WO2016154389A1 - Compositions and use of isl-1+c-kit+cells for cardiovascular repair - Google Patents

Compositions and use of isl-1+c-kit+cells for cardiovascular repair Download PDF

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WO2016154389A1
WO2016154389A1 PCT/US2016/023919 US2016023919W WO2016154389A1 WO 2016154389 A1 WO2016154389 A1 WO 2016154389A1 US 2016023919 W US2016023919 W US 2016023919W WO 2016154389 A1 WO2016154389 A1 WO 2016154389A1
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isl
kit
progenitor cells
cells
cpcs
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French (fr)
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Mary Kearns-Jonker
Nahidh HASANIYA
Leonard Bailey
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Loma Linda University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body

Definitions

  • the present disclosure relates to compositions and use of a cloned population of isl-l+c-kit+ progenitor cells for cardiovascular repair and regeneration in treating cardiac tissue damage.
  • a method for treating a patient having damage within a region of cardiac tissue comprising transplanting into the region or into cardiac tissue surrounding the region, a clonal population of isl-l+c-kit+ cardiac progenitor cells.
  • the clonal population of isl-l+c-kit+ progenitor cells is preconditioned under hypoxic conditions prior to transplantation.
  • a composition comprising a cell type for cell therapy treatment for treating cardiac damage.
  • the composition comprises: clonal population of isl-l+c-kit+ progenitor cells configured to differentiate into one or more cardiovascular lineages.
  • the isl-l+c-kit+ progenitor cells comprise autologous or allogeneic cells.
  • a method for providing a therapeutic cell matrix for use in regenerative cardiovascular medicine comprises obtaining a cloned population of isl-l+c-kit+ progenitor cells; and culturing the isl-l+c-kit+ progenitor cells on a support structure which may be removed either prior to or after implantation of the cells into the patient.
  • the support structure is an implantable biodegradable matrix configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the matrix into an infarct zone, wherein the isl-l+c-kit+ progenitor cells are capable of regenerating damaged heart tissue.
  • a method is disclosed for providing a therapeutic cell suspension for use in regenerative cardiovascular medicine.
  • the method for providing a therapeutic cell suspension for use in regenerative cardiovascular medicine comprises obtaining a cloned population of isl-l+c-kit+ progenitor cells; and suspending the isl-l+c-kit+ progenitor cells in a pharmaceutically acceptable medium configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the suspension into an infarct zone, wherein the isl-l+c-kit+ progenitor cells are capable of regenerating damaged heart tissue.
  • the cloned population of isl-l+c-kit+ progenitor cells are obtained from the atrial appendage, epicardium, or ventricle of one or more human tissue donors. In some embodiments, the cloned population of isl-l+c-kit+ progenitor cells are configured to differentiate into one or more cardiovascular lineages. In some embodiments, the cloned population of isl-l+c-kit+ progenitor cells comprises autologous or allogeneic cells. In some embodiments, the cloned population of isl-l+c-kit+ progenitor cells is preconditioned under hypoxic conditions.
  • a composition for treating cardiac damage comprises a cloned population of isl-l+c-kit+ progenitor cells; wherein the isl-l+c-kit+ progenitor cells are adapted to promote repair of damaged heart tissue when transplanted into a region of cardiovascular tissue comprising damaged heart tissue.
  • the cloned population of isl-l+c-kit+ progenitor cells are obtained from the atrial appendage, epicardium, or ventricle of one or more human tissue donors.
  • the cloned population of isl-l+c-kit+ progenitor cells are configured to differentiate into one or more cardiovascular lineages.
  • the clonal population of isl-l+c-kit+ progenitor cells comprises autologous or allogeneic cells.
  • the composition further comprises one or more cytokines, growth factors or other agents that promote survival, proliferation and/or differentiation of the isl-l+c-kit+ progenitor cells.
  • the composition further comprises a support structure which may be removed either prior to or after implantation of the cells into the patient.
  • the support structure is an implantable biodegradable matrix associated with the isl-l+c-kit+ progenitor cells, wherein the biodegradable matrix promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c-kit+ progenitor cells.
  • the composition further comprises a biocompatible nutrient medium that promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c- kit+ progenitor cells.
  • a use of a composition for treating damaged cardiac tissue in a patient in need thereof comprises a cloned population of isl-l+c-kit+ progenitor cells.
  • the composition is configured for transplantation into a region of cardiovascular tissue comprising damaged cardiac tissue.
  • the cloned population of isl-l+c- kit+ progenitor cells are obtained from the atrial appendage, epicardium, or ventricle of one or more human tissue donors.
  • the cloned population of isl-l+c-kit+ progenitor cells are configured to differentiate into one or more cardiovascular lineages.
  • the clonal population of isl-l+c-kit+ progenitor cells comprises autologous or allogeneic cells.
  • the composition further comprises one or more cytokines, growth factors or other agents that promote survival, proliferation and/or differentiation of the isl-l+c-kit+ progenitor cells.
  • the composition further comprises a support structure which may be removed either prior to or after implantation of the cells into the patient.
  • the support structure is an implantable biodegradable matrix associated with the isl-l+c-kit+ progenitor cells, wherein the biodegradable matrix promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c-kit+ progenitor cells.
  • the composition further comprises a biocompatible nutrient medium that promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c-kit+ progenitor cells.
  • Figures 1A-B show the isl-l+ckit+ cardiovascular progenitor cells uniquely express markers of early cardiogenic mesoderm, thereby indicating multipotency to differentiate into a variety of cardiovascular derivatives.
  • Figure IB shows the results of flow cytometry used to show expression of markers of early cardiogenic mesoderm.
  • FIG. 2A-B illustrate steps of selecting (FIG. 2A) and isolating (FIG. 2B) cardiac progenitor cells.
  • Figures 3A-C show characterization of the CPCs.
  • Figure 3D shows the results of gel electrophoresis to demonstrate that c-kit+isl-l+ cells express Mesp-1 the early cardiovascular progenitor cell marker, in addition to low levels of Brachyury and PDGF receptor alpha.
  • Figure 4 shows flow cytometry used to identify phenotypes of neonatal and adult progenitor cells.
  • Figure 5 shows histocompatibility antigens on cardiac progenitors.
  • Figure 6 shows a cell cycle analysis comparing neonatal and adult CPCs.
  • Figure 7 shows a higher frequency of adult progenitors are in the Gl phase, while a higher frequency of neonatal progenitors are in the S and G2 phases.
  • Figure 8 illustrates the Transwell invasion assay.
  • Figure 9A shows a higher number of neonatal CPCs invade in response to
  • FIG. 9B shows that SDF-1 receptors CXCR4 and CXCR7 were adequately expressed on both neonatal and adult CPCs.
