred to a new container and centrifuged at 800 x g for 15- 20 minutes at room temperature.
• the pelleted platelet-like cells are then washed twice without resuspension to avoid platelet activation.
• Cells were immunostained by first resuspending cells in incubation buffer and adding primary antibody according to the manufacturer's recommended dilution. Cells were incubated with primary antibody for 1 hour at room temperature, followed by three washes with incubation buffer. Cells were then resuspended in incubation buffer with conjugated secondary antibody at the manufacturer's recommended dilution for 30 minutes at room temperature, followed by three washes in incubation buffer. Stained cells were then analyzed by flow cytometry.
• a standard immunofluorescence protocol was used. Briefly, adherent cells were fixed with 4% formaldehyde diluted in warm PBS for 15 minutes at room temperature. The fixative was aspirated and the cells washed three times with PBS for 5 minutes each. Cells were blocked with blocking buffer for 60 minutes at room temperature. Blocking buffer was then aspirated and a solution of primary antibody diluted according to the manufacturer's instructions was incubated with the cells overnight at 4o C. Cells were then rinsed three times with PBS for 5 minutes each, and subsequently incubated with fluorochrome conjugated secondary antibody diluted according to the manufactures instructions for 1-2 hours at room
• Cells were then treated with 1% osmium tetroxide in phosphate buffer for 1 -2 hours at 4o C, and then washed a least 5 times in distilled water. Cells were then stained with 2% aqueous uranyl acetate for 2 hours at 4o C in the dark. Cells were then dehydrated through the following series of acetone washes: 30% acetone for 15 minutes; 50% acetone for 15 minutes; 70% acetone for 15 minutes; 90 acetone for 15 minutes; 100% acetone for 30 minutes three times.
• a Standard Western blotting protocol was employed. Briefly, cells were lysed with cold lysis buffer and centrifuged to pellet cellular debris. Protein concentration of the supernatant was determined by a protein quantification assay (e.g., Bradford Protein Assay, Bio-Rad Laboratories). The lysate supernatant was then combined with an equal volume of 2X SDS sample buffer and boiled at 100o C for 5 minutes. Equal amounts of protein in sample buffer were loaded into the wells of an SDS-PAGE gel along with a molecular weight marker, and electrophoresed for 1 -2 hours at 100 V. Proteins were then transferred to a nitrocellulose or PVDF membrane. The membrane was then blocked for 1 hour at room temperature using blocking buffer.
• a protein quantification assay e.g., Bradford Protein Assay, Bio-Rad Laboratories
• the membrane was then incubated with appropriate dilutions of primary antibody in blocking buffer according to the manufacturer's instructions, followed by three washes in 20 Mn Tris, Ph 7.5; 150 mM NaCI, 0.1% Tween 20 (TBST) for 5 minutes.
• the membrane was then incubated with conjugated secondary antibody at manufacturer recommended dilutions in blocking buffer for 1 hour at room temperature, followed by three washes in TBST for 5 minutes each. Images of the blot were obtained using dark room development techniques for chemiluminesence detection, or using image scanning techniques for colorimetric or fluorescent detection.
• RNA samples were extracted from cells using the Quiagen kit (Valencia CA), followed by first strand cDNA synthesis using random hexamer primers (Fermentas, Hanover MD).
• Real-time PCR was performed on each sample using the Mx3000p Quantitative PCR system (Stratagene, La Jolla, CA), for 40 cycles using validated gene specific RT-PCR primer sets for each gene of interest. Relative expression level of each transcript was corrected for that of the house keeping gene beta-actin as an internal control.
• Example 1 Expression of embryonic stem (ES) cell markers and human islet cell-specific transcription factors in cord blood and adult peripheral blood platelet-like cells.
• ES embryonic stem
• Example 2 Functional modulation of blood immune cells by platelets through the adherence of platelets to immune cells
• CD41 b is a ⁇ chain of glycoprotein Mb (known as CD41) which is associated with glycoprotein Ilia (or integrin ⁇ 3, CD61) and forms the heterodimeric gpllb/gpllla complex present on human megakaryocytes and platelets;
• CD42a GP-IX CD42a
• platelet glycoprotein GPIX GP9
• CD42a-d complex the receptor for von Willebrand factor and thrombin, mediates adhesion of platelets to subendothelial matrices that are exposed in damaged endothelium and amplifies platelet response to thrombin.
