WO2015164506A1 - Methods of enhancing stem cell engraftment - Google Patents
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
- A61K31/5578—Eicosanoids, e.g. leukotrienes or prostaglandins having a pentalene ring system, e.g. carbacyclin, iloprost
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
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- A61P9/00—Drugs for disorders of the cardiovascular system
Definitions
- This disclosure relates to methods of enhancing stem cell engraftment. More specifically, it relates to compositions and methods of using biologically active compounds to enhance stem cell engraftment.
- PAD peripheral arterial disease
- atherosclerotic disease associated with diabetes, hypertension, hypercholesterolemia, and coronary artery disease.
- PAD affects 12- 14% of the general population, and its incidence is accelerating because of the increase in the elderly population. More than 10 million people in the United States have PAD.
- CLI critical limb ischemia
- Conventional treatments for PAD such as angioplasty, stent deployment, and peripheral bypass surgery, are less effective when PAD progresses and causes obstruction of arterioles.
- Exogenous prostacyclin (PGI2 or PG3 ⁇ 4 replacement therapy offers a therapeutic alternative for patients who are poor candidates for surgical revascularization, such as high- risk patients (e.g., the elderly).
- PGI2 therapy is efficacious, but because PGI2 is an unstable compound with a circulating half-life of 1-2 minutes, this approach requires continuous intravenous or intraarterial infusion, which is associated with side effects and several potential complications. While continuous intravenous PGI2 therapy is effective, this approach is inconvenient for PAD patients, as PGI2 must be administered by using a continuous pump with an indwelling catheter. This delivery system is cumbersome and greatly reduces the patient's quality of life.
- FIG. 1 illustrates a schematic of biosynthesis of prostanoids (e.g., prostaglandins, such as prostaglandin D 2 (PGD 2 ), E 2 (PGE 2 ), F 2 (PGF 2 ), and I 2 (PGI 2 ) (prostacyclin), or thromboxane A 2 (TXA 2 )) through coupling reactions of upstream cyclooxygenases (COXs) and downstream individual synthases;
- PGD 2 prostaglandin D 2
- PGE 2 E 2
- F 2 PEF 2
- I 2 PGI 2
- COXs upstream cyclooxygenases
- Figure 2A displays laser Doppler images of local treatment of mouse ischemic limbs with carbaprostacyclin (CPGI2) as compared to control (saline);
- CPGI2 carbaprostacyclin
- Figure 2B displays a graph of a quantitative analysis of perfusion recovery of mouse ischemic limbs with CPGI2 treatment as compared to saline treatment;
- Figure 3 displays live images of distinct arterial growth of mouse ischemic limbs treated with CPGI2, wherein more intraarteriolar connections (solid line arrows) and corkscrew extensions of arterioles (dashed line arrows) developed in the CPGI2-treated versus the saline-treated group;
- Figure 4A displays a histogram of mean blood vessel size distribution in a quantitative micro-CT analysis
- Figure 4B displays micro-CT images of microvascular network in CPGI2 -treated and saline-treated ischemic legs; the red dashed circles show the vasculature of the thigh muscle where CPGI2 or saline was administered;
- Figure 5A displays western blot images of COX-l-lOaa-PGIS and COX-1 expression in human mesenchymal stem cells (hMSC or hMSCs);
- Figure 5B displays a graph of PGI2 production levels in hMSCs engineered to overexpress PGI2 (PGI2 -hMSCs) versus control;
- Figure 5C displays endothelial cell tube formation incubated with PGI2-hMSC conditioned medium
- Figure 5D displays endothelial cell tube formation incubated with control medium
- Figure 6A displays a schematic representation of the lentiviral vector encoding herpes virus thymidine kinase (HSVl-tk), mCherry fluorophore, and firefly luciferase reporter genes;
- Figure 6B displays representative in vitro bioluminescent imaging (BLI) images of hMSCs transduced with lentiviruses;
- Figure 6C displays a representative photomicrograph and its corresponding fluorescence image showing the expression of red mCherry fluorescent protein in transduced hMSCs;
- Figure 6D displays a graph of high efficiency lentiviral transduction in hMSCs as confirmed by flow cytometry analysis
- Figure 7A displays representative BLI images of NOD-SCID mice 3 days after PGI2 -hMSCs or 3.1-hMSCs were injected into the gastrocnemius muscle of the ischemic hindlimb;
- Figure 7B displays a quantitative analysis of the BLI images of Figure 7A
- Figure 8A displays BLI images of NOD-SCID mice over a 14 day period after PGI2 -hMSCs or 3.1-hMSCs were injected into the gastrocnemius muscle of the ischemic hindlimb;
- Figure 8B displays a quantitative analysis of the BLI images of Figure 8A
- Figure 9A displays BLI images of NOD-SCID mice over a 5 day period after a hMSCs injection combined with daily cicaprost or CW501516 treatments;
- Figure 9B displays a quantitative analysis of the BLI images of Figure 9A;
- Figure 10A displays a graph of systolic blood pressure in mice at 3 days after injection with PGI2-hMSC and 3.1-hMSC;
- Figure 10B displays a graph of mean arterial pressure in mice at 3 days after injection with PGI2-hMSC and 3.1-hMSC;
- Figure 11 A displays a graph of functional recovery of ischemic hindlimbs in mice at 21 days after injection with PGI2-hMSC and 3.1-hMSC;
- Figure 1 IB displays a graph of functional recovery of ischemic hindlimbs in mice at 28 days after injection with PGI2-hMSC and 3.1-hMSC;
- Figure 12 displays endogenous Ki67 + cells spread within the hMSC injection area
- Figure 13 displays confocal images indicating of endogenous proliferating (Ki67 + ) cells only rarely seen in regions further away from both 3.1-hMSC and PGI 2 -I1MSC injection site;
- Figure 14A displays representative confocal images of endogenous Ki67 + Sca-l + and Ki67 + Sca-1 " cells;
- Figure 14B displays a quantitative analysis of Ki67 + Sca-l + cells surrounding PGI 2 - hMSCs injection sites as compared to 3.1-MSC sites;
- Figure 14C displays a quantitative analysis of Ki67 + Sca-l " cells surrounding PGI 2 - hMSCs injection sites as compared to 3.1-MSC sites;
- Figures 15A-F display HI 9 RNA levels along with cell viability in C2C12 myoblasts in various coculture environments
- Figures 15G-I display HI 9 RNA levels along with cell viability in C2C12 myoblasts after specific knock down with HI 9 siRNA (HI 9 KD) compared to negative control siRNA;
- Figures 16A-F display HI 9 RNA levels along with cell viability in primary myoblasts
- Figure 16G-I display HI 9 RNA levels along with cell viability in primary myoblasts after specific knock down with HI 9 siRNA (HI 9 KD) compared to negative control siRNA;
- Figure 16J displays representative images of HI 9 RNA fluorescence in situ hybridization in gastrocnemius muscle sections at 3 days after 3.1-hMSC or PG 2 -hMSC injections.
- composition comprising a stem cell, a stem cell engraftment enhancer, and a carrier fluid, for use in the treatment of an individual having a disease or at risk of developing a disease, wherein the disease is a vascular-associated disease and/or a muscular disease.
- compositions comprising PGI2-overexpressing human mesenchymal stem cells (PGI2-hMSCs), and a carrier fluid; wherein an effective amount of the composition is administered via a single treatment stream as an intramuscular injection to an individual having a disease or at risk of developing a disease, wherein the disease is a vascular-associated disease and/or a muscular disease, and wherein stem cell engraftment is enhanced in said individual by greater than about 200%, when compared to stem cell engraftment in an individual treated with a composition lacking the stem cell engraftment enhancer.
- PGI2-overexpressing human mesenchymal stem cells PGI2-overexpressing human mesenchymal stem cells
- compositions comprising: human mesenchymal stem cells (hMSCs), Iloprost, and a carrier fluid; wherein the composition is administered to an individual having a disease or at risk of developing a disease, wherein the disease is a vascular- associated disease and/or a muscular disease, and wherein the composition is administered via multiple treatment streams comprising: a stem cell treatment stream, and a stem cell engraftment enhancer treatment stream; wherein the stem cell treatment stream comprises hMSCs and is administered via an intramuscular injection; and wherein the stem cell engraftment enhancer treatment stream comprises Iloprost and is administered via inhalation.
- hMSCs human mesenchymal stem cells
- Iloprost vascular- associated disease and/or a muscular disease
- composition for stem cell engraftment comprises a stem cell, wherein the stem cell comprises human mesenchymal stem cells (hMSCs), endothelial progenitor cells (EPCs), hematopoietic stem cells (HSCs), cardiac progenitor cells (CPCs), satellite cells, or combinations thereof, a stem cell engraftment enhancer, wherein the stem cell engraftment enhancer comprises prostacyclin (PGI2), a PGI2 precursor, a peroxisome proliferator-activated receptor ⁇ / ⁇ isoform (PPAR8) agonist, a cAMP inducer, a phosphodiesterase inhibitor, an endothelin receptor antagonist, a nitrous oxide modulating agent, a prostacyclin receptor (IP) agonist, a non-prostanoid IP receptor agonist, or combinations thereof, and a carrier fluid.
- hMSCs human mesenchymal stem cells
- EPCs endothelial progenitor cells
- compositions for stem cell engraftment designated a CSCE, and methods of using the same.
- engraftment may be defined as (i) a process by which transplanted stem cells are retained within a tissue and/or (ii) a process by which upon transplantation of stem cells within a tissue, beneficial effects of stem cell transplantation (e.g., tissue healing; tissue repair; up- regulating lnc-RNA H 19 in a host cell environment; host cell stimulation; improved exercise; etc.) are retained within the tissue, even when the stem cells themselves or a portion thereof are not retained within the tissue.
- tissue healing e.g., tissue repair; up- regulating lnc-RNA H 19 in a host cell environment; host cell stimulation; improved exercise; etc.
- the CSCE may be used for targeted delivery of stem cells in specific body areas, wherein the stem cells may engraft and provide a repair function (e.g., a tissue repair function).
- the CSCE may be used for targeted delivery of prostacyclin (PGI2 or PGI2) in specific body areas, such as for example ischemic areas.
- PGI2 prostacyclin
- compositions for stem cell engraftment can comprise other types of cells, such as for example cells that have been engineered to produce prostacyclin (e.g., fibroblasts, endothelial cells, etc.).
- the cells can comprise any cells compatible with the disclosed methods and materials.
- the CSCE comprises a stem cell, a stem cell engraftment enhancer (designated a SEE), and a carrier fluid.
- the stem cell may produce the SEE (e.g., PGI2).
- the SEE may be supplied exogenously.
- PGI2 peripheral arterial disease
- the CSCEs will be discussed in detail in the context of peripheral arterial disease (PAD), it should be understood that treatment for other diseases is also contemplated, wherein enhanced engraftment of stem cells in the presence of a SEE may be useful.