  • Figure 10 shows activation of the AKT pathway is higher during differentiation of neonatal CPCs.
  • Figure 1 1 shows transcripts SDF- la and IGF-1 are expressed at higher levels in neonatal CPCs.
  • Figures 12A-B illustrates that AKT signaling is increased in neonatal CPCs in response to SDF- la.
  • FIG. 13A-B shows that AKT signaling was increased in neonatal CPCs with IGF- 1 treatment and AKT signaling was unchanged in adult CPCs with IGF- 1 treatment.
  • Figures 14 and 15 identify isl-1 derivatives in the heart.
  • Figure 16 shows that endogenous CPCs are heterogeneous.
  • Figure 17 shows that isl-1 + CPC clones express KDR, PDGFR and CXCR4.
  • Figure 18 illustrates isl-1 +c-kit+ PDGFRa +IGF1R +CPC clones.
  • Figure 19 illustrates the time to transplant, for a representative neonatal clone, from 1 cell to 1x10 cells.
  • Figure 20 shows the distribution of markers for cell types illustrating that both neonatal adult CPCs differentiate into all three cardiac lineages. This data was captured using the dexamethasone protocol.
  • Figure 21 illustrates the differentiation into cardiomyocytes and the timeline of the differentiation procedure.
  • Figure 22 shows the differentiation of progenitor clones.
  • Figures 23A-C illustrates that the CPCs form endothelial cells in a tube formation assay.
  • Figures 24A-B shows that neonatal and adult CPCs form capillary-like networks on Matrigel.
  • Figure 25 shows that 42 significant microRNA expression differences were identified when comparing neonatal and adult cardiac progenitors.
  • Figure 26 shows that microRNAs that promote proliferation are highly expressed in neonatal CPCs.
  • Figure 27A shows that microRNAs that prevent senescence are more highly expressed in adult CPCs.
  • Figure 27B shows that microRNAs associated with senescence are more highly expressed in adult CPCs.
  • Figure 28A shows isl- 1 expression was significantly elevated when cultured ovine isl- l+c-kit+ CPC were cultured on a scaffold.
  • Figure 28B shows the results of cell cycle analysis performed on scaffold cultured CPC where an elevated number of cells are in G2 after scaffold culture.
  • FIGS 28C-H show that cardiac stem cells show elevated expression of isl- 1 after culture on a scaffold and continue to proliferate actively as shown by Ki67 labeling of sections and quantitation using Image Pro software.
  • Figure 29A-F shows representative histograms of TropT-positive and vWF-positive cells, demonstrating that scaffold culturing induces differentiation of cardiac stem cells.
  • Figure 29G-L shows the results of confocal microscopy, which demonstrates that TropT and vWF were induced by differentiation of scaffold-cultured cells.
  • Figure 30 A shows fewer scaffold cultured cardiac stem cells are in the Gl phase as shown by flow cytometry.
  • Figure 30B shows the expression of TropT and vWF was significantly increased in scaffold cultured cardiac stem cells.
  • Figure 31 A shows a significant decrease in ERK phosphorylation as verified by flow cytometry.
  • Figure 3 IB shows elevated AKT phosphorylation shown by flow cytometry.
  • Figure 32A shows a significant increase HGF, and SDF- ⁇ expression, and an increase in IGF-1 expression in scaffold cultured cells.
  • Figure 32B shows the amplified cDNA products of the statistically significant growth factors on an agarose gel.
  • Figure 33 shows microRNAs associated with enhanced capacity to invade are elevated in neonatal CPCs. MicroRNAs that inhibit invasion are more highly expressed in adult CPCs.
  • FIG. 34 shows alterations in CPC after short term hypoxia treatment.
  • Gene expression changes associated with the AKT pathway were induced in neonatal CPC (FIG. 34A) and adult CPC (FIG. 34B).
  • Akt phosphorylation measurements taken by flow cytometry analyzed as a function of age reveals that hypoxia-mediated Akt activation occurs more readily in neonatal hCPCs (FIG. 34C).
  • Elevated levels of phosphorylated AKT after short term hypoxia in adult CPC identified by Western blot are shown in (FIG.
  • FIG. 34D elevated transcripts encoding prosurvival genes after short term hypoxia (FIG. 34E), and elevated phosphorylation of AKT after short term hypoxia are shown by flow cytometry (FIG. 34F).
  • Heat shock proteins 40 and 90 are elevated after short term hypoxia (FIG. 34G), apoptosis (FIG. 34H) and differentiation (FIG. 341) are not induced by short term hypoxia.
  • Figure 35 shows hypoxia-pretreated hCPCs exhibit significantly improved invasion as measured by Transwell invasion assay.
  • Figure 36 shows the progenitor cell distribution in sheep.
  • Figure 37 shows the subpopulations of sheep CPC that include progenitors which are phenotypically comparable to those isolated from humans.
  • Figure 38 shows CPC clone used in cardiovascular stem cell transplantation.
  • Figure 39 shows CFSE labeling of CPC prior to transplantation.
  • Figure 40 shows the heart of a sheep harvested 2 months after CPC injection.
  • Figure 41 shows sectioning of frozen tissues harvested from the sheep heart.
  • Figure 42 shows the procedure for preparation and fluorescent IHC staining of frozen tissue sections.
  • Figure 43 shows the results of gel electrophoresis used to compare gene expression of CXCR4, CXCL12, and IGF1 at non-infarcted and infarcted sites.
  • Figure 44 shows the results of real-time PCR analysis used to compare transcripts of genes associated with paracrine effects after transplantation of isl-l+c-kit cells at infarcted and non-infarcted sites.
  • Figure 45 shows the results of real time PCR used to show transcripts of genes in the Akt pathway were elevated at the site of infarction after transplantation with isl- l+c-kit+ progenitors.
  • Figure 46 shows the results of real time PCR used to show Notch transcripts were elevated at the site of infarction after transplantation with isl-l+c-kit+ progenitors.
  • Figure 47A-E show the results of real time PCR used to show heat shock protein transcripts were elevated at the site of infarction after transplantation with isl-l+c- kit+ progenitors.
  • Figure 48 shows the results of real time PCR used to show pro survival transcripts were elevated at the site of infarction after transplantation with isl-l+c-kit+ progenitors.
  • Figure 49 shows the results of real time PCR used to show transcription factors were elevated at the site of infarction after transplantation with isl-l+c-kit+ progenitors.
  • Figure 50A shows the results of real time PCR used to show superoxide dismutase 2 transcripts were elevated at the site of infarction after transplantation with isl- l+c-kit+ progenitors, and Figure 50B shows the results were verified using gel electrophoresis.
  • Figure 51A shows the results of real time PCR used to show c-Kit transcripts were elevated at the site of infarction after transplantation with isl-l+c-kit+ progenitors
  • Figure 5 IB shows the results were verified using gel electrophoresis.