• platelet-like cells adhere to most of CD 14+ monocytes and CD66b+ granulocytes, as well as to some of CD4+ T cells, coa+ T cells, CD19+ B cells, and CD56+ NK cells ( Figure 2A-C). After being treated with the cell dissociation reagents trypsin-EDTA (0.25%), the percentage of
• the data suggest that the platelet-like cells adhere more strongly to monocytes than to granulocytes and other immune cells.
• monocytes/macrophages could de-differentiate into pluripotent stem cells (designated as fibroblast-like ⁇ s, f- ⁇ ) after ex vivo treatment with inducers (Zhao Y, Glesne D, Huberman E: A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc Natl Acad Sci USA 2003, 100: 2426-2431).
• PBMCs peripheral blood mononuclear cells
• Fig. 3D shows a zoomed out (upper panel) and zoomed in (lower panel) image of the close association of macrophages ( ⁇ s) with platelet-like cells (P) by electron microscopy.
• the lower panel represents the image outlined by the dashed box in the upper panel.
• the letter “N” indicates the nuclei of the ⁇ s.
• the membrane boundary of the macrophage ( ⁇ ) appears to have merged with the membrane boundary of the platelet-like cell (P) (See junction of apparent fusion indicated by arrow, lower panel).
• Fig. 3D shows a zoomed out (upper panel) and zoomed in (lower panel) image of the close association of macrophages ( ⁇ s) with platelet-like cells (P) by electron microscopy.
• the lower panel represents the image outlined by the dashed box in the upper panel.
• the letter “N” indicates the nuclei of the ⁇ s.
• the membrane boundary of the macrophage ( ⁇ ) appears to
• FIG. 3E shows a series of zoomed images of increasing magnification from upper left, to right, to lower left, showing the interaction of a ⁇ and a platelet-like cell.
• the membranes of the ⁇ and platelet-like cell appear to be fused together (See apparent fused membrane indicated by arrows, right panel, lower left panel). In some places, the cell membrane boundaries have disappeared (See Fig. 3E, right and lower left panels, indicated by triangular arrow).
• Fig. 2F also shows a zoomed out (left panel) and zoomed in (right panel) image of the close association between a ⁇ and a platelet-like cell.
• the membrane of the ⁇ and platelet-like cell are closely associated or appear to be fused (See e.g. arrow, right panel).
• Fig. 3G shows a zoomed out (left panel) and zoomed in (right panel) image of a macrophage that is in close contact with/appears to have engulfed a platelet-like cell (P).
• the undulating nucleus of the macrophage is indicated (N).
• the platelet-like cell is completely surrounded by the boundary of the ⁇ membrane.
• Confocal microscopy data confirmed the presence of CD41 b, CD42 markers and sternness markers within ⁇ cells. Specifically, confocal data confirmed the distribution of CD42a+OCT3/4+ and CD42a+NANOG+ inside of ⁇ s and the translation of OCT3/4 and NANOG into the nucleus of ⁇ ( Figure 3H). As shown in Fig. 3H, ⁇ s were immunoflourescently stained for either CD42a and OCT3/4 (upper panels) or CD42a and NANOG (lower panels). The nucleus was stained with DAPI for each group. Fig.
• monocytes/macrophages could de-differentiate into pluripotent stem cells, designated as fibro blast-like ⁇ s (f- ⁇ ), after ex vivo treatment with inducers (Zhao Y, Glesne D, Huberman E: A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc Natl Acad Sci USA 2003, 100: 2426-2431 ), the pluripotency of f- ⁇ derived from the sternness of platelet-like cells was
• Macrophages ( ⁇ s) contacted with a platelet rich fraction of cord blood were treated with trypsin/EDTA or left untreated. Trypsinized and untreated ⁇ s were cultured in the presence of 50 ng/ml macrophage colony stimulating factor (M-CSF) for two days and then visually evaluated for the presence or absence of f- ⁇ morphology. As shown in Fig. 4A, the percentage of f- ⁇ s significantly decreased in the group of macrophages treated with trypsin/EDTA. Without being limited by theory, this data suggests that the pluripotency of f- ⁇ is derived from contact with, and the sternness of, components within the platelet rich fraction of blood that confer sternness characteristics on the f- ⁇ .
• M-CSF macrophage colony stimulating factor
• phase contrast microscopy images showing the difference in the total cell population of trypsin/EDTA treated (left panel) and untreated (right panel) cells are shown in Fig. 4B.