- each ingredient/component of the CSCE may perform more than one function (e.g., stem cells may be both the stem cell component as well as the SEE, wherein the stem cells may be engineered to express or overexpress the SEE).
- stem cells may be both the stem cell component as well as the SEE, wherein the stem cells may be engineered to express or overexpress the SEE.
- stem cells may comprise stem cells and/or progenitor cells.
- stem cells comprise natural stem cells, induced pluripotent stem cells, engineered adult stem cells, or combinations thereof.
- natural stem cells refer to stem cells that are present in an organism (e.g., human) and may be isolated and used without further modification.
- induced pluripotent stem cells refer to stem cells (e.g., human adult stem cells) that have been modified (e.g., genetically modified) to provide pluripotent stem cells.
- Nonlimiting examples of stem cells suitable for use in the present disclosure include human mesenchymal stem cells (hMSC or hMSCs), endothelial progenitor cells (EPCs), hematopoietic stem cells (HSCs), cardiac progenitor cells (CPCs), satellite cells (e.g., myosatellite cells, skeletal muscle progenitor cells, etc.), or combinations thereof.
- hMSC or hMSCs human mesenchymal stem cells
- EPCs endothelial progenitor cells
- HSCs hematopoietic stem cells
- CPCs cardiac progenitor cells
- satellite cells e.g., myosatellite cells, skeletal muscle progenitor cells, etc.
- the stem cells comprise hMSCs.
- Human mesenchymal stem cells offer advantages as vehicles for therapeutic gene transfer. Stem cell therapy is emerging as a novel and promising therapeutic approach for PAD. Clinical studies in PAD patients have shown that hMSCs are attractive candidates for stem cell-based strategies for tissue repair and gene therapy.
- hMSCs can be easily isolated and expanded to large numbers in vitro or ex vivo.
- hMSCs show low immunogenicity after allogeneic transplantation and provide paracrine factors for repairing damaged tissue.
- hMSCs accumulate at sites of injury to protect against inflammation and promote revascularization. These unique properties make hMSCs an excellent choice for exogenous gene delivery.
- hMSCs can be modified to express therapeutic genes before being administered directly to damaged tissues. This combined hMSC-gene therapy approach eliminates the need for repetitive or continuous gene delivery because hMSCs are able to self-renew.
- hMSCs may be engineered to augment production of specific desired proteins, thereby enhancing the therapeutic benefits provided by native hMSCs.
- hMSCs may be engineered to produce PGI2, thereby offering a novel, targeted PGI2 replacement therapy for treating PAD, as will be described in more detail later herein.
- EPCs generally comprise a population of rare cells that circulate in the blood or reside in vasculatures. EPCs have the ability to differentiate into endothelial cells (e.g., cells that make up the lining of blood vessels).
- EPCs may be engineered to augment production of specific desired proteins, thereby enhancing the therapeutic benefits provided by native EPCs.
- EPCs may be engineered to produce PGI2.
- HSCs generally comprise a heterogeneous population of blood cells. HSCs are derived from mesoderm and have the ability to give rise to all the other blood cells. In an embodiment, HSCs may be engineered to augment production of specific desired proteins, thereby enhancing the therapeutic benefits provided by native HSCs. In an embodiment, HSCs may be engineered to produce PGI2.
- CPCs generally comprise a population of resident cardiac stem cells. CPCs are thought to account for the physiological turnover of cardiac myocytes and vascular endothelial cells. In an embodiment, CPCs may be engineered to augment production of specific desired proteins, thereby enhancing the therapeutic benefits provided by native CPCs. In an embodiment, CPCs may be engineered to produce PGI2.
- Satellite cells generally comprise small mononuclear progenitor cells with virtually no cytoplasm found in mature muscle. Satellite cells are precursors to skeletal muscle cells, able to give rise to satellite cells or differentiated skeletal muscle cells.
- the stem cells may be included within the CSCE in a suitable amount.
- the stem cells may be present within the CSCE in an amount of from about 5 million cells/mL to about 600 million cells/mL, alternatively from about 10 million cells/mL to about 500 million cells/mL, or alternatively from about 25 million cells/mL to about 400 million cells/mL, based on the total volume of the CSCE.
- the stem cells may be present within the CSCE in an amount of about 200 million cells/mL, based on the total volume of the CSCE.
- the term "about,” when used in conjunction with a percentage or other numerical amount means plus or minus 10% of that percentage or other numerical amount.
- the term "about 400 million cells” would encompass 400 million cells plus or minus 40 million cells.
- the CSCE can comprise a SEE.
- the SEE can enhance (e.g., increase) (a) an ability of the stem cells to engraft (e.g., be retained) in a tissue upon transplantation into the tissue and/or (b) retention of beneficial effects of stem cell transplantation (e.g., tissue healing; tissue repair; up-regulating lnc-RNA H19 in a host cell environment; host cell stimulation; improved exercise; etc.) in the tissue, even when the stem cells themselves or a portion thereof are not retained within the tissue.
- a host cell refers to a cell present in a location (e.g., tissue location) where the stem cells are transplanted.
- engraftment plays a role in co-stimulation of the host cells to proliferate and regenerate due to the stem cells being retained long enough to stimulate host cells and the new growth of muscle and blood vessels.
- the SEE may comprise PGI2; PGI2 stable precursors or analogues (e.g., Cicaprost, Iloprost, Beraprost, Carbaprostacyclin, Trepostinil, Epoprostenol, etc.); a peroxisome proliferator-activated receptor ⁇ / ⁇ isoform (PPAR8) agonist (e.g., GW501516, also known as GW-501,516, GW1516, GSK-516, Endurobol, etc.); a cAMP inducer (e.g., forskolin, also known as coleonol, 8-bromo-cAMP, etc.); a phosphodiesterase inhibitor (e.g., sildenafil citrate (VIAGRA ® ), tadalafil (CIALIS ® ), vardenafil (LEVITRA ® ), etc.); an endothelin receptor antagonist (e.g., bosentan (TRA), TRA
- the SEE may be a biologically or pharmacologically active compound.
- a biologically active compound can be defined as a compound that interacts in some fashion with any living cell, tissue, and/or organism.
- PGI2, PGI2 precursors or analogues, PPAR8 agonists, cAMP inducers, phosphodiesterase inhibitors, endothelin receptor antagonists, nitrous oxide modulating agents, IP agonists, and non-prostanoid IP receptor agonists are biologically active compounds.
- the SEE comprises PGI2.
- PGI2 a member of the prostaglandin family, is synthesized from arachidonic acid (AA) in a multistep process involving the enzymes cyclooxygenase- 1 (COX-1) or cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS).
- AA arachidonic acid
- COX-1 cyclooxygenase- 1
- COX-2 cyclooxygenase-2
- PGIS prostacyclin synthase
- PGI2 has multiple favorable properties for treating PAD.
- PGI2 inhibits thrombosis and platelet aggregation.
- PGI2 PGI2 receptor
- IP PGI2 receptor
- PGI2 may enhance functional benefits of human stem cell therapy. Accumulating evidence indicates a critical role for PGI2 in controlling stem cell recruitment and survival and in promoting angiogenesis.
- Patients with critical limb ischemia (CLI) have reduced numbers of circulating progenitor cells; however, after 4 weeks of treatment with a PGI2 analogue, such patients show increased levels of progenitor cells and pain relief.
- Human outgrown EPCs may produce PGI2 and endogenous secretion of PGI2 by EPCs may facilitate vascular regeneration. In contrast, inhibiting PGI2 production in EPCs may reduce their proliferation, survival, and angiogenic capacity in ischemic hindlimbs.
- PGI2 signaling promotes the migration and recruitment of EPCs to injured vessels.
- PGI2 may have the ability to enhance the natural abilities of stem cells.
- the cell-protective property of PGI2 in vivo may attenuate cell loss by stimulating their plasticity to adapt to unfavorable environments.
- biosynthesis also known as biogenesis or anabolism, is a multi-step, enzyme-catalyzed process, wherein substrates are converted into more complex products.
- biosynthesis simple compounds are modified, converted into other compounds, or joined together to form macromolecules.
- FIG. 1 shows a schematic of the biosynthesis of prostanoids.
- Biosynthesis of prostanoids generally comprises prostaglandins and thromboxane, formed via the COX pathway from arachidonic acid (AA) in three catalytic (tri-catalytic) steps (represented by some of the thin line arrows in Figure 1).
- AA may traverse across an endoplasmic reticulum (ER) membrane (e.g., from a first or cytoplasmic side of the ER membrane to a second or luminal side of the ER membrane) and be converted in catalytic step 1 to prostaglandin G 2 (PGG 2 ) by COX isoform-1 (COX-1) and/or COX-2, wherein COX- 1 and COX-2 may be located on the luminal side of the ER membrane.
- PGG 2 may be further converted to prostaglandin endoperoxide (prostaglandin H 2 (PGH 2 )) by COX-1 and/or COX-2.
- PGH 2 may traverse across the ER membrane (e.g., from the luminal side of the ER membrane to the cytoplasmic side of the ER membrane).
- PGH 2 may be further isomerized to biologically active end-products (prostaglandin D 2 (PGD 2 ), E 2 (PGE 2 ), F 2 (PGF 2 ), and I 2 (PGI 2 (prostacyclin) or thromboxane A 2 (TXA 2 ) by individual synthases (PGD 2 synthase (PGDS), PGE 2 synthase (PGES), PGF 2 synthase (PGFS), and PGI 2 synthase (PGIS), or TXA 2 synthase (TXAS), respectively, as depicted in Figure 1) in tissue specific manners, wherein such individual synthases may be located on the cytoplasmic side of the ER membrane.
- PGD 2 synthase PGE 2 synthase
- PGES PGE 2 synthase
- PGFS
- Prostanoids act as local hormones in the vicinity of their production site to regulate hemostasis and smooth muscle functions. Unlike the stable level of COX-1 expression, COX-2 expression is inducible and it responds to the stimuli of pro-inflammatory and other pathogenic factors.
- TXA 2 produced from PGH 2 by TXA 2 synthase (TXAS) has been implicated in various pathophysiological conditions as a proaggregatory and vasoconstricting mediator.
- PGI 2 is the main AA metabolite in vascular walls and has opposing biological properties to TXA 2 , representing the most potent endogenous vascular protector acting as an inhibitor of platelet aggregation and a strong vasodilator on vascular beds.
- PGE 2 exhibits a variety of biological activities in inflammation.
- Aspirin and non-steroidal anti-inflammatory drugs SAID
- SAID non-steroidal anti-inflammatory drugs
- COX-1 and COX-2 activities inhibit both COX-1 and COX-2 activities to reduce the production of all prostanoids, which leads to thinning of the blood by reducing TXA 2 production and the suppression of inflammation through decreasing PGE 2 production.