  • Figure 52A shows the results of real time PCR used to show Connexin-43 transcripts were elevated at the site of infarction after transplantation with isl-l+c-kit+ progenitors
  • Figure 52B shows a Connexin-43 stained image showing connexin staining between the newly introduced cardiovascular cells in the myocardium.
  • Figure 53A-D shows the results of real-time PCR used to identify gene expression changes in the infarct zone of four sheep after transplantation with isl-l+c-kit+ progenitors.
  • Figure 53E shows the pooled real-time PCR data.
  • Figures 54A-H show the retention of transplanted CFSE-labeled allogeneic neonatal cardiac progenitor cells in the sheep ventricle at 2 months post-injection.
  • Figures 55A-B show transplanted CSFE-labeled CPCs remain in the infarct zone 2 months post-injection.
  • Figures 56A-D show that transplanted CPCs are actively dividing in the infarct zone 60 days post-injection.
  • Figures 57A-F show introduced sheep cardiac progenitor cells are capable of differentiation into cardiac and endothelial cells.
  • Figures 58A-D show endothelial cells are recruited into the infarct zone after CPC injection.
  • Figures 59A-E show that CPCs form capillary-like networks on Matrigel.
  • Cardiovascular disease is the number 1 cause of death and disability worldwide. Numerous clinical trials are underway to study the efficacy of stem cell-based cardiac therapy. Initial human clinical trials have shown that endogenous cardiac progenitor cells, isolated from the heart itself, show promise to repair cardiovascular damage, including e.g., myocardial infarction.
  • Cardiovascular progenitor cells were examined for myocardial repair, and were found to be capable of safe and effective regeneration of myocardial tissue in vivo. Key features required for effective myocardial repair were studied including: retention at the site of injection; differentiation into endothelial cells in vivo and differentiation into cardiac myocytes in vivo; recruitment of endogenous progenitors to the site of injury; and examination of arrhythmias was conducted. In vivo, progenitor cells are retained, differentiate and proliferate at the infarct site. There is evidence for recruitment of endogenous endothelial cells into the infarct zone.
  • cloned isl-l+c-kit+ endogenous cardiovascular progenitors represent a cell product that may benefit patients after cell transplantation for the repair of any cardiovascular damage, including e.g., myocardial infarction.
  • the cell product may be provided for regenerative cardiovascular medical treatment as: (1) cells cultured on a support structure which may be removed either prior to or after implantation of the cells into the patient, or (2) a therapeutic cell suspension comprising a cloned population of isl-l+c-kit+ progenitor cells in a biocompatible nutrient medium, where the suspension is configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the suspension into an infarct zone.
  • the support structure is an implantable biodegradable matrix comprising a cloned population of isl-l+c-kit+ progenitor cells, where the matrix is configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the matrix into an infarct zone.
  • Isl- 1+ are a renewable, rare subset of master heart progenitor cells. These multipotent cells give rise to over two thirds of the heart as well as the three major cell types of the heart including: cardiac muscle; smooth muscle; and endothelium cells.
  • Figures 14 and 15 display colored dots to illustrate the contribution of progenitors that express isl-1 to the coronary vasculature, valves, pulmonary artery/aorta, endothelial/ smooth muscle cell layers of the proximal area of the great vessels, atrioventricular myocardium, conduction system, and cardiac ganglia. The use of these cells requires: 1) migration & retention; 2) proliferation; and 3) differentiation including cardiomyocytes and endothelial cells.
  • FIG. 54A-H show that CPCs, labeled with CFSE and injected into four sheep approximately one month after infarction, were retained for at least 55 days post-injection.
  • Figure 55A displays a representative section showing CFSE+ CPCs retained in the infarct zone
  • Figure 55B displays a graph of the percentage of total cells in each sheep that are retained CFSE+ CPCs.
  • Figures 56A-D show that transplanted CPCs are actively dividing in the heart.
  • Figures 56A-B display representative sections from the infarct zone of two sheep, stained with anti-Ki-67 to identify the dividing cells.
  • the CFSE+ cells shown in green, are the introduced CPCs.
  • the sections were stained with anti-Ki-67 to identify dividing cells shown in red.
  • the dividing CPCs are co-localized and image yellow.
  • Figure 56C shows a graph of the percentage of CFSE+ CPCs which are actively dividing in each animal.
  • Figure 56D illustrates a compiled percentage of the CFSE+CPCs which were actively dividing among all four sheep.
  • FIG. 57A shows that non-infarct sections exhibit Troponin I staining
  • Figure 57B shows infarct zone staining with anti-Troponin I, illustrating co- localization (yellow) of anti-Troponin I (red) labeling and the CFSE (green) labeling of the introduced cells.
  • Figure 57C shows that the introduced CPCs exhibited cardiac differentiation in all four sheep studied.
  • Figure 57D shows the non-infarct section of the left ventricle exhibits von Willebrand Factor (vWF) staining (red) indicative of endothelial cells.
  • vWF von Willebrand Factor
  • Figure 57E shows that the infarct zone section exhibits CFSE labeled introduced CPCs (green), von Willebrand Factor labeling (red) and introduced CPCs which have differentiated into endothelial cells (colocalized - yellow, and indicated by arrows).
  • Figure 57F shows that the introduced CPCs exhibited endothelial cell differentiation in three of the four sheep studied.
  • Figure 58A shows a representative section of the non-infarct area of the left ventricle, showing vascularization. Endothelial cells are stained with anti-vWF (red).
  • Figure 58B shows a representative section of the infarct area of the left ventricle, showing vascularization. Endothelial cells are stained with anti-vWF (red). The CPCs are labeled with CFSE (green).
  • Figure 58C shows the infarct area contains a large number of vWF positive endothelial cells (red).
  • Figure 58D shows that there is an increase in the total percentage of cells which are vWF+ in the infarct zone in all four sheep, illustrating recruitment of endogenous endothelial cells.
  • Time to transplant from 1 cell to 1x10 cells, is illustrated in Figure 19.
  • Cardiac progenitor cells were isolated from a 1-month-old patient. Begin with 0.8 cells/well in a 96 well plate. One clone expanded to determine time from single cell to enough for a patient. At day 0, about one cell can be present. At day 24, about 1.38x10 total cells can be present. Therefore, the time to transplant can be about 3.5 weeks. In some embodiments, the time to transplant can be about 2.5 to 4.5 weeks, for example, the time to transplant can be about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 4 weeks, about 4.5 weeks, about 5 weeks, about 5.5 weeks, about 6 weeks, or greater than 6 weeks.
  • endogenous cardiac progenitor cells represent a heart's endogenous regenerative potential.
  • These cells have been isolated as isl-l+c-kit+ cells which are clonogenic and can also be grown as cardiospheres.