• the data indicates that the formation of f- ⁇ was reduced after the (at least partial) removal of attached components in the platelet rich fraction of blood from contact with the ⁇ s.
• the data show that the potential for differentiation of f- ⁇ into epithelial-like cells was decreased after removal of platelet rich blood fraction components via treatments with trypsin/EDTA.
• ⁇ s were obtained from cord blood and treated with trypsin/EDTA or left untreated. The treated and untreated ⁇ s were cultured in 100 ng/ml epithelial growth factor (EGF) for 10 days,
• the platelet rich fraction of cord blood comprising platelet-like cells may provide ES- related transcription factors that, by contacting ⁇ / ⁇ , leads to the reprogramming of ⁇ / ⁇ and the proliferation and differentiation of f- ⁇ .
• Example 3 Generation of functional islet ⁇ cells through plateletlike cell mediated cell reprogramming in humans
• Type 1 diabetes is a T cell-mediated autoimmune disease that causes a deficit of pancreatic islet cells.
• Islet transplantation, drug-mediated promotion of -cell regeneration, and transplantation of functional islet cells differentiated from human induced pluripotent stem cell (hiPSC) or embryonic stem (ES) cell lines have been proposed and tested as likely approaches for treating T1 D (Pagliuca FW, et al., Generation of functional human pancreatic beta cells in vitro. Cell 2014, 159: 428- 439; Quiskamp N, et al., Differentiation of human pluripotent stem cells into beta- cells: Potential and challenges.
• reprogramming adult cells with a platelet rich fraction of cord blood via cell contact may be capable of generating platelet- induced pluripotent stem cells (Pi PS) that can subsequently differentiate into functional islet cells.
• Pi PS platelet- induced pluripotent stem cells Due to safety concerns involved in the generation of iPS cells by viral- or drug-induced transduction, the components of platelet rich fractions of cord blood can function as a vehicle for protein and mRNA delivery and
• Insulin a potent antilipolytic (inhibiting fat breakdown) hormone, is known to reduce blood glucose levels by accelerating transport of glucose into insulin-sensitive cells and facilitating its conversion to storage compounds via glycogenesis (conversion of glucose to glycogen) and lipogenesis (fat formation) within the islets of Langerhans of the pancreas, ⁇ -cells produce insulin.
• Glucagon a hormone that also plays a role in glucose homeostasis, is produced by a-cells within the islets of Langerhans in response to low normal glucose levels or hypoglycemia, and acts to increase glucose levels by accelerating glycogenolysis and promoting gluconeogenesis. After a glucose-containing meal, glucagon secretion is inhibited by hyperinsulinemia, which contributes to
• Incretins which include glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), are also involved in regulation of blood glucose, in part by their effects on insulin and glucagon [Drucker D.J. et al., "The incretin system: glucagon-like peptide- 1 receptor agonists and dipeptidyl peptidase- 4 inhibitors in type 2 diabetes", Lancet, Vol. 368: 1696-1705, (2006)]. Both GLP-1 and GIP are considered glucose-dependent hormones, meaning they are secreted only when glucose levels increase above normal fasting plasma glucose levels.
• these hormones are released in response to meals and, by activating certain receptors on pancreatic ⁇ -cells, they aid in stimulation of insulin secretion.
• glucose levels are low, however, GLP-1 and GIP levels (and their stimulating effects on insulin secretion) are diminished [Drucker D.J., "The biology of incretin hormones", Cell Metab. Vol. 3: 153-165, (2006)].
• the preproglucagon-derived peptides glucagon, GLP1 and GLP2 are encoded by the preproglucagon gene, which is expressed in the central nervous system, intestinal L-cells, and pancreatic and gastric a-cells.
• a post-translational cleavage by prohormone convertases (PC) is responsible for the maturation of the preglucagon hormone that generates all these peptides.
• PC prohormone convertases
• proprotein convertase subtilisin/kexin type 2 leads to production of glucagon together with the products glicentin, glicentin- repeated pancreatic polypeptide, intervening peptide 1 and the major proglucagon fragment [Dey A. et al., "Significance of prohormone convertase 2, PC2, mediated initial cleavage at the proglucagon interdomain site, Lys70-Arg71 , to generate glucagon", Endocrinol., Vol. 146: 713-727, (2005)].
• PCSK1/3 enzymes cleave the preproglucagon hormone to generate GLP1 and GLP2 along with glicentin, intervening peptide 1 and oxyntomodulin [Mojsov S., "Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing", J. Biol. Chem., Vol. 261 : 11880-11889 (1986)].