- the selective COX-2 inhibiting drugs exhibit anti-inflammatory effects similar to aspirin and NSAIDs, but they may also promote strokes and heart attacks by decreasing the production of PGI 2, and increasing the production of TXA 2 .
- PGI 2 has also been determined to be a ligand for the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR).
- PPARa nuclear hormone receptor peroxisome proliferator-activated receptor
- ⁇ / ⁇ nuclear hormone receptor peroxisome proliferator-activated receptor
- ⁇ Three PPAR-isoforms, PPARa, ⁇ / ⁇ and ⁇ have been cloned and implicated in the regulation of the expression of genes involved in lipid metabolism.
- PGI 2 and PGI2 agonists e.g., carbaprostacyclin, iloprost, etc.
- PGI 2 acting as a ligand for PPAR8, induces increased expression of PPAR8 in the arterial wall after a balloon injury, suggesting that PGI 2 effects vasodilation and anti-platelet aggregation through the IP receptor and PPAR8. It has also been speculated that PGI 2 , as a ligand for PPAR8, induces anti-inflammatory activity in vascular diseases, such as atherosclerosis.
- peroxisome proliferator-activated receptor-beta/delta can be a potential regulator of PGI2 signaling.
- PPAR8 In the search for endogenous targets for PGI2 signaling, PPAR8 was found to colocalize with COX-2/PGIS and actively respond to PGI2 agonists.
- PPAR8 is a ligand-activated nuclear hormone receptor that is ubiquitously expressed in various tissues. It forms heterodimers with retinoid X receptor, which binds to the peroxisome proliferator response element in the promoter region of target genes to control transcription. Emerging evidence suggests that PPAR8 plays a critical role in stem cell survival and neovascularization.
- PPAR8 activation of PPAR8 by PGI2 may promote stem cell-mediated vascular regeneration in ischemic hindlimbs. Inhibition of PPAR8 by selective antagonists or specific siRNA in human progenitor cells may reduce PGI2-induced regenerative ability and blood vessel formation.
- PGI2 in partnership with PPAR8, accelerates embryo implantation and blastocyst hatching.
- PPAR8 is important in adaptive responses to environmental changes.
- PPAR8 regulates several metabolic genes involved in cellular homeostasis. PPAR8 may play a critical role in mitochondrial function.
- PGI2-PPAR8 axis may affect the ability of stem cells to adjust to environmental changes (e.g., may affect the viability of stem cells introduced to certain body areas, such as for example ischemic areas), thus might affect the ability of stem cells to engraft.
- the SEE comprises a PPAR8 agonist, such as for example GW501516, also known as GW-501,516, GW1516, GSK-516, Endurobol, etc.
- the carrier fluids that may be used in the CSCE include any carrier fluid suitable for delivery of stem cells in vivo.
- the carrier fluid comprises a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier is meant to encompass any carrier that does not interfere with effectiveness of a biological activity of any active ingredient (e.g., stem cell, stem cell engraftment enhancer) and that is not toxic to an individual to which it is administered.
- “Pharmaceutically acceptable” as used herein adheres to the U.S. Food and Drug Administration guidelines.
- the CSCE may comprise an aqueous carrier fluid.
- the aqueous carrier fluid comprises deionized water and a variety of additives that may promote the viability and health of the stem cells of the CSCE.
- the carrier fluid comprises a saline solution (e.g., phosphate buffer saline).
- Nonlimiting examples of additive suitable for use in the carrier fluid in the present disclosure include nutritional supplements, growth factors, proteins (e.g., human serum albumin or HSA), and the like, or combinations thereof.
- the carrier fluid may be included within the CSCE in a suitable amount.
- PGI2 may be delivered by stem cells that may be engineered (e.g., programmed) to overexpress PGI2, e.g., express high levels of PGI2 or express PGI2 levels that are higher than the PGI2 levels expressed by the same stem cells prior to being engineered.
- stem cells may be engineered (e.g., programmed) to overexpress PGI2, e.g., express high levels of PGI2 or express PGI2 levels that are higher than the PGI2 levels expressed by the same stem cells prior to being engineered.
- a system that increases PGI2 biosynthesis in cells of the ischemic areas would help prevent the adverse events caused by conventional PGI2 delivery methods.
- effective and stable biosynthesis of PGI2 requires an increase in the expression of COX-1 or COX-2 in conjunction with PGIS, as illustrated in Figure 1.
- the SEE may comprise a PGI2 precursor.
- the PGI2 precursor may comprise a triple catalytic enzyme, a PGI2-overexpressing stem cell (PGI2-SC), a DNA sequence encoding for the triple catalytic enzyme, a cDNA sequence encoding for the triple catalytic enzyme, a host cell containing an expressible DNA sequence encoding for the triple catalytic enzyme, a vector comprising a DNA sequence encoding for the triple catalytic enzyme, a plasmid comprising a DNA sequence encoding for the triple catalytic enzyme, a fusion gene encoding for the triple catalytic enzyme, a synthetic PGI2 analogue, and the like, or combinations thereof.
- PGI2 precursor may comprise a triple catalytic enzyme, a PGI2-overexpressing stem cell (PGI2-SC), a DNA sequence encoding for the triple catalytic enzyme, a cDNA sequence encoding for the triple catalytic enzyme, a host cell containing an expressible DNA sequence encoding for the
- Nonlimiting examples of synthetic PGI2 analogues suitable for use in the present disclosure include Iloprost, Carbaprostacyclin, Treprostinil, Cicaprost, Beraprost, Epoprostenol, and the like, or combinations thereof.
- stem cells such as hMSCs may be engineered to overexpress an active triple catalytic enzyme to promote PGI2 expression (e.g., release PGI2).
- the PGI2 overexpression by hMSCs may provide a means for local PGI2 delivery in body areas such as ischemic areas (e.g., ischemic tissue) and may concurrently enhance the natural ability of hMSCs to mediate repair in ischemic tissue.
- prostacyclin and/or prostacyclin analogues may alleviate hindlimb ischemia by improving perfusion and promoting arteriolar growth
- this approach is not clinically practical because an invasive catheter-connected pump carrying a prostacyclin and/or prostacyclin analogues solution is generally subcutaneously implanted.
- a triple catalytic enzyme may enhance the expression of PGI2 in stem cells, such as for example hMSCs, EPCs, HSCs, CPCs, satellite cells, or combinations thereof.
- the triple catalytic enzyme may be characterized by a formula COX-linker-ES, wherein COX comprises a cyclooxygenase (COX) amino acid sequence, such as for example COX-1 or COX-2; wherein ES comprises an eicosanoid-synthesizing (ES) enzyme amino acid sequence; wherein the linker comprises from about 10 to about 22 amino acid residues of a transmembrane sequence; wherein the linker may be disposed between the COX and the ES; and wherein the linker may directly connect the COX to the ES.
- the triple catalytic enzyme comprises a hybrid protein or hybrid peptide.
- the linker (e.g., linker peptide) may function as a transmembrane linker in a cell, such that folding ability and function of each enzyme (e.g., COX, ES) of the triple catalytic enzyme may be substantially unaltered compared to the folding ability and function of respective native enzymes.
- the linker is a peptide, since it comprises a relatively short sequence of amino acids.
- linker and linker peptide can be used interchangeably.
- the linker (e.g., linker sequence) comprises His-Ala-Ile-Met- Gly-Val-Ala-Phe-Thr-Trp (SEQ ID NO. 1) or His-Ala-Ile-Met-Gly-Val-Ala-Phe-Thr-Trp- Val-Met-Ala-Leu-Ala-Cys-Ala-Ala-Pro-Pro-Leu-Val (SEQ ID NO. 2).
- the linker sequence comprises residues 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1- 17, 1-18, 1-19, 1-20 or 1-21 of SEQ ID NO. 2.
- the linker peptide provides approximately 10 A separation between the catalytic sites of the COX and the ES enzyme.
- the connected enzymes e.g., COX, ES
- the connected enzymes are preferably capable of substantially normal folding and enzymatic activity compared to the native folding and enzymatic activity of the native COX and ES enzymes.
- the triple catalytic enzyme may be characterized by a faster turnover rate when compared to a mixture of the native COX and ES enzymes.
- the hybrid protein e.g., COX-linker-ES
- COX-linker-ES does not only possess the individual enzymes' activities, but has a faster turnover rate as compared to a mixture of separate COX and ES enzymes.
- the ES may comprise PGIS or a downstream synthase thereof.
- the PGIS downstream synthase may comprise prostaglandin E synthase (PGES), prostaglandin D synthase (PGDS), or prostaglandin F synthase (PGFS).
- PGES prostaglandin E synthase
- PGDS prostaglandin D synthase
- PGFS prostaglandin F synthase
- the triple catalytic enzyme may be characterized by formulas COX-linker- PGIS, COX-linker-PGES, COX-linker-PGDS, or COX-linker-PGFS, wherein COX comprises COX-1 or COX-2.
- the triple catalytic enzyme combines the enzymatic functions of COX (e.g., COX-1, COX-2) and ES (e.g., PGIS, PGES, PGDS, PGFS) in a single hybrid protein.
- COX e.g., COX-1, COX-2
- ES e.g., PGIS, PGES, PGDS, PGFS
- the triple catalytic enzyme may be characterized by a formula COX-linker-PGIS, wherein COX comprises COX-1 or COX-2.
- the COX- linker-PGIS may adopt the functions of COX and PGIS.
- the COX-linker- PGIS may be able to continually convert AA into prostaglandin G2 (catalytic step 1), prostaglandin H 2 (catalytic step 2) and prostacyclin (PGI2; catalytic step 3), wherein the catalytic steps have been described previously herein.
- Such conversion of AA into PGI2 may be even faster than coupling reactions using unlinked, co-expressed COX and PGIS.
- the triple catalytic enzyme may be characterized by a formula COX-l-linker-PGIS.
- the triple catalytic enzyme may catalyze the three catalytic steps (e.g., three key reactions) in the biosynthesis of PGI2, thereby enhancing the expression of PGI2 (e.g., increasing the production of PGI2).
- the triple catalytic enzyme links COX-1 to PGIS and catalyzes three key reactions for efficient production of PGI2 from AA.
- the COX-l-linker-PGIS protein may comprise an 130 kDa recombinant protein, wherein the recombinant protein may be constructed by linking together human cyclooxygenase (COX) isoform-1 (COX-1) and PGIS via a linker.
- the linker may comprise from 10 to 22 amino acid residues of a transmembrane sequence, as previously described herein.
- the COX-l-linker-PGIS protein may comprise COX-l-lOaa-PGIS, wherein the linker comprises a 10 amino acid (lOaa) transmembrane sequence (e.g., SEQ ID NO. 1).
- the triple catalytic enzyme may be characterized by a formula COX-l-lOaa-PGIS.