  • These cells are HLA Class I positive and HLA Class II negative as shown in Figure 5. They have been shown to express various growth factors such as IGF-1, SDF- ⁇ , and HGF.
  • the cells also express Mesp- 1 and low levels of Brachyury as shown in Figures 1A-C. They are therefore very early cardiovascular progenitors that have the capability to proliferate and produce additional early cardiovascular progenitor cells as well as differentiate in vivo into all lineages necessary for cardiovascular repair in vivo.
  • FIG 16 shows that endogenous CPCs are heterogeneous.
  • FIG 17 shows that isl-l+ CPC clones express KDR, PDGFR and CXCR4.
  • Figure 18 illustrates isl-1 +c-kit+ PDGFRa +IGF1R +CPC clones.
  • PDGFR and IGF1R are present on subpopulations of c-kit+ progenitors with superior regenerative capacity as shown in Figure 18.
  • neonatal CPCs have been seen to regenerate the heart more effectively than adult CPCs.
  • the mechanism of how this heterogeneity and the aging process impact the regenerative capacity of cardiac progenitors has been studied.
  • microRNAs that promote proliferation were highly expressed in neonatal cardiac progenitors.
  • Cell cycle analysis shows that a higher percentage of neonatal vs. adult CPC were actively dividing.
  • MicroRNAs that promote senescence were highly expressed in adult CPC.
  • Neonatal CPCs further have increased AKT autocrine/paracrine signaling as shown by enhanced levels of AKT activation.
  • isl- 1 +c-kit+ endogenous CPC represent a promising new population of cardiovascular progenitors that can be expanded rapidly and studied using single cell, clonal populations isolated from human neonates and adults. Furthermore, these cells are suitable for a repairing a wide variety of cardiovascular damage, including myocardial infarction among other types of damage.
  • Isl- l+c-kit cardiac progenitor cells are useful for cardiac repair
  • Cardiac progenitor cells suitable for cardiac repair can be obtained from a variety of sources. Cardiovascular progenitor cells residing within the heart co-express isl-1 and c-kit. In some embodiments, endogenous stem cell-derived cardiomyocytes are isolated from humans. In some embodiments, CPCs are isolated from the atrial appendage, epicardium or ventricle of human patients. Isolation and selection of cardiac stem cells may be conducted as depicted in Figures 2A-B. Further characterization of the CPCs, as shown in Figures 3A-B, shows that some clones co-express the markers isl-1 and c-kit. As can be seen in Figure 3 A, flow cytometry was used to verify the cells positively express isl-1.
  • autologous stem cells can be used for cardiovascular repair. These can include bone marrow-derived cells and mesenchymal stem cells as are used in early clinical trials. These cells can be safe but have modest, if any, functional improvement.
  • resident cardiac progenitor cells can be used for cardiac repair. Resident cardiac progenitor cells exhibit heterogeneity, including phenotype and regenerative capacity cell type differences and age-related differences. There is evidence that aging plays a role in the quality of stem cells that are isolated from the heart. Cells isolated from cardiac biopsies and expanded as c-kit+ cells or cardiosphere-derived cells can improve cardiac function in animal models.
  • human embryonic stem cell-derived cardiovascular cells can be used for cell-based therapy. These are available "off the shelf and it has been shown that these cells can improve cardiac function after a myocardial infarction in animals. Cell retention and survival is limited and the long-term efficacy may be improved. Additionally, safety concerns that allogeneic cells may initiate an immune response may be addressed.
  • Cardiac progenitor cells can be isolated using various markers.
  • these progenitors are c-kit positive cells or cardiosphere-derived cells.
  • the cells are safe and have been reported to reduce scar size and increase cardiac muscle mass.
  • the newly introduced cells can be used to effectively regenerate the damaged heart tissue. While there is a wide range of expression of cell markers in CPCs, some markers were found to be expressed in all the clones such as ckit, pdgfr, kdr. Some markers were found to be selectively expressed, such as SSEA-4, where some clones were positive and some negative.
  • Flow cytometry may be used to identify phenotypes of neonatal and adult progenitor cells, and to identify phenotypically similar clones as shown in Figure 4.
  • each column represents one clone and three representative neonatal and adult clones are shown.
  • clones that were most similar when comparing neonatal and adults CPCs were selected for further differentiation and functional studies.
  • the isl-l+ckit+ cardiovascular progenitor cells uniquely express markers of early cardiogenic mesoderm, thereby indicating multipotency to differentiate into a variety of cardiovascular derivatives.
  • mesoderm differentiates into cardiogenic mesoderm, which has the capacity to become several types of cardiac cells, including cardiomyocytes, endothelium, smooth muscle cells, and sinoatrial nodal cells. While cardiogenic mesoderm and more committed cells express Isll and c-kit, cardiovascular progenitors that precede the cardiogenic mesoderm stage express Mesp-1 , Brachyury, KDR/Flk-1, and PDGFRa. The expression of markers of early cardiogenic mesoderm was assessed by visualizing amplicons of genes of interest using gel electrophoresis (Figure IB) and by flow cytometry ( Figure 1C).
  • the differentiation protocol used can be similar to the differentiation protocol described in Smits AM, van Vliet P, Metz CH, Korfage T, Sluijter JP, et al. (2009) Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology. Nat Protoc 4: 232-243, which is hereby incorporated by reference in its entirety and should be considered a part of this specification.
  • Gene expression during differentiation can be identified.
  • RT-PCR was used to identify gene expression changes during differentiation of these clones.
  • Trop T was acquired and other markers such as Oct 4 were lost. This along with other gene expression data demonstrates that these clones can be differentiated into cardiomyocytes. Both neonates and adults can be differentiated in adult cardiomyocytes. Their differentiation markers are the same or similar.
  • Figure 22 shows the differentiation of progenitor clones.
  • Figures 23A-C illustrates that the CPCs form endothelial cells in a tube formation assay.
  • Figure 23A illustrates the 96 well plate with plating 10,000 cells in medium containing VEGF and coat with Matrigel.
  • Figure 23B shows an image tube formation 5 hours post-plating. Quantifying using Wimasis software is illustrated with Figure 23C including: branching points; tube length, numbers, and statistics; loop numbers, areas, and perimeters; and cell-covered area.
  • FIG. 20 shows that both neonatal and adult CPCs differentiate into all three cardiac lineages.
  • Figures 59A-D show that CPCs form capillary-like networks on Matrigel.
  • 24A-B shows that neonatal and adult CPCs form capillary-like networks on Matrigel.
  • Figure 24A shows neonatal CPCs at about 5 hours.
  • Figure 24B shows adult CPCs at about 5 hours.