• islet a cells are an extraintestinal site for GLP-1 production [Portha B. et al., "Activation of the GLP-1 receptor signalling pathway: a relevant strategy to repair a deficient beta-cell mass", Exptl Diabetes Res. Article 376509: 1- 11 , (2011 )].
• One of the many observed cellular effects of GLP-1 is the inhibition of ⁇ -cell KATP channels, which initiates Ca2+ influx through voltage-dependent calcium channels and triggers the exocytotic release of insulin [MacDonald P.E. et al., "The multiple actions of GLP-1 on the process of glucose-stimulated insulin secretion", Diabetes, Vol. 51 (Suppl. 3): S434-S442, (2002)].
• glucose Since glucose cannot readily diffuse through all cell membranes, it requires assistance from both insulin and a family of transport proteins (facilitated glucose transporter [GLUT] molecules) in order to gain entry into most cells [Bryant, et al, Nat. Rev. Mol. Cell Biol. "Regulated transport of the glucose transporter GLUT 4", Vol. 3(4): 267-277, (2002)].
• GLUTs act as shuttles, forming an aqueous pore across otherwise hydrophobic cellular membranes, through which glucose can move more easily.
• GLUT4 is considered the major transporter for adipose, muscle, and cardiac tissue, whereas GLUTs 1 , 2, 3, and 8 facilitate glucose entry into other organs (eg, brain, liver). Activation of GLUT4 and, in turn, facilitated glucose diffusion into muscle and adipose tissue, is dependent on the presence of insulin, whereas the function of other GLUTs is more independent of insulin [Uldry M. et al., "The SLC2 family of facilitated hexose and polyol
• the liver does not require insulin to facilitate glucose uptake, it does need insulin to regulate glucose output. Thus, for example, when insulin concentrations are low, hepatic glucose output rises. Additionally, insulin helps the liver store most of the absorbed glucose in the form of glycogen.
• the kidneys play a role in glucose homeostasis via release of glucose into the circulation (gluconeogenesis), uptake of glucose from the circulation to meet renal energy needs, and reabsorption of glucose at the proximal tubule.
• the kidneys also aid in elimination of excess glucose (when levels exceed approximately 180 mg/dL, though this threshold may rise during chronic hyperglycemia) by facilitating its excretion in the urine.
• insulin-containing ⁇ -cells intermingle with other cell types within the islet, i.e., insulin-, glucagon-, and somatostatin-containing cells are found distributed throughout the human islet [Cabrera O. et al., "The unique cytoarchitecture of human pancreatic islets has implications for islet cell function", Proc. Natl Acad. Sci. U.S., Vol. 103: 2334-2339, (2006)].
• Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia. Chronic hyperglycemia is associated with long-term damage, dysfunction, and potential failure of organs, including the eyes, kidneys, nerves, heart and blood vessels. The ideal therapeutic agent for treating diabetes has yet to be developed.
• T1 D Type 1 Diabetes mellitus
• LDL low density lipoprotein
• T1D Four pathological characteristics of T1D are blood glucose levels, hemoglobin A1C, glucagon and C-peptide
• T1 D The immune dysfunction in T1 D is complicated, with effects both in pancreatic islets and outside the pancreas.
• Different components of the immune system e.g., CD4+, CD8+ T cells, T regulatory cells (Tregs), B cells, dendritic cells (DCs), monocyte/macrophages ( ⁇ / ⁇ s), natural killer T cells (NKTs)] are all envisioned to actively contribute to auto-immune responses in T1 D, thus
• T1 D-related dysfunction in the immune system has been traced to dysfunctions in multiple cell types and targets including T cells, B cells, regulatory T cells (Tregs), monocytes/macrophages, dendritic cells (DCs), natural killer (NK) cells, and natural killer T (NKT) cells [Lehuen A. et al., "Immune cell crosstalk in type I diabetes", Nat Rev Immunol. Vol. 10: 501 -513, (2010)].
• Type 2 diabetes is a hyperglycemic disorder in which ⁇ -cells are present, thus distinguishing it from type 1 diabetes.
• T2D Type 2 diabetes
• the central defects are inadequate insulin secretion (insulin deficiency) and/or diminished tissue responses to insulin (insulin resistance) at one or more points in the complex pathways of hormone action [Triplitt C.L., "Examining the mechanisms of glucose regulation", Am. J. Manag. Care, Vol. 18 (1 Suppl) S4-S10, (2012)]. Insulin deficiency and insulin resistance frequently coexist, though the contribution to hyperglycemia can vary widely along the spectrum of T2D.