- the triple catalytic enzyme may be chemically synthesized. In other embodiments, the triple catalytic enzyme may be recombinantly produced.
- the triple catalytic enzyme and methods of producing and/or using same are described in more detail in U.S. Publication No. 20100015120 Al, which is incorporated by reference herein in its entirety.
- PGI2 may be delivered by stem cells (SCs) that may be engineered (e.g., programmed) to express high levels of PGI2.
- SCs stem cells
- Stem cells that overexpress PGI2 may be referred to as PGI2-SCs, such as for example PGI2-hMSCs, PGI2-EPCs, PGI2- HSCs, PGI2-CPCs, PGI2-(satellite cells), etc.
- the PGI2 precursor comprises a PGI2-SC.
- the COX-linker-ES may be introduced in stem cells via any suitable transfection methods, such as nucleofection. Nucleofection is a nonviral transfection technique. As will be appreciated by one of skill in the art, and with the help of this disclosure, stable expression of COX-linker-ES may be confirmed via a variety of biochemical methods, such as for example Western Blot, genomic PCR, RT- PCR, and the like, or combinations thereof.
- SCs comprise a DNA sequence encoding for a COX, a transmembrane linker peptide, and an ES.
- COX comprises COX-1.
- COX comprises COX-2.
- ES comprises PGIS.
- the linker directly connects the COX to the ES.
- SCs e.g., PGI2-SCs
- SCs comprise a DNA sequence encoding for the triple catalytic enzyme, and such DNA sequence may be referred to as a "fusion gene.”
- stem cells may be transfected by introducing a plasmid expressing the triple catalytic enzyme that links COX to ES (e.g., COX-l-lOaa-PGIS).
- a plasmid expressing the triple catalytic enzyme that links COX to ES (e.g., COX-l-lOaa-PGIS).
- Such plasmid may comprise a promoter and an antibiotic resistance gene for selection of stable cell lines.
- Nonlimiting examples of promoters suitable for use in the present disclosure include a human cytomegalovirus promoter; endothelial-specific promoters (e.g., tie gene promoter, Tie2 gene promoter also known as Tek gene promoter, ICAM-2 (intercellular adhesion molecule-2) promoter, Flk-1 (fetal liver kinase- 1) promoter, Flt-1 (fms-like tyrosine kinase) promoter, thrombomodulin promoter, vWf (von Willebrand factor) promoter, VE-cadherin promoter, etc.); cardiomyocyte specific promoters (e.g., alpha-MHC (myosin heavy chain) promoter; troponin promoter); smooth muscle cell specific promoters (e.g., SM22alpha promoter); human muscle specific promoter; human muscle creatinine kinase promoter; human a-skeletal actin promoter; human desmin promoter; human troponin I promoter; and
- Nonlimiting examples of antibiotic resistance genes suitable for use in the present disclosure include a neomycin resistance gene (e.g., resistant to antibiotic G418); a puromycin resistance gene; an ampicillin resistance gene; a kanamycin resistance gene; a blasticidin resistance gene; a hygromycin resistance gene; a gentamicin resistance gene; a spectinomycin resistance gene; a streptomycin/spectinomycin resistance gene; and the like; or combinations thereof.
- transfected cells may be grown (e.g., cultured) for selection for a time period of from about 1 week to about 4 weeks. Then, cell clusters may be selected for further subculture, propagated and examined for PGI2 and/or COX-linker-ES expression. Subcultures that overexpress PGI2 comprise PGI2-SCs.
- a vector may comprise a DNA sequence encoding for the triple catalytic enzyme.
- the vector comprises an expression vector, such as for example a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, etc.
- the DNA sequence encoding for the triple catalytic enzyme may be introduced into SCs (e.g., a host cell) via transduction.
- the SCs comprise a host cell containing an expressible DNA sequence encoding for the triple catalytic enzyme.
- the triple catalytic enzyme may be produced by a host cell containing an expressible DNA sequence encoding for the triple catalytic enzyme.
- the host cell may be transfected with a vector comprising the DNA sequence encoding for the triple catalytic enzyme to produce host cell containing an expressible DNA sequence encoding for the triple catalytic enzyme.
- the host cell comprises a SC.
- the host cell does not comprise a SC.
- the host cell may produce the triple catalytic enzyme.
- the host cell may be cultured under conditions suitable for expression of the DNA sequence encoding the triple catalytic enzyme, and then the triple catalytic enzyme may be recovered.
- the triple catalytic enzyme comprises enzymatically active cyclooxygenase, transmembrane linker, and enzymatically active prostacyclin synthase.
- a cDNA may comprise a sequence encoding for the triple catalytic enzyme.
- the cDNA may be used for COX gene therapy.
- the CSCE may be prepared via any suitable method or process.
- the components of the CSCE e.g., stem cells, SEE, carrier fluid
- the stem cells and/or SEE may be suspended in a saline solution comprising HSA. More details regarding stem cell preparation for administering as a treatment are available in Cytotherapy, 2010, 12(5), pp 684-691; and JAMA, 2011, 306(19), p 21 10-2119; each of which is incorporated by reference herein in its entirety.
- the CSCE may be used for the treatment of an individual having a disease or at risk of developing a disease, wherein the disease can be a vascular-associated disease and/or a muscular disease, and wherein the CSCE may be a pharmaceutical composition.
- the CSCE may be used for the treatment of an individual having a vascular-associated disease or at risk of developing a vascular-associated disease, wherein the CSCE may be a pharmaceutical composition.
- the vascular-associated disease may comprise PAD, peripheral vascular disease, thrombosis, ischemia, CLI, heart attack, acute myocardial infarction, congestive heart failure, pulmonary arterial hypertension, acute lung injury, stroke, inflammation in an organ or vessel of a vascular system, chronic kidney disease, leukemia, bone marrow transplant, metabolic diseases, diabetes, and the like, or combinations thereof.
- the CSCE may be used for the treatment of an individual having a muscular disease or at risk of developing a muscular disease, wherein the CSCE may be a pharmaceutical composition.
- a method of treating an individual having a disease or at risk of developing a disease may comprise administering to the individual an effective amount of the CSCE, wherein the CSCE may be a pharmaceutical composition, to enhance stem cell engraftment in said individual, thereby ameliorating, deterring and/or preventing the disease in said individual.
- an "effective amount" of CSCE may be defined as an amount of CSCE that produces a therapeutic response or desired effect (e.g., increase PGI2 levels in a body area) in some fraction of individuals to which it is administered.
- a method of treating an individual having a vascular-associated disease or at risk of developing a vascular-associated disease may comprise administering to the individual an effective amount of the CSCE, wherein the CSCE may be a pharmaceutical composition, to enhance stem cell engraftment in said individual, thereby ameliorating, deterring and/or preventing the vascular-associated disease in said individual.
- a method of treating an individual having a muscular disease or at risk of developing a muscular disease may comprise administering to the individual an effective amount of the CSCE, wherein the CSCE may be a pharmaceutical composition, to enhance stem cell engraftment in said individual, thereby ameliorating, deterring and/or preventing the muscular disease in said individual.
- a method of treating an individual having a disease or at risk of developing a disease may comprise administering to the individual a pharmaceutical composition comprising an effective amount of the CSCE, to enhance stem cell engraftment in said individual, thereby ameliorating, deterring and/or preventing the disease in said individual.
- a method of treating an individual having a vascular-associated disease or at risk of developing a vascular-associated disease may comprise administering to the individual a pharmaceutical composition comprising an effective amount of the CSCE, to enhance stem cell engraftment in said individual, thereby ameliorating, deterring and/or preventing the vascular-associated disease in said individual.
- a method of treating an individual having a muscular disease or at risk of developing a muscular disease may comprise administering to the individual a pharmaceutical composition comprising an effective amount of the CSCE, to enhance stem cell engraftment in said individual, thereby ameliorating, deterring and/or preventing the muscular disease in said individual.
- the CSCE may be a pharmaceutical composition.
- a pharmaceutical composition generally refers to any composition that may be used on or in a body to prevent, deter, diagnose, alleviate, treat, and/or cure a disease in humans or animals.
- the stem cell engraftment in an individual treated with a CSCE may be enhanced by greater than about 200%, alternatively by greater than about 300%, alternatively by greater than about 400%, or alternatively by greater than about 500%, when compared to stem cell engraftment in an individual treated with an otherwise similar composition lacking the SEE.
- the stem cell engraftment in an individual treated with a CSCE may be enhanced by from about 200% to about 500%, when compared to stem cell engraftment in an individual treated with an otherwise similar composition lacking the SEE.
- stem cell engraftment may be defined as retention of the stem cells by a tissue, subsequent to administering stem cells to an individual.
- the components of the CSCE may be administered at the same time and via a single treatment stream (e.g., a single injection, such as intramuscular injection, intra-arterial injection, etc.). In other embodiments, the components of the CSCE may be administered at the same time and via multiple treatment streams. In yet other embodiments, the components of the CSCE may be administered at different times and via multiple treatment streams.
- a single treatment stream e.g., a single injection, such as intramuscular injection, intra-arterial injection, etc.
- the components of the CSCE may be administered at the same time and via multiple treatment streams.
- a method of treating an individual having a disease or at risk of developing a disease may comprise administering to the individual an effective amount of the CSCE via a single treatment stream (e.g., single stream CSCE treatment).
- a single treatment stream e.g., single stream CSCE treatment
- the single stream CSCE treatment may comprise PGI2-SCs and a carrier fluid.
- a method of treating an individual having a vascular-associated disease or at risk of developing a vascular-associated disease may comprise administering to the individual an effective amount of the CSCE via a single treatment stream (e.g., single stream CSCE treatment).
- a single treatment stream e.g., single stream CSCE treatment
- the single stream CSCE treatment may comprise PGI2-SCs and a carrier fluid.
- a method of treating an individual having a muscular disease or at risk of developing a muscular disease may comprise administering to the individual an effective amount of the CSCE via a single treatment stream (e.g., single stream CSCE treatment).
- a single treatment stream e.g., single stream CSCE treatment
- the single stream CSCE treatment may comprise PGI2-SCs and a carrier fluid.
- the single stream CSCE treatment may comprise SCs, a SEE, and a carrier fluid; wherein the SEE may be a biologically active compound and wherein the SEE may be exogenously supplied to the SCs.
- the single stream CSCE treatment may comprise SCs, a SEE comprising PGI2 and/or PGI2 precursor, and a carrier fluid; wherein the SCs do not overexpress PGI2; and wherein the PGI2 may be exogenously supplied to the SCs in the form of PGI2 and/or PGI2 precursor.
- the single stream CSCE treatment may comprise SCs, a SEE comprising a PPAR8 agonist, and a carrier fluid; wherein the PPAR8 agonist may be exogenously supplied to the SCs.
- the single stream CSCE treatment may comprise engineered SCs, a SEE, and a carrier fluid, wherein the SEE may be a biologically active compound.