  • Neonatal cells provide superior regenerative capacity [0087] Functional differences were observed between neonatal and adult CPCs, including cell cycling and proliferation as well as motility and invasion. Therefore, the regulatory mechanisms that govern the differences in functional ability between neonatal and adult CPCs was explored. For example, differences in cell cycle and invasion, signaling in response to cytokines and growth factors, gene expression, and microRNA were examined.
  • the cell cycle includes the Gl , S, G2 and M phases.
  • the cell cycle can be analyzed using ethanol fixation, RNAse (Remove Cellular RNA), propidium iodide (intercalates into DNA), and run on Flow Cytometry.
  • RNAse Remove Cellular RNA
  • propidium iodide intercalates into DNA
  • Flow Cytometry As illustrated in Figure 6, it has been shown that a greater percentage of adult cardiac progenitors are in Gl. Conversely, as shown in Figure 7, a greater percentage of neonatal cardiac progenitors are actively dividing, as shown by the significantly higher frequency of neonatal CPC in the S and G2 phase.
  • MicroRNAs regulate gene expression. MicroRNAs can be small 20 nucleotide molecules. They can regulate cardiac development. MicroRNA is conserved across species and gene expression is controlled through microRNA degradation or inhibition of translation. MicroRNA can target multiple genes. MicroRNA profiling was studied. 42 significant microRNA expression differences were identified when comparing neonatal and adult cardiac progenitors as illustrated in Figure 25.
  • microRNA expression pattern of neonatal cardiac progenitors was more similar to that of human embryonic stem cells.
  • Figure 26 shows that microRNAs that promote proliferation are highly expressed in neonatal CPCs.
  • Figures 27A-B shows the microRNA expression and senescence.
  • Figure 27A shows that microRNAs that prevent senescence are elevated in neonatal CPC.
  • Figure 27B shows that miR -371-3p is upregulated in senescent stem cells and is highly expressed in adult CPC.
  • microRNA expression is associated with enhanced capacity to invade in neonatal CPCs. Neonatal CPCs have higher levels of expression of microRNAs that promote invasion and lower levels of microRNAs that inhibit invasion.
  • IGF-1 Insulin-like growth factor- 1
  • SDF-1 stromal cell-derived factor 1
  • IGF-1 Insulin-like growth factor-1
  • SDF-1 stromal cell-derived factor 1
  • Stem cell-based therapy may thus be improved by understanding how growth factors promote cellular expansion.
  • Autocrine and paracrine actions of the transplanted stem cells may promote better cardiac regeneration.
  • exogenous growth factors, including IGF- 1 and SDF-1 administered together with the stem cells or in conjunction with (before or after) administration of the stem cells may promote better stem cell proliferation and cardiac regeneration.
  • Neonatal clones could be further distinguished by their differential response to SDF- 1 and IGF-1.
  • the response of isl- l+ c-kit+ co-expressing neonatal and adult CPCs to IGF- 1 and SDF- 1 was compared.
  • Cardiomyocytes from neonates responded to IGF- 1 treatment with a nearly two-fold increase in AKT phosphorylation while cells from adults showed no response.
  • Flow cytometry revealed that IGF-1 receptor levels were present on both neonatal and adult CPCs. Accordingly, age impacts cardiovascular progenitor cell signaling and may account for the inability of cardiovascular progenitor cells to mobilize to the site of injury and repair the heart in older adults.
  • Neonatal clones could be further distinguished by their differential response to SDF- 1.
  • the adult cells were not stimulated to invade in response to SDF- 1 and there was no evidence of AKT activation, although the receptors were present on the cells as identified by flow cytometry.
  • flow cytometry was used to show SDF-1 receptors CXCR4 and CXCR7 were adequately expressed on both neonatal and adult CPCs as shown in Figure 9B.
  • Figure 11 shows transcripts SDF-1 a and IGF-1 are expressed at higher levels in neonatal CPCs when compared to adults.
  • AKT is well documented to increase cell survival and induce antiapoptotic effects, to promote cardiomyocyte cycling and expansion, and also increase cell motility and migration. Accordingly, the AKT pathway was studied to look at the differences between neonatal and adult CPCs.
  • FIG. 12A-B illustrates that AKT signaling is increased in neonatal CPCs in response to SDF-1 a.
  • Figure 12 shows that AKT signaling is increased in neonatal CPCs and not in adult CPCs in response to SDF-1 a.
  • AKT signaling was increased in neonatal CPCs with SDF-1 a treatment.
  • AKT was not phosphorylated in adult CPCs after SDF- 1 a treatment.
  • FIG. 13A-B shows that AKT signaling was increased in response to IGF- 1 treatment in neonatal CPCs, but not in adult CPCs. This indicates a possible mechanism for the observed differences noted between neonatal and adult CPCs.
  • hypoxic pretreatment can be beneficial to these cells and may benefit cardiac regeneration in vivo, as evidence suggests that short-term hypoxia activates the AKT signaling pathway as shown in Figures 34A-D and 34F. Accordingly, hypoxic pretreatment significantly elevates AKT phosphorylation and upregulates the expression of genes that promote proliferation and survival, as shown in Figures 34E and 34G.
  • the additional benefits of hypoxia preconditioning include anti-apoptotic effects and improved invasion capabilities in vitro.
  • hypoxic pretreatment has no significant effect on the differentiation process or cell cycle progression in isl-l+ hCPCs. In some embodiments, the cells may undergo hypoxic pretreatment prior to administration.
  • the hypoxic pretreatment comprises subjecting the CPCs to reduced levels of oxygen, for a period of time.
  • the reduced level of oxygen may range from about 0.1% to about 10%, such as for example, about 0.5%, about 1%, about 2%, about 4%, about 6%, about 8%, about 10% and overlapping ranges thereof.
  • the dwell time for hypoxic pretreatment is variable depending on the embodiment.
  • the reduced oxygen levels are maintained for a period of time ranging from about 10 minutes to about 10 hours, such as for example, about 15 minutes, about 30 minutes, about 1 hour, about 2 hour, about 4 hours, about 6 hours, about 8 hours, about 10 hours, and overlapping ranges thereof.
  • Neonatal CPCs have an increased ability to proliferate and expand compared to CPCs isolated from an adult source.
  • Neonatal CPCs were more invasive than adult CPCs in response to SDF- ⁇ in vitro despite similar levels of expression of SDF-1 receptors CXCR4 and CXCR7.
  • Neonatal CPCs have increased AKT autocrine/paracrine signaling and an increased intracellular response to growth factor as seen by enhanced levels of AKT activation. The impact of these differences on the ability of adult cardiac progenitors to repair the heart in vivo was determined in further studies.
  • a "patient” as used herein may refer to a human or a non-human mammal, e.g. ., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.
  • a human or a non-human mammal e.g. ., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.