• T2D includes an autoimmune component that initiates inflammation affecting pancreatic islet ⁇ -cells, which provides new insight into the mechanism and potential treatment of insulin resistance through immune modulation.
• Some clinical studies showed increasing levels of IL-17 production in T2D patients [Jagannathan-Bogdan M. et al., "Elevated proinflammatory cytokine production by a skewed T cell compartment requires monocytes and promotes inflammation in type 2 diabetes", J Immunol, Vol. 186: 1162-1172, (2011)] and obese patients [Sumarac-Dumanovic M.
• Platelet-like cells were examined for human islet cell-specific markers including transcription factors (NKX6.1 and MAFA) and KATP channel proteins (Sur1 and Kir6.2). Platelet-like cells from cord blood were isolated as described above and analyzed by real-time PCR and Western blot.
• Real time PCR data revealed the expressions of Sur1 and Kir6.2 mRNAs in platelet-like cells derived from seven different samples (Fig. 5A). Numbers to the right of the DNA gel images represent the average quantified amount of PCR DNA observed in arbitrary intensity units (Fig. 5A, right margin). The real-time PCR data also revealed weak expression of insulin mRNA (Fig. 5A).
• Cord blood platelet-like cells were also found to comprise mRNA from the pancreatic islet ⁇ cell-released hormone somatostatin (Fig. 5A). [00303] Cord blood platelet-like cells were also examined for markers via Western blot analysis. Platelet-like cells were found to display the transcription factors NKX6.1 and MAFA at protein levels (Fig. 5B), suggesting a higher potential to promote the differentiation and regeneration of islet cells. Numbers to the right of each blot represent the average quantified amount of protein identified (Fig. 5B).
• PB-IPC peripheral blood insulin-producing cells
• Example 4 Platelet-like cells display expression of immune tolerance-related molecular markers.
• the platelet-rich fraction from cord blood comprising platelet-like cells was isolated from cord blood (Fig. 6A-D) or adult peripheral blood (Fig. 6E-I), as described above. Platelet-like cells were then examined for various immune- relevant markers. [00307] Flow cytometry showed that both cord blood derived (Fig. 6A) and adult blood derived (Fig. 6E) platelet-like cells display co-inhibitory
• Fig. 6A human cord blood platelet-like cells express CD270/CD41 and CD270/CD61.
• Fig. 6 B intracellular staining of permeabilized cord blood platelet-like cells showed co- expression of TGF 1/CD41 and TGF i/CD42.
• Human cord blood platelet-like cells express the autoimmune regulator, AIRE (Fig. 6 C and D).
• AIRE autoimmune regulator
• FIG. 6C western blot analysis revealed that each of seven different samples of cord blood platelet-like cells comprised the AIRE protein. Numbers to the right of each blot represent the average quantified amount of protein in arbitrary relative intensity units.
• FIG. 6D over 85% of cord blood platelet-like cells positive for platelet marker CD41 were also positive for AIRE protein (right panel). lgG-FITC/lgG-PC7 served as negative controls.
• Galectin 9 is a tandem-repeat type galectin with two carbohydrate-recognition domains, which was first identified as an eosinophil chemoattractant and activation factor; it modulates a variety of biological functions, including cell aggregation and adhesion, as well as apoptosis of tumor cells.
• Galectin 9 has an immunomodulatory role towards lymphocytes, where it shows specific interactions with TIM-3, and can negatively regulate Th1 immunity.
• lgG-FITC/lgG-PC7 upper left panel
• IgG-PE/lgG-APC lower left panel
• lgG-APC/lgG-PC7 served as negative controls.
• lgG-FITC/lgG-PC7 served as negative controls.
• a low percentage of human adult platelets were identified as comprising high levels of CXCL10 (over 9%), CCR4 (over 2%), CCR5 (over 6%), CCR7 (over 27%), CXCR1 (over 9%), CXCR2 (over 10%), CXCR3 (over 22%), and CD62L (over 4%). Virtually none of the adult platelets expressed high levels of CXCL1 (less than 1%).
• the x- axis for each of the flow cytometry charts represents the marker CD41.
• adult mononuclear cells that contact the plasma rich fraction of cord blood comprising platelet-like cells can induce immune tolerance via transfer of, or induction of, immune regulatory molecules.

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