- the single stream CSCE treatment may comprise PGI2-SCs, a SEE comprising PGI2 and/or PGI2 precursor, and a carrier fluid; wherein the SCs overexpress PGI2; and wherein PGI2 may also be exogenously supplied to the PGI2-SCs in the form of PGI2 and/or PGI2 precursor.
- a method of treating an individual having a disease or at risk of developing a disease may comprise administering to the individual an effective amount of the CSCE via multiple treatment streams.
- the CSCE may be administered via a stem cell treatment stream and via a SEE treatment stream; wherein the stem cell treatment stream comprises stem cells and a carrier fluid; wherein the SEE treatment stream may comprise a SEE and a carrier fluid; and wherein the SEE may be a biologically active compound.
- the CSCE may be administered via a stem cell treatment stream and via a PGI2 treatment stream; wherein the stem cell treatment stream comprises stem cells and a carrier fluid; and wherein the PGI2 treatment stream may comprise a PGI2 and/or PGI2 precursor and a carrier fluid.
- the stem cell treatment stream may be administered prior to, concurrent with, and/or subsequent to administering the SEE treatment stream.
- the SEE treatment stream may be administered concurrent with and subsequent to administering the stem cell treatment stream.
- a method of treating an individual having a vascular-associated disease or at risk of developing a vascular-associated disease may comprise administering to the individual an effective amount of the CSCE via multiple treatment streams.
- the CSCE may be administered via a stem cell treatment stream and via a SEE treatment stream; wherein the stem cell treatment stream comprises stem cells and a carrier fluid; wherein the SEE treatment stream may comprise a SEE and a carrier fluid; and wherein the SEE may be a biologically active compound.
- the CSCE may be administered via a stem cell treatment stream and via a PGI2 treatment stream; wherein the stem cell treatment stream comprises stem cells and a carrier fluid; and wherein the PGI2 treatment stream may comprise a PGI2 and/or PGI2 precursor and a carrier fluid.
- the stem cell treatment stream may be administered prior to, concurrent with, and/or subsequent to administering the SEE treatment stream.
- the SEE treatment stream may be administered concurrent with and subsequent to administering the stem cell treatment stream.
- a method of treating an individual having a muscular disease or at risk of developing a muscular disease may comprise administering to the individual an effective amount of the CSCE via multiple treatment streams, as disclosed herein.
- the PGI2-SCs may be administered to an individual, upon engraftment of such stem cells, the PGI2-SCs may consistently produce PGI2, which may then be secreted into surrounding areas/tissues.
- the PGI2-SCs may be locally injected (e.g., intramuscular injection) in ischemic areas or tissues, such as for example ischemic heart tissue, ischemic kidney tissue, ischemic limb tissue, ischemic lung tissue, ischemic brain tissue, ischemic pancreas tissue, and the like.
- the PGI2-SCs may consistently release PGI2 and may engraft into the ischemic tissue, thereby enhancing tissue vascularization and restoring blood flow into at least a portion of the ischemic tissue.
- the PGI2-SCs may comprise a vehicle for direct delivery of PGI2 to ischemic tissue.
- a method of treating an individual having a disease or at risk of developing a disease, wherein the disease can be a vascular-associated disease and/or a muscular disease, comprising administering to the individual an effective amount of a CSCE can further comprise up-regulating a long non-coding RNA H19 (lnc-RNA H19) in a host environment.
- lnc-RNA H19 long non-coding RNA H19
- a method of treating an individual having a muscular disease or at risk of developing a muscular disease comprising administering to the individual an effective amount of a CSCE can further comprise up-regulating a long non-coding RNA HI 9 (lnc-RNA HI 9) in a host environment.
- a method of treating an individual having a vascular-associated disease or at risk of developing a vascular-associated disease comprising administering to the individual an effective amount of a CSCE can further comprise up-regulating a long non- coding RNA HI 9 (lnc-RNA HI 9) in a host environment.
- up-regulating the long non-coding RNA H19 in the host environment can promote host cell growth (e.g., can promote endogenous progenitor cell activity under a hostile environment, such as for example under tissue damage and/or ischemia).
- a host environment refers to a cellular environment in a location (e.g., tissue location) where the stem cells are transplanted.
- a location e.g., tissue location
- long non- coding RNAs are an array of non-protein coding transcripts over 200 nucleotides long and have emerged as critical transcriptional or post-transcriptional regulators of cellular activity.
- the IncRNA H19 is a maternally imprinted gene that is abundantly expressed during embryonic development. After birth, H19 expression is reduced except in skeletal muscle. While up-regulation of H19 in myoblasts has been proposed to promote differentiation and myogenesis, the cytoprotective properties ⁇ 19 on progenitor cells have not been elucidated.
- HI 9 up-regulation can promote progenitor cell (e.g., myogenic progenitor cell) survival under hypoxia, as supported by targeted H19 knock down leading to an increase in nonviable cells.
- progenitor cell e.g., myogenic progenitor cell
- H19 may act as an early regulatory element in augmenting cellular adjustment to environmental stress, thereby mobilizing protection mechanisms and increasing resistance to stress.
- H19 may promote cellular proliferation by modulating downstream target genes.
- paracrine effects e.g., paracrine signaling
- CSCE e.g., PGI2- hMSCs
- progenitor cells can be achieved by modulating lncRNA H19.
- paracrine signaling is a form of cell-cell communication in which a cell produces a signal to induce changes in nearby cells, altering the behavior or differentiation of those cells.
- CSCE e.g., PGI2-hMSCs
- target cells e.g., host environment, host cells, host cell environment etc.
- the up-regulation of HI 9 RNA in target cells can be accompanied by a simultaneous reduction in progenitor cell death.
- a method of treating an individual having a vascular-associated disease or at risk of developing a vascular-associated disease may comprise administering to the individual an effective amount of PGI2-hMSCs, to enhance stem cell engraftment in said individual, thereby ameliorating, deterring and/or preventing the vascular-associated disease.
- the vascular-associated disease may comprise PAD and the PGI2-hMSCs may be administered by local injection (e.g., intramuscular injection) into the ischemic tissue.
- a method of treating an individual having a vascular-associated disease or at risk of developing a vascular-associated disease may comprise administering to the individual an effective amount of PGI2-hMSCs, to enhance stem cell engraftment in said individual, thereby ameliorating, deterring and/or preventing the vascular-associated disease.
- the vascular-associated disease may comprise PAD and the PGI2-hMSCs may be administered by intra-arterial injection.
- a method of treating an individual having a vascular- associated disease or at risk of developing a vascular-associated disease may comprise administering to the individual an effective amount of hMSCs along with an effective amount of a PGI2 precursor, to enhance stem cell engraftment in said individual, thereby ameliorating, deterring and/or preventing the vascular-associated disease.
- the vascular-associated disease may comprise PAD; the hMSCs may be administered by local injection (e.g., intramuscular injection) into the ischemic tissue; and the PGI2 precursor may comprise a PGI2 analogue, such as for example iloprost. In an embodiment, iloprost may be administered by inhalation.
- a method of treating an individual having a vascular- associated disease or at risk of developing a vascular-associated disease may comprise administering to the individual an effective amount of hMSCs along with an effective amount of a PGI2 precursor, to enhance stem cell engraftment in said individual, thereby ameliorating, deterring and/or preventing the vascular-associated disease.
- the vascular-associated disease may comprise diabetes; the hMSCs may be administered by local injection (e.g., intramuscular injection) into ischemic limb tissue; and the PGI2 precursor may comprise a PGI2 analogue, such as for example iloprost. In an embodiment, iloprost may be administered by inhalation.
- the method of treating an individual having a disease or at risk of developing a disease, wherein the disease can be a vascular-associated disease and/or a muscular disease, as disclosed herein advantageously displays improvements in one or more outcomes when compared to a treatment method with an otherwise similar composition lacking the SEE.
- stem cell engraftment in an individual treated with a CSCE may be increased when compared to stem cell engraftment in an individual treated with an otherwise similar composition lacking the SEE.
- stem cell engraftment in an individual treated with a CSCE may be increased when compared to stem cell engraftment in an individual treated with an otherwise similar composition lacking the PGI2 and/or the PGI2 precursor.
- PGI2-SCs e.g., PGI2-hMSCs
- PGI2-hMSCs may advantageously display an enhanced ability to promote angiogenesis when compared to otherwise similar stem cells that lack the ability to overexpress PGI2.
- the method of treating an individual having a disease or at risk of developing a disease, wherein the disease can be a vascular-associated disease and/or a muscular disease, as disclosed herein may have several advantages over current standard PGI2 therapies.
- PGI2-SCs e.g., PGI2-hMSCs
- the treatment method may advantageously deliver PGI2 directly to ischemic tissue.
- PGI2-SCs e.g., PGI2-hMSCs
- the treatment method may advantageously and consistently provide a high level of PGI2 to ischemic tissues.
- the treatment method when PGI2-SCs (e.g., PGI2-hMSCs) are used as a vehicle for PGI2 delivery, the treatment method may advantageously enhance the capability of stem cells (e.g., hMSCs) to repair the damaged tissue.
- stem cells e.g., hMSCs
- the treatment method as disclosed herein may advantageously and effectively alleviate tissue ischemia and improve functional recovery.
- the PGI2-SCs may advantageously provide a way to specifically increase the biosynthesis of the vascular protector PGI2 in ischemic tissue, and as such is believed to be an important development in pharmacology.
- PGI2-SCs may advantageously allow for direct in vivo synthesis of the potent vascular protector, PGI2, from AA with a high efficiency, which may be used to prevent and rescue patients from vascular-associated diseases (e.g., PAD, peripheral vascular disease, thrombosis, ischemia, CLI, heart attack, acute myocardial infarction, congestive heart failure, pulmonary arterial hypertension, acute lung injury, stroke, inflammation in an organ or vessel of a vascular system, chronic kidney disease, leukemia, bone marrow transplant, metabolic diseases, diabetes, etc.) and/or muscular diseases through specifically increasing PGI2 production in target areas, such as for example in ischemic tissue.
- vascular-associated diseases e.g., PAD, peripheral vascular disease, thrombosis, ischemia, CLI, heart attack, acute myocardial infarction, congestive heart failure, pulmonary arterial hypertension, acute lung injury, stroke, inflammation in an organ or vessel of a vascular system, chronic kidney disease, leukemia
- the method of treating an individual having a disease or at risk of developing a disease, wherein the disease can be a vascular-associated disease and/or a muscular disease, with a CSCE (e.g., PGI2-SCs, PGI2-hMSCs, etc.) as disclosed herein may advantageously allow for improved recovery (e.g., tissue healing, tissue repair, etc.) of such individual when compared to recovery of an individual treated with an otherwise similar composition lacking the SEE, and for maintained the improved recovery after stopping therapy with the CSCE.
- a CSCE e.g., PGI2-SCs, PGI2-hMSCs, etc.