  • an "effective amount” or a “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent that is effective to relieve, to some extent, or to reduce the likelihood of onset of, one or more of the symptoms of a disease or condition, and includes curing a disease or condition.
  • "Treat,” “treatment,” or “treating,” as used herein refers to administering a compound or pharmaceutical composition to a subject for prophylactic and/or therapeutic purposes.
  • prophylactic treatment refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition.
  • therapeutic treatment refers to administering treatment to a subject already suffering from a disease or condition.
  • a therapeutic cell matrix may be an implantable biodegradable matrix configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the matrix into any region of cardiovascular damage, such as an infarct zone, wherein the isl-l+c-kit+ progenitor cells are capable of regenerating damaged heart tissue.
  • a therapeutic cell suspension may be administered intramuscularly into cardiovascular tissue.
  • the combination may be provided to caregivers as a mixture, or the caregivers may mix the two agents prior to administration, or the two agents may be administered separately.
  • the actual dose of the stem cells or active compounds described herein depends on the specific cells or compounds, and on the condition to be treated; the selection of the appropriate dose is well within the knowledge of the skilled artisan.
  • the number of cells to be administered may range from less than 10 3 to at least 1010.
  • the number of cells to be administered may be 10 3 J , 106°, 108°, 108°, 1010, and overlapping ranges thereof.
  • the number of cells to be administered may be 10 .
  • Some embodiments include the combination of compounds, therapeutic agents, and/or pharmaceutical compositions described herein. In such embodiments, the two or more agents may be administered at the same time or substantially the same time.
  • the two or more agents are administered sequentially.
  • the agents are administered through the same route (e.g. orally) and in yet other embodiments, the agents are administered through different routes (e.g. one agent is administered orally while another agent is administered intravenously).
  • Conditional language used herein such as, among others, “can,” “could,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and/or steps. Thus, such conditional language is not generally intended to imply that features, elements and/or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and/or steps are included or are to be performed in any particular embodiment.
  • Example 1 Isolation and culture of Isll + human cardiac progenitor cells
  • hCPCs Human Isll + cardiac progenitor cells
  • This clonal model of CPCs has the benefit of being pure and thus the cells can be well characterized. They can be examined for efficacy and safety in large animal models, and are capable of becoming "available off-the-shelf.” A total of 240 cardiovascular clones were isolated from a number of patients.
  • Antibodies used in this study include: Phospho-Akt (S473) (D9E) Rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA), FITC goat anti-rabbit IgG polyclonal antibody (BD Biosciences, San Jose, CA) and Flourescein-anti-BrdU (PRB-1) monoclonal antibody (Phoenix Flow Systems, San Diego, CA).
  • Cycler settings were set to 94°C for 10 minutes, 94°C for 15 seconds, 52-56°C (depending on the primer) for 60 seconds, and 72°C for 30 seconds for a total of 45 cycles.
  • Human primers were created using NCBI primer blast as listed in Table 1. Relative gene expression data was analyzed using the comparative d method.
  • HSP105 TTATCA GCC AGC CGC CGC TG CCT GCC TGC TTC TCC TGC CG
  • the cardiac progenitor cells were labeled with Br-dUTP (Phoenix Flow Systems, San Diego, CA) and re-suspended in Antibody Solution containing the Flourescein anti-BrdU antibody (Phoenix Flow Systems, San Diego, CA). Population analysis was performed using flow cytometry.
  • Cultrex® basement membrane extract (Trevigen, Gaithersburg, MD) was applied to the upper chamber of a Corning HTS Transwell® plate (8.0 ⁇ pore size, Venlo, Limburg).
  • Human cardiac progenitor cells were suspended in starvation media composed of 98.5% IMDM with GlutaMAXTM (Life Technologies, Carlsbad, CA), 1.0% Insulin- Transferrin-Selenium (Life Technologies, Carlsbad, CA), and 0.5% fetal bovine serum (Thermo Scientific, Waltham, MA) then plated onto the coated wells at a density of 50,000 cells per well.
  • Stromal cell-derived factor 1-a (SDF-la, Life Technologies, Carlsbad, CA), a chemoattractant, was diluted with growth media to a final concentration of lOOng/ml and administered to the lower chamber. After forty-eight hours of incubation at 37°C, the cells in the lower chamber were dissociated, stained with calcein AM (BD Biosciences, San Jose, CA), and analyzed using an FLx800 Microplate Fluorescence Reader (BioTek Instruments, Winooski, VT).
  • a population of hCPCs was subjected to hypoxic preconditioning to determine the effect of short-term hypoxia on Akt phosphorylation.
  • a sample of hCPCs was obtained through the procedure detailed in Example 1. The day before hypoxic pretreatment, the hCPC samples fresh culture media, and if necessary, passaged to achieve 80% confluency within 24 hours. Experimental hCPCs were then placed in a Heracell 150 tri-gas incubator (Thermo Scientific, Waltham, MA) set to 1.0% 0 2 , 5.0% C0 2 , and 94% N 2 for 6 hours at 37°C. Control CPC conditions were 37°C and 5.0%CO 2 . The effects of short term hypoxic preconditioning on hCPCs were analyzed with Flow Cytometry, Quatitative Real-Time PCR, Transwell Invasion Assay, TUNEL Assay, and Cell Cycle analysis. Flow cytometry analysis
  • Pretreated hCPCs displayed a 6.5-fold overall increase of B-cell lymphoma 2 (Bcl-2) and a 5-fold increase of Hemoxygenase 1 (Hmoxl), two genes known to regulate apoptosis.
  • Bcl-2 B-cell lymphoma 2
  • Hmoxl Hemoxygenase 1
  • Example 8 Neonatal CPC are more responsive to growth factors than adult CPCs
  • CPCs human neonatal and adult cardiovascular progenitor cells
  • cardiovascular progenitor cells residing within the heart co-express isl- 1 and c-kit.
  • CPCs produce and are activated by IGF- 1 and SDF-1 and exist as a heterogeneous population.
  • Age impacts cardiovascular progenitor cell signaling and may account for the inability of cardiovascular progenitor cells to mobilize to the site of injury and repair the heart in older adults.
  • Example 9 A greater percentage of neonatal CPCs are actively dividing as compared to adult CPCs.
  • Propidium iodide based cell-cycle analysis and flow cytometry was used to define the number of cells in each stage of the cell cycle.
  • a greater percentage of neonatal CPCs are actively dividing as shown in Figure 6. It was found that a higher frequency of adult progenitors were identified in the Gl phase while a higher frequency of neonatal progenitors were found in the S and G2 phase as shown in Figure 7. This revealed that a greater percentage of neonatal progenitors are actively dividing than compared to adult CPCs.
  • Example 10 Neonatal CPCs invade more readily in response to growth factors such as SDF-la
  • a Transwell invasion assay was also employed to see if the adults have a lesser capacity to respond to a site of injury.
  • Figure 8 illustrates the Transwell invasion assay. The ability to respond and invade is important because cardiac regeneration in vivo requires CPCs to move from their stem cell niche and invade to the site of injury.
  • a Transwell insert with 8 ⁇ pores was coated with a basement membrane extract.
  • SDF-la a growth factor known to be secreted in the heart to recruit CPCs to the site of injury, was selected as the chemoattractant. After 24 hours, the number of cells that migrated through the membrane by flow cytometry was quantified. Components included: Starved media 24 hours; SDF-la lOOng/mL of M199 + EGM-2; Coated with: Cultrex basement extract from Trevigen.
  • Neonatal CPCs invade more readily in response to SDF-la. What was found was that a higher number of neonatal CPCs invade in response to SDF as shown in Figure 9 A. The possibility that this finding was due to a lack of SDF- 1 receptors on the adult progenitors was ruled out. By flow cytometry, it was found that SDF-1 receptors CXCR4 and CXCR7 were adequately expressed on both neonatal and adult CPCs as shown in Figure 9B.
  • Example 11 Neonatal CPCs exhibit increased phosphorylation of the AKT pathway as compared to adult CPCs, leading to increased proliferation and motility.
  • AKT AKT is well documented to increase cell survival and induce antiapoptotic effects, to promote cardiomyocyte cycling and expansion, and also increase cell motility and migration.
  • the observed functional differences between neonatal and adult CPCs include cell cycling and proliferation as well as motility and invasion.
  • Activation of the AKT pathway is higher during differentiation of neonatal CPCs as shown in Figure 10.
  • RT-PCR it was found that genes associated with AKT pathway are induced at higher levels during differentiation of neonatal CPCs than adult.
  • Figure 1 1 shows transcripts SDF- ⁇ and IGF-1 are expressed at higher levels in neonatal CPCs. Furthermore, transcripts for SDF-la and IGF1 were expressed at higher levels in neonatal CPCs when compared to adults. These findings suggest elevated autocrine and paracrine signaling in neonatal CPCs which aid in cell survival, proliferation, and migration.
  • FIG. 12A-B illustrates that AKT signaling is increased in neonatal CPCs in response to SDF-la.
  • AKT protein phosphorylation in CPCs when exposed to growth factor was measured.
  • cells were treated with growth factors SDF-la or IGF-1 for 30 minutes. The samples were lysed and western blots were performed to measure AKT activation or phosphorylation.
  • AKT signaling was found to be increased in neonatal CPCs in response to both SDF-la while signaling was found to be reduced in adult CPCs.
  • Figures 12A-B show that AKT signaling was increased in neonatal CPCs with SDF-la treatment and AKT signaling was reduced in adult CPCs with SDF-la treatment. Included: AKT phos on ser473; lOOng/mL SDF-la; +some neonates also have a decreased or no AKT phosphorylation.
  • FIG. 13A-B shows that AKT signaling was increased in neonatal CPCs with IGF- 1 treatment and AKT signaling was unchanged in adult CPCs with IGF- 1 treatment. This indicates a possible mechanism for the observed differences noted between neonatal and adult CPCs. Included: lOOng/mL IGF-1.
  • Neonatal CPCs have an increased ability to proliferate and expand compared to CPCs isolated from an adult source.
  • Neonatal CPCs were more invasive than adult CPCs in response to SDF-la in vitro despite similar levels of expression of SDF-1 receptors CXCR4 and CXCR7.
  • Neonatal CPCs have increased AKT autocrine/paracrine signaling and an increased intracellular response to growth factor as seen by enhanced levels of AKT activation. The impact of these differences on the ability of adult cardiac progenitors to repair the heart in vivo was determined in further studies.
  • Example 12 The mechanism responsible for differences in regenerative capacity of adult vs neonatal CPCs
  • Figure 26 shows that microRNAs that promote proliferation are highly expressed in neonatal CPCs.
  • Figures 27A-B shows the microRNA expression and senescence.
  • Figure 27A shows that microRNAs that prevent senescence are elevated in neonatal CPC.
  • Figure 27B shows that miR-371-3p is upregulated in senescent stem cells and is highly expressed in adult CPC.
  • MicroRNAs that promote proliferation were highly expressed in neonatal cardiac progenitors.
  • Cell cycle analysis shows that a higher percentage of neonatal CPCs were actively dividing as compared to adult CPCs.
  • MicroRNAs that promote senescence were highly expressed in adult CPC.
  • Neonatal CPCs have increased AKT autocrine/paracrine signaling as shown by enhanced levels of AKT activation.
  • Example 13 Flow cytometry and RT-PCR indicates CPCs co-express isl-1 and c-kit
  • Example 14 Flow Cytometry indicates CPCs are HLA Class I positive and HLA Class II negative
  • Figure 5 shows histocompatibility antigens on cardiac progenitors. Flow cytometry characterization was used to identify similar phenotypic populations of CPC for analysis of microRNA expression and functional analysis. It was found that CPCs are HLA Class I positive and HLA Class II negative as shown in Figure 5.
  • Example 15 Effects of isl-l+ c-kit+ CPCs at site of infarction in sheep model
  • Tissue was harvested from the right atrium of neonatal sheep and cut into 1 mm cubed pieces. The pieces were then digested with collagenase for about 2 hours at 37°C. A 40 mm cell strainer was used to isolate the CPCs. The solution was diluted to 0.8 cells per well and single cell clones were expanded and confirmed to be isll+ c-Kit+ CPCs by flow cytometry and PCR. The neonatal clones selected for cardiac repair were expanded to ten million cells which was the number of cells administered into the infarct zone of each animal. The CPC were labeled with CFSE on the day of injection.
  • FIG. 40 shows a harvested sheep heart.
  • the left ventricle (LV) was cut into 1.5 cm pieces, and each piece saved for further study was labeled according to the location.
  • the pieces intended for IHC were wrapped in aluminum foil and frozen with dry ice and liquid nitrogen. The pieces were stored in a -80°C freezer until sectioned.
  • the pieces intended for RNA extraction were placed in RNAlater® solution and stored in a 4°C refrigerator.
  • Tissue Tek® O.C.T. compound was added to surround the LV tissue in a Leica® CM1900 cryostat set to -20°C to ensure clean 6 ⁇ microtome cuts.
  • the smoothest tissue slices were placed against charged Surgipath® X-tra® slides. Extra tissue in O.C.T. compound from cutting was saved in 15 mL tubes for later RNA extraction.
  • the frozen slides were warmed to room temperature for 30 minutes then put into cold PBS for 10 minutes to rinse off the O.C.T. compound.
  • the slide were then were fixed with cold acetone/methanol 1 : 1 solution for 20 minutes on ice, then washed three times with cold PBS.
  • the slides were left to dry enough to circle the sections with a PAP pen.
  • the slides were blocked for 30 minutes in 5% goat serum in PBS and dilutions of the primary antibody (mouse monoclonal 79010 to Connexin 43 from Abeam®) were prepared in aliquots of the same blocking solution.
  • the blocking solution was aspirated off from the slides and the diluted primary antibody ( 1 :50 to 1 : 1000 concentration) was added.
  • the slides were incubated overnight with wet Kimwipes® at 4°C. The next day the primary antibody was aspirated off carefully and then washed in cold PBS three times for 10 minutes each. The slides were then washed in cold blocking buffer twice for 10 minutes each.
  • the supernatant was pipetted into a new sterile microfuge tube and diluted 2: 1 in 100% EtOH and mixed with pipetting. ⁇ 00 ⁇ L ⁇ of the supernatant in EtOH was transferred to an RNeasy Mini-spin column in a 2 mL collection tube. It was then centrifuged at 23 °C for 15 seconds at 9,000g. Flow through was discarded. The remainder of the supernatant in EtOH solution was transferred to an RNeasy Mini-spin column in a 2 mL collection tube. It was then centrifuged at 23 °C for 15 seconds at 9,000g. Flow through was discarded. 350 ⁇ L ⁇ of RWl Buffer was added to the column and centrifuged for 15 seconds at 9,000g.
  • oligo (dT)20 50 ⁇ was added to a nuclease-free microfuge tube. 2 ⁇ g of RNA in solution up to 11 ⁇ L ⁇ was added. 1 ⁇ L ⁇ of lOmM dNTP Mix was added and then nuclease free water was added to bring the total volume to 13 ⁇ . The mixture was heated to 65 °C for 5 minutes with a heat block then put on ice for at least one minute. The contents of the tube were collected via centrifugation then 4 ⁇ L ⁇ 5X First-Strand Buffer and ⁇ 0.1 M DTT were added.
  • Mastermix was prepared so that each used well of a 96 well plate contained 12.5 ⁇ L ⁇ SYBR Green, 10.3 ⁇ L ⁇ nuclease-free water, and (20/96) ⁇ L ⁇ cDNA. An extra 10% mastermix was included to account for pipetting error. Each primer set was run in triplicate. 3 ⁇ L ⁇ of each forward and reverse primer was used. After loading the plate, the wells were covered with 8-strip caps and spun with a plate spinner. After placing the plate into a iQ5 real-time PCR cycler the camera was allowed to warm up while the temperature rose to 94°C for 10 minutes.
  • the denaturation temperature was set to 94°C for 15 seconds, the annealing temperature was set to 52°C for 60 seconds, and the elongation temperature was set to 72°C for 30 seconds. This cycle would be repeated 45 times. When the cycles completed the cycle threshold for each well was then recorded.
  • the agarose gels were composed of 1% or 2% Bio-Rad agarose in 60 mL of IX TBE and 6 ⁇ L ⁇ SYBR Safe from Invitrogen. 5 ⁇ L ⁇ of a plate well of interest was run with loading dye and a low mass ladder for 45 minutes at 70V. The gel was imaged with a Bio-Rad Gel Doc XR+ with Image Lab software. The noninfarct and infarct zone bands were then compared for each gene of interest.
  • Figure 43 shows exemplary gel electrophoresis results used to compare expression of CXCR4, CXCL12 and IGF-1 at infarcted and non- infarcted sites.
  • Paracrine factors are the cell-to-cell mechanism for promoting regeneration after infarction.
  • IGF1, CXCL12, HGF, VEGF, CXCR4, IL10, and TGFB 1 were measured at infarct and non-infarct cites in four animals as shown in Figures 53A-D.
  • VEGF and HGF were important to examine because they promote angiogenesis to restore blood flow to the damaged region.
  • PCR was used to detect significantly elevated levels of VEGF in three of four animals.
  • HGF transcripts were also elevated to a significant extent (8-26 fold) in two animals when comparing the non-infarct zone and the infarct zone in four animals.
  • IGF-1 was examined in the context of the regenerating myocardium because it activates Akt signaling for cell survival. IGF-1 levels in the infarct zone were elevated to a significant extent in one animal. CXCL12 and its receptor CXCR4 recruit progenitors to promote angiogenesis and CXCL12 was significantly elevated in two animals while CXCR4 was significantly elevated in three of four animals (up to 4 fold). Elevated transcripts for IL-10 noted in two of four animals could potentially contribute to preventing immune-mediated rejection of allogeneic cells.
  • IGF-1 , HGF, VEGF, and CXCR4 were significantly more expressed in the infarct zone on average, as shown in Figure 53E.
  • TGF- ⁇ (0.84-0.96), levels were not changed
  • PCR results were verified by agarose gel electrophoresis.
  • CXCR4, CXCL12, and IGF-1 bands, for example, were shown to be the correct size in Figure43.
  • GAPDH products taken from the matching PCR plates were run as loading controls, as shown in Figure 43.
  • AKT pathway The importance of the AKT pathway is that it upregulates anti-apoptosis proteins to promote survival of cells in the infarct zone. Wnt signaling contributes to renewal and differentiation of Isll+ CPCs after their injection to continue regeneration of the infarct area. The average transcripts from the AKT pathway genes were increased significantly in the infarct zone except for c-Myc, as shown in Figure 45.
  • Notch ligands are important for cardiac progenitor survival and differentiation to continue regeneration after myocardial infarction (MI). Notchl infarct fold changes were significantly elevated up to 12 fold. Notch2 expression was elevated, but the levels were not found to be significant due to animal-to-animal variation when the data was pooled from all animals, as shown in Figure 46.
  • Heat shock protein 70 offers cytoprotection during MI, which compliments the process of regeneration.
  • Noninfarct and infarct zone transcripts were recorded for each sheep, as shown in Figures 47A-D, and averaged as shown in Figure 47E.
  • HSP40 transcripts were elevated in two of four animals and the fold change ranged from 0-5.
  • HSP70 expression was significantly elevated in two animals but the change was not higher than two fold, as shown in. Figures 47A-D.
  • SOD2 superoxide dismutase
  • the receptor tyrosine kinase protein c-kit is a surface receptor for cytokines and it is also a surface marker. C-kit is critical for cell survival and proliferation in the myocardium and this is why cardiac progenitors that express c-kit a leading choice for regenerative therapy after MI.
  • the transcript levels of c-kit were elevated in the infarct zone, as shown in Figures 51A-B. The infarct zone in individual sheep examined for this study saw a fold change of 23 fold at the maximum. One of the four sheep did not have a significant c- kit rise.
  • GATA4 is an important cardiac transcription factor that positively affects cell survival and growth factor secretion in an infarcted area to stimulate regeneration.
  • Isl