- the method of treating an individual having a disease or at risk of developing a disease wherein the disease can be a vascular-associated disease and/or a muscular disease, with a CSCE (e.g., PGI2-SCs, PGI2-hMSCs, etc.) as disclosed herein may advantageously confer pro-survival benefits to host cells (e.g., proliferating myogenic progenitor cells).
- the method can comprise up-regulating the lnc-RNA HI 9 in host cell environment (e.g., host cell stimulation).
- the method of treating an individual having a disease or at risk of developing a disease, wherein the disease can be a vascular-associated disease and/or a muscular disease, with a CSCE (e.g., PGI2-SCs, PGI2-hMSCs, etc.) as disclosed herein may advantageously retain benefits/effects of stem cell transplantation even when not all the transplanted stem cells are retained at a stem cell transplantation location.
- the individual may advantageously display an improved ability to exercise even when not all the transplanted stem cells are retained at a stem cell transplantation location. Additional advantages of the CSCE and treatment methods of using same may be apparent to one of skill in the art viewing this disclosure.
- hMSCs containing pcDNA 3.1 were referred to as 3.1 -hMSCs and those containing pCOX-l-lOaa- PGIS were referred to as PGI 2 -hMSCs.
- Genomic PCR was isolated and purified from native hMSCs, 3.1 -hMSCs, and PGL-hMSCs according to the manufacturer's protocol (DNeasy Blood and Tissue Kit, QIAGEN, Germantown, MD). PCR was performed using total DNA (200ng/sample), COX-l-lOaa-PGIS specific primers, and platinum Taq DNA polymerase (Invitrogen). Cell lysates prepared from hMSCs, 3.1 -hMSCs, and PGL-hMSCs were used to assess the expression of fusion protein (COX-l-lOaa-PGIS) by Western blot.
- COX-l-lOaa-PGIS fusion protein
- the absorbance was read using a microplate reader (Safire II, Tecan, Triangle Park, NC), and the concentration (pg/ml) of 6-keto prostaglandin Fla was calculated for each sample by using XFluor4 Safire II, V4.62n software.
- Lentiviral transduction of hMSCs Lentiviral transduction was performed on hMSCs and quantitative flow cytometry was used to assess the transduction efficiency. Because the lentiviral particles contain triple reporter genes, including herpes virus lthymidine kinase (HSVl-tk), mCherry fluorophore, and firefly luciferase, transduced cells were tracked by using multiple types of imaging modalities.
- HSVl-tk herpes virus lthymidine kinase
- mCherry fluorophore mCherry fluorophore
- firefly luciferase firefly luciferase
- the belt was set at a slow speed (6 meters/min), and the treadmill velocity was increased 2 meters every 2 minutes for the initial 12 minutes and held constant (18 m/min) thereafter. Exhaustion was defined as the point when mice spent more than 10 consecutive seconds on the shock grid without trying to reengage the treadmill. Maximal running time and distances were recorded.
- the sections were then incubated with corresponding secondary antibodies: Alexa Fluor- 647 donkey or anti-rabbit IgG, Alexa Fluor-488 goat anti-rabbit IgG, or Alexa Fluor-488 donkey anti-rat IgG (all from Invitrogen). Nuclei were counterstained with DAPI. A confocal laser scanning microscope (Leica TCS SP5II, Buffalo Grove, IL) was used to obtain fluorescence images of stained sections. Image processing and quantitative analysis were performed by using the ImageJ software (http://imagei.nih.gov/ii/). To quantify Ki67 + Sca-1 + and Ki67 + Sca-1 " cells, a total 10 high power fields were analyzed.
- GM DMEM medium
- FBS ATCC
- penicillin-streptomycin Lonza
- Transwell inserts (0.4 ⁇ pore size; BD Biosciences; San Jose, CA) containing either 3.1- hMSCs or PGI 2 -hMSCs (5* 10 4 cells/well in C2C12 coculture or 4x l0 4 cells/well in primary myoblast coculture) were placed into each well.
- the cells were cocultured in GM in a hypoxic incubator (1.5% (3 ⁇ 4, New Brunswick Galaxy 14 S, Eppendorf, Enfield, CT) for 24 or 48 hours.
- C2C12 cells were grown in the absence of any treatment or in the presence of iloprost (100 nM). the inserts were removed and the myoblasts were processed for viability assays, RT-qPCR, or other analyses.
- Cell viability assay Trypan blue exclusion assay was used to obtain counts of viable and nonviable myoblasts. The assay was performed according to the online protocol provided by Life Technologies (http://www.lifetechnologies.com/us/en/home/references/gibco-cell- culture-basics/cell-culture-protocols/trypan-blue-exclusion.html). All experiments were performed in quadruplicate in 3 independent experiments.
- RNA isolation RNase Plus Micro Kit, QIAGEN.
- Total RNA ⁇ g was reverse transcribed by using high capacity RNA-to-cDNA kit (Invitrogen) and T100 thermal cycler (Bio-rad, Hercules, CA).
- qPCR was performed by using TaqMan Gene Expression Master Mix (Invitrogen) and 7900HT Fast Real-Time PCR System (Life Technologies, Grand Island, NY). HI 9 specific primers/probes and 18S rRNA endogenous control (VIC/MGB Probe) were purchased from Life Technologies.
- the relative expression of RNA was calculated using RQ Manager 1.2.1 (the AACt method). All experiments were performed in triplicate in 3 independent experiments.
- RNAiMAX transfection reagent purchased from Invitrogen.
- HI 9 siRNA was transfected into myoblasts according to the manufacturer's procedures.
- Negative control siRNA was transfected in parallel.
- Mouse C2C12 myoblasts (2x l0 4 /well) and primary myoblasts (4x l0 4 cells) were seeded and cultured with GM in a 5% CC-2 incubator at 37°C for 24 hours (day 1) prior to transfection.
- H19 siRNA (n253569,100 pmol/siRNA) was transfected into myoblasts at day 2 and then H19 siRNAs (n253569 and n253570, 100 pmol/per siRNA) at day 3 to ensure sufficient knock down.
- the cells were transferred to a hypoxic incubator (1.5% 02) for an additional 42 hours before being harvested for analyses. All experiments were performed in triplicate in 3 independent experiments.
- EXFO X-cite 120PC Mercury lamp
- FIG. 2A Blood perfusion was measured before ligation of femoral artery, 24 hours after drug treatment, and up to 14 days thereafter, and the results are shown in Figures 2A and 2B, wherein "NI” denotes “nonischemic legs;” “Isch” denotes “ischemic legs;” and “Pre-S” denotes "before surgery.”
- Figure 2A displays representative laser Doppler images that illustrate perfusion of ischemic (left) legs versus nonischemic contralateral limbs.
- Figure 2B displays a quantitative analysis of ischemic hindlimb perfusion recovery following CPGI2 treatment.
- FIG. 3 More intra-arteriolar connections (solid line arrows, as shown in Figure 3) and corkscrew extensions of arterioles (dashed line arrows, as shown in Figure 3) developed in CPGI2- treated versus saline-treated groups.
- Arteriolar networks were identified by their branching out from a large feeder femoral artery or from a saphenous branch of descending genicular artery. Quantitative confocal analysis indicated that a number of microvessels ranging in size from 15-50 ⁇ in diameter was significantly higher in the CPGI2 group than in the vehicle group (38.00 ⁇ 2.41/high-power field [HPF] versus 18.69 ⁇ 2.12/HPF; O.01).
- CPGI2 a prostacyclin analogue
- ischemic hindlimbs were investigated. More specifically, remodeling of microvascular network in ischemic hindlimbs was monitored in conjunction with CPGI2 treatment. High-definition, volumetric, quantitative micro-CT was used to assess the overall microvascular geometry of ischemic and contralateral non-ischemic legs 14 days after femoral occlusion and constant local administration of CPGI2 or saline. Supporting the perfusion data in Example 1, it was found that a vascular volume of CPGI2 -treated legs was significantly higher than that of saline-treated legs (41.28 ⁇ 2.22 versus 27.11 ⁇ 2.85 mm 3 ; O.05).
- CPGI2- treated legs had significantly more blood vessels (0.16 ⁇ 0.014 versus 0.09 ⁇ 0.011 1/mm; ⁇ 0.05) and less distance between vessels (6.60 ⁇ 0.52 versus 10.15 ⁇ 1.14 mm; ⁇ 0.05) than did saline-treated legs.
- CPGI2-treated ischemic legs showed a significant increase in small vessels, with vessel diameter bins ranging from 40-60 ⁇ ( ⁇ 0.05; as shown in Figure 4A).
- Figure 4A displays a histogram of mean blood vessel size distribution showing a marked increase in arterioles between 40-60 ⁇ in the CPGI2-treated group.
- FIG. 4B displays representative micro-CT images of the microvascular network in CPGI2 -treated and saline-treated ischemic legs. Referring to Figure 4B, the red dashed circles show more vasculature in the thigh muscle where CPGI2 was administered as compared to the similar area where saline was administered. Contralateral non-ischemic legs were similarly evaluated and no significant differences in vessel distribution were found between the CPGI2 -treated and saline-treated groups.
- CPGI2 improves perfusion in ischemic legs.
- CPGI2 treatment positively affects remodeling of the microvascular network in ischemic hindlimbs.
- Quantitative micro-CT analyses indicate CPGI2 augments vascular growth.
- a triple catalytic enzyme e.g., COX-l-lOaa-PGIS that links COX-1 to PGIS and catalyzes 3 key reactions for the efficient production of PGI2 from arachidonic acid (AA, as illustrated in Figure 1) was created, and the procedure is described in more detail in U.S. Publication No. 20100015120 Al.
- the effective and stable biosynthesis of PGI2 requires an increase in the expression of COX-1 or COX-2 in conjunction with PGIS.
- COX-1 -lOaa- PGIS was introduced into hMSCs via nucleofection, to produce PGI2 -hMSCs.
- FIG. 5A displays western blots showing the overexpression of COX-l-lOaa- PGIS fusion protein (130 kD) in PGI2 -hMSCs and endogenous COX-1 protein levels in hMSCs.
- CM from PGI2- hMSCs markedly stimulated endothelial cell tube formation, as seen in Figure 5C, as compared to endothelial cell tube formation in CM from 3.1-hMSCs as seen in Figure 5D, indicating that the paracrine effects of PGI2 release included protective vascular activities.
- Figure 5C displays a representative image of endothelial cell tubes incubated with CM from PGI2-hMSC
- Figure 5D displays a representative image of endothelial cell tubes incubated with CM from 3.1-hMSC.
- the injected cells ubiquitously expressed human herpes simplex virus type 1 -thymidine kinase, mCherry fluorophore protein, and firefly luciferase reporter genes driven by the human ubiquitin promoter, as shown in Figures 6A-6D.
- Figure 6A displays a diagrammatic representation of the lentiviral vector encoding herpes virus thymidine kinase (HSVl-tk), mCherry fluorophore, and firefly luciferase genes;
- Figure 6B displays representative in vitro bioluminescent imaging (BLI) images of hMSCs transduced with the lentiviral vector, wherein cells were consecutively diluted in a 6-well plate; it displays the positive relationship between bioluminescent intensity and cell numbers.
- HSVl-tk herpes virus thymidine kinase
- mCherry fluorophore mCherry fluorophore
- Figure 6C displays a representative photomicrograph and its corresponding fluorescence image showing the expression of red mCherry fluorescent protein in transduced hMSCs; and Figure 6D displays a flow cytometry analysis graph confirming high efficiency of lentiviral transduction in hMSCs.
- hMSCs were efficiently transduced with a lentiviral vector containing triple fusion reporters (>99%). Direct correlation between the numbers of hMSCs and luciferase activity was confirmed, as shown in Figures 6A-6D.
- Luciferase catalyzes light-emitting photochemical reactions of luciferin in live cells, allowing for whole-body imaging to track the distribution and engraftment of transplanted cells.
- In vivo BLI was performed using the Xenogen IVIS 200 system (Xenogen, Alameda, CA).
- Xenogen Xenogen, Alameda, CA.
- D- luciferin 150 mg/kg
- the mice were scanned at 1, 3, 5, 7 and 14 day post- injection of 3.1-hMSCs or PGI2-hMSCs. Imaging signals were quantified in units of maximum photons/s/cm 2 /steridian (photons/ s/ cm 2 / sr).
- FIG. 7A displays representative in vivo BLI images of NOD-SCID mice 3 days after PGI2-hMSCs or 3.1-hMSCs were injected into the gastrocnemius muscle of the ischemic hindlimb.
- Figure 8A displays in vivo BLI images of NOD-SCID mice over a 14 day time period after PGI2 -hMSCs or 3.1-hMSCs were injected into the gastrocnemius muscle of the ischemic hindlimb.
- the BLI signal was the strongest at 3 days after the cell injections, and it started to decay after day 3.
- Figure 8B displays a quantitative analysis of the BLI images in Figure 8A.
- hMSCs were preconditioned with either prostacyclin receptor agonist Cicaprost ( ⁇ ) or PPAR /5 agonist GW501516 (100 nM) for 4 days in vitro. Cells were then injected into the gastrocnemius of ischemic hindlimbs of NOD/SCID mice.
- IP intra peritoneal
- In vivo BLI was performed on cell-drug treated mice. The mice were scanned at 1, 3, and 5 day post- injection of cells and drug treatment, and the data is shown in Figure 9A.
- Figure 9B displays a quantitative analysis of the BLI images in Figure 9A.
- Local PGI2-hMSCs treatment did not significantly alter mouse blood pressure parameters.
- the mice that were treated with PGI2-hMSC performed overall better at both 21 days and 28 days than the mice that were treated with 3.1-hMSC.
- Figure 13 displays confocal images indicating that endogenous proliferating (Ki67 + ) cells were rarely detected in tissue that was located more than 250 ⁇ away from the hMSC injection site at day 3 in both 3.1-hMSC and PGI 2 -hMSC- treated mice.
- Ki67 + cells expressed stem cell antigen-1 (Sca-1), a common marker on stem/progenitor cells.
- Sca-1 stem cell antigen-1
- Figure 14A displays representative confocal images illustrating the distribution of endogenous Ki67 Sca- 1 + and Ki67 + Sca-l " cells.
- Figure 15B indicates that the number of viable C2C12 myoblasts was not different after 24 hours of coculture with PGI 2 -hMSCs or 3.1-hMSCs, but PGI 2 -hMSC coculture significantly reduced nonviable cells compared with 3.1-hMSC coculture at 24 hours ( Figure 15C).
- H19 silencing caused a small, albeit significant, reduction in the number of viable cells at the end of 3 days of siRNA treatment (7.72 ⁇ 0.07 vs 6.67 ⁇ 0.10xl0 4 cells in negative control siRNA-transfected vs HI 9 siRNA-transfected myoblasts; ⁇ 0.01 ; Figure 15H).
- Figure 15H indicates that HI 9 silencing significantly reduced the numbers of viable cells.
- a concurrent increase of myoblast death was also detected after H19 siRNA treatment (4.12 ⁇ 0.27 vs 6.70 ⁇ 0.45xl0 3 cells, negative control siRNA vs H19 siRNA-transfected cells; O.01 ; Figure 151).
- Figure 151 indicates that HI 9 silencing increased cell death.
- siRNA approach described in Example 10 was used to evaluate the survival or growth benefit of H19 in primary myoblasts.
- Downregulation of HI 9 RNA (40%, ⁇ 0.02) caused a 23% reduction of viable cells (4.89 ⁇ 0.16xl0 4 vs 3.97 ⁇ 0.09xl0 4 cells; negative control siRNA vs HI 9 siRNA-transfected myoblasts; O.01 ; Figure 16H) and a 34%> increase of nonviable cells (3.85 ⁇ 0.32xl0 3 vs 5.87 ⁇ 0.27xl0 3 cells; O.01 ; Figure 161).
- H19 silencing significantly reduced the number of viable cells (Figure 16H) and increased cell death (Figure 161).
- RNA-FISH fluorescence in situ hybridization
- RNA-FISH studies showed an increase of H19 RNA levels in the cytoplasm and within the nucleus of endogenous cells surrounding PGI2-hMSC injection sites as compared with 3.1-hMSC injection sites at 3 days after cell administration (Figure 16G). These results demonstrate endogenous HI 9 RNA upregulation induced by PG ⁇ -hMSCs. Figure 16G indicated that HI 9 silencing by siRNA significantly reduced H19 levels in primary myoblasts.
- Figure 16J displays representative images of HI 9 RNA fluorescence in situ hybridization in gastrocnemius muscle sections at 3 days after 3.1-hMSC or PG 2 -I1MSC injections.
- a fluorescein-tagged H19 FISH probe that specifically targets endogenous H19 RNA was used, resulting in intense intracellular green fluorescent particles.
- a higher expression of host HI 9 RNA was found in PGI 2 -hMSC-treated muscles than in 3.1-hMSC- treated muscles. All sections were counterstained with DAPI to localize nuclei. *P ⁇ 0.05; ** P ⁇ 0.01.
- a first embodiment which is an effective amount of a composition comprising a stem cell, a stem cell engraftment enhancer, and a carrier fluid, for use in the treatment of an individual having a disease or at risk of developing a disease, wherein the disease is a vascular- associated disease and/or a muscular disease.
- a second embodiment which is an effective amount of a composition comprising a stem cell, a stem cell engraftment enhancer, and a carrier fluid, for use in the treatment of an individual having a vascular-associated disease or at risk of developing a vascular-associated disease.
- a third embodiment which is an effective amount of a composition comprising a stem cell, a stem cell engraftment enhancer, and a carrier fluid, for use in the treatment of an individual having a muscular disease or at risk of developing a muscular disease.
- a fourth embodiment which is the composition of any of the first and the second embodiments wherein the vascular-associated disease comprises peripheral arterial disease, peripheral vascular disease, thrombosis, ischemia, critical limb ischemia, heart attack, acute myocardial infarction, congestive heart failure, pulmonary arterial hypertension, acute lung injury, stroke, inflammation in an organ or vessel of a vascular system, chronic kidney disease, leukemia, bone marrow transplant, metabolic diseases, diabetes, or combinations thereof.
- the vascular-associated disease comprises peripheral arterial disease, peripheral vascular disease, thrombosis, ischemia, critical limb ischemia, heart attack, acute myocardial infarction, congestive heart failure, pulmonary arterial hypertension, acute lung injury, stroke, inflammation in an organ or vessel of a vascular system, chronic kidney disease, leukemia, bone marrow transplant, metabolic diseases, diabetes, or combinations thereof.
- a fifth embodiment which is the composition of any of the first through the fourth embodiments wherein the stem cell comprises human mesenchymal stem cells (hMSCs), endothelial progenitor cells (EPCs), hematopoietic stem cells (HSCs), cardiac progenitor cells (CPCs), satellite cells, or combinations thereof.
- hMSCs human mesenchymal stem cells
- EPCs endothelial progenitor cells
- HSCs hematopoietic stem cells
- CPCs cardiac progenitor cells
- satellite cells or combinations thereof.
- a sixth embodiment which is the composition of any of the first through the fifth embodiments wherein the stem cell overexpresses prostacyclin (PGI2).
- a seventh embodiment which is the composition of any of the first through the sixth embodiments wherein the stem cell engraftment enhancer comprises PGI2, a PGI2 precursor, a peroxisome proliferator-activated receptor ⁇ / ⁇ isoform (PPAR8) agonist, a cAMP inducer, a phosphodiesterase inhibitor, an endothelin receptor antagonist, a nitrous oxide modulating agent, a prostacyclin receptor (IP) agonist, a non-prostanoid IP receptor agonist, or combinations thereof.
- PGI2 precursor a peroxisome proliferator-activated receptor ⁇ / ⁇ isoform (PPAR8) agonist
- a cAMP inducer a phosphodiesterase inhibitor
- an endothelin receptor antagonist an endothelin receptor antagonist
- a nitrous oxide modulating agent a prostacyclin receptor (IP) agonist
- IP prostacyclin receptor
- An eighth embodiment which is the composition of the seventh embodiment wherein the PGI2 precursor comprises a triple catalytic enzyme, a PGI2-overexpressing stem cell (PGI2-SC), a DNA sequence encoding for the triple catalytic enzyme, a cDNA sequence encoding for the triple catalytic enzyme, a host cell containing an expressible DNA sequence encoding for the triple catalytic enzyme, a vector comprising a DNA sequence encoding for the triple catalytic enzyme, a plasmid comprising a DNA sequence encoding for the triple catalytic enzyme, a fusion gene encoding for the triple catalytic enzyme, a synthetic PGI2 analogue, or combinations thereof.
- PGI2 precursor comprises a triple catalytic enzyme, a PGI2-overexpressing stem cell (PGI2-SC), a DNA sequence encoding for the triple catalytic enzyme, a cDNA sequence encoding for the triple catalytic enzyme, a host cell containing an expressible DNA sequence encoding for the triple catalytic enzyme,
- a ninth embodiment which is the composition of the eighth embodiment wherein the synthetic PGI2 analogue is selected from the group consisting of Iloprost, Carbaprostacyclin, Treprostinil, Cicaprost, Beraprost, and Epoprostenol.
- a tenth embodiment which is the composition of the eighth embodiment wherein the triple catalytic enzyme is characterized by a formula COX-linker-ES, wherein COX comprises a cyclooxygenase (COX) amino acid sequence; ES comprises an eicosanoid- synthesizing (ES) enzyme amino acid sequence; and the linker comprises from about 10 to about 22 amino acid residues of a transmembrane sequence; wherein the linker is disposed between the COX and the ES, and wherein the linker directly connects the COX to the ES.
- COX comprises a cyclooxygenase (COX) amino acid sequence
- ES comprises an eicosanoid- synthesizing (ES) enzyme amino acid sequence
- the linker comprises from about 10 to about 22 amino acid residues of a transmembrane sequence; wherein the linker is disposed between the COX and the ES, and wherein the linker directly connects the COX to the ES.
- An eleventh embodiment which is the composition of the any of the eighth through tenth embodiments wherein the triple catalytic enzyme is characterized by a formula COX-1- lOaa-PGIS; wherein COX-1 is cyclooxygenase isoform-1; the linker comprises a 10 amino acid (lOaa) transmembrane sequence; and PGIS is prostacyclin synthase.
- a twelfth embodiment which is the composition of any of the first through the eleventh embodiments further comprising a PGI2-overexpressing human mesenchymal stem cell (PGI2-hMSC).
- PGI2-hMSC PGI2-overexpressing human mesenchymal stem cell
- a thirteenth embodiment which is the composition of any of the first through the twelfth embodiments administered via an intramuscular injection.
- a fourteenth embodiment which is the composition of any of the first through the thirteenth embodiments having PGI2-SCs and a carrier fluid wherein the composition is administered via a single treatment stream.
- a fifteenth embodiment which is the composition of any of the first through fourteenth embodiments comprising stem cells, a PPAR8 agonist, and a carrier fluid wherein the composition is administered via a single treatment stream.
- a sixteenth embodiment which is the composition of any of the first through the thirteenth embodiments administered via multiple treatment streams comprising:
- stem cell treatment stream comprises stem cells and a carrier fluid
- stem cell engraftment enhancer treatment stream comprises a stem cell engraftment enhancer and a carrier fluid
- a seventeenth embodiment which is the composition of the sixteenth embodiment wherein the stem cell treatment stream comprises hMSCs and wherein the stem cell engraftment enhancer treatment stream comprises PGI2, a PGI2 precursor, or both.
- An eighteenth embodiment which is the composition of any of the first through the seventeenth embodiments wherein a stem cell engraftment in an individual treated with the composition is enhanced by greater than about 200%, when compared to stem cell engraftment in an individual treated with a composition lacking the stem cell engraftment enhancer.
- a nineteenth embodiment which is the composition of any of the first through the eighteenth embodiments wherein the composition up-regulates a long non-coding RNA HI 9 in a host environment.
- a twentieth embodiment which is the composition of the nineteenth embodiment wherein up-regulating the long non-coding RNA H19 in the host environment promotes host cell growth.
- a twenty-first embodiment which is a composition comprising PGI2- overexpressing human mesenchymal stem cells (PGI2 -hMSCs), and a carrier fluid; wherein an effective amount of the composition is administered via a single treatment stream as an intramuscular injection to an individual having a disease or at risk of developing a disease, wherein the disease is a vascular-associated disease and/or a muscular disease, and wherein stem cell engraftment is enhanced in said individual by greater than about 200%, when compared to stem cell engraftment in an individual treated with a composition lacking the stem cell engraftment enhancer.
- PGI2- overexpressing human mesenchymal stem cells PGI2- overexpressing human mesenchymal stem cells (PGI2 -hMSCs)
- a carrier fluid wherein an effective amount of the composition is administered via a single treatment stream as an intramuscular injection to an individual having a disease or at risk of developing a disease, wherein the disease is a vascular-associated disease and/or a muscular
- a twenty-second embodiment which is a composition comprising PGI2- overexpressing human mesenchymal stem cells (PGI2 -hMSCs), and a carrier fluid; wherein an effective amount of the composition is administered via a single treatment stream as an intramuscular injection to an individual having a vascular-associated disease or at risk of developing a vascular-associated disease, and wherein stem cell engraftment is enhanced in said individual by greater than about 200%, when compared to stem cell engraftment in an individual treated with a composition lacking the stem cell engraftment enhancer.
- PGI2- overexpressing human mesenchymal stem cells PGI2- overexpressing human mesenchymal stem cells (PGI2 -hMSCs)
- a carrier fluid wherein an effective amount of the composition is administered via a single treatment stream as an intramuscular injection to an individual having a vascular-associated disease or at risk of developing a vascular-associated disease, and wherein stem cell engraftment is enhanced in said individual by greater than about 200%,
- a twenty-third embodiment which is a composition comprising PGI2- overexpressing human mesenchymal stem cells (PGI2-hMSCs), and a carrier fluid; wherein an effective amount of the composition is administered via a single treatment stream as an intramuscular injection to an individual having a muscular disease or at risk of developing a muscular disease, and wherein stem cell engraftment is enhanced in said individual by greater than about 200%, when compared to stem cell engraftment in an individual treated with a composition lacking the stem cell engraftment enhancer.
- PGI2- overexpressing human mesenchymal stem cells PGI2- overexpressing human mesenchymal stem cells (PGI2-hMSCs)
- a carrier fluid wherein an effective amount of the composition is administered via a single treatment stream as an intramuscular injection to an individual having a muscular disease or at risk of developing a muscular disease, and wherein stem cell engraftment is enhanced in said individual by greater than about 200%, when compared to stem cell engraftment in an individual treated with
- a twenty-fourth embodiment which is a composition comprising human mesenchymal stem cells (hMSCs), Iloprost, and a carrier fluid; wherein the composition is administered to an individual having a disease or at risk of developing a disease, wherein the disease is a vascular-associated disease and/or a muscular disease, and wherein the composition is administered via multiple treatment streams comprising: a stem cell treatment stream, and a stem cell engraftment enhancer treatment stream; wherein the stem cell treatment stream comprises hMSCs and is administered via an intramuscular injection; and wherein the stem cell engraftment enhancer treatment stream comprises Iloprost and is administered via inhalation.
- hMSCs human mesenchymal stem cells
- Iloprost vascular-associated disease and/or a muscular disease
- a twenty-fifth embodiment which is a composition comprising human mesenchymal stem cells (hMSCs), Iloprost, and a carrier fluid; wherein the composition is administered to an individual having a vascular-associated disease or at risk of developing a vascular-associated disease via multiple treatment streams comprising: a stem cell treatment stream, and a stem cell engraftment enhancer treatment stream; wherein the stem cell treatment stream comprises hMSCs and is administered via an intramuscular injection; and wherein the stem cell engraftment enhancer treatment stream comprises Iloprost and is administered via inhalation.
- hMSCs human mesenchymal stem cells
- Iloprost vascular-associated disease
- carrier fluid comprising: a stem cell treatment stream, and a stem cell engraftment enhancer treatment stream; wherein the stem cell treatment stream comprises hMSCs and is administered via an intramuscular injection; and wherein the stem cell engraftment enhancer treatment stream comprises Iloprost and is administered via inhalation.
- a twenty-sixth embodiment which is a composition comprising human mesenchymal stem cells (hMSCs), Iloprost, and a carrier fluid; wherein the composition is administered to an individual having a muscular disease or at risk of developing a muscular disease via multiple treatment streams comprising: a stem cell treatment stream, and a stem cell engraftment enhancer treatment stream; wherein the stem cell treatment stream comprises hMSCs and is administered via an intramuscular injection; and wherein the stem cell engraftment enhancer treatment stream comprises Iloprost and is administered via inhalation.
- hMSCs human mesenchymal stem cells
- Iloprost a carrier fluid
- the composition is administered to an individual having a muscular disease or at risk of developing a muscular disease via multiple treatment streams comprising: a stem cell treatment stream, and a stem cell engraftment enhancer treatment stream; wherein the stem cell treatment stream comprises hMSCs and is administered via an intramuscular injection; and wherein the stem cell engraftment enhance
- a twenty-seventh embodiment which is a composition for stem cell engraftment, wherein the composition for stem cell engraftment comprises a stem cell, wherein the stem cell comprises: human mesenchymal stem cells (hMSCs), endothelial progenitor cells (EPCs), hematopoietic stem cells (HSCs), cardiac progenitor cells (CPCs), satellite cells, or combinations thereof; a stem cell engraftment enhancer, wherein the stem cell engraftment enhancer comprises: prostacyclin (PGI2), a PGI2 precursor, a peroxisome proliferator- activated receptor ⁇ / ⁇ isoform (PPAR8) agonist, a cAMP inducer, a phosphodiesterase inhibitor, an endothelin receptor antagonist, a nitrous oxide modulating agent, a prostacyclin receptor (IP) agonist, a non-prostanoid IP receptor agonist, or combinations thereof; and a carrier fluid.
- PGI2
- a twenty-eighth embodiment which is the composition of the twenty-seventh embodiment wherein the stem cells overexpress PGI2.
- R R u +k* (R u -Ri), wherein k is a variable ranging from 1 percent to 100 percent with a 1 percent increment, i.e., k is 1 percent, 2 percent, 3 percent, 4 percent, 5 percent, 50 percent, 51 percent, 52 percent, , 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or 100 percent.
- any numerical range defined by two R numbers as defined in the above is also specifically disclosed.
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CA2946747A CA2946747A1 (en) | 2014-04-23 | 2015-04-22 | Methods of enhancing stem cell engraftment |
AU2015249751A AU2015249751A1 (en) | 2014-04-23 | 2015-04-22 | Methods of enhancing stem cell engraftment |
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CN112494651A (en) * | 2020-09-30 | 2021-03-16 | 吉林大学 | Application of lncRNA H19 as molecular target in atherosclerosis treatment |
EP4015623A1 (en) | 2020-12-17 | 2022-06-22 | Universite De Montpellier | Pre-treatment of msc with ppar beta/delta agonist for treatment of ischemia-reperfusion injury |
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US20130216507A1 (en) * | 2006-03-24 | 2013-08-22 | The General Hospital Corporation | Methods for promoting hsc engraftment |
US20130344038A1 (en) * | 2011-01-13 | 2013-12-26 | Michael Freissmuth | Method for enhancing engraftment of haematopoetic stem cells |
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US20130344038A1 (en) * | 2011-01-13 | 2013-12-26 | Michael Freissmuth | Method for enhancing engraftment of haematopoetic stem cells |
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Cited By (3)
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CN112494651A (en) * | 2020-09-30 | 2021-03-16 | 吉林大学 | Application of lncRNA H19 as molecular target in atherosclerosis treatment |
EP4015623A1 (en) | 2020-12-17 | 2022-06-22 | Universite De Montpellier | Pre-treatment of msc with ppar beta/delta agonist for treatment of ischemia-reperfusion injury |
WO2022129468A1 (en) | 2020-12-17 | 2022-06-23 | Universite De Montpellier | Pre-treatment of msc with pparβ/δ agonist for treatment of ischemia-reperfusion injury |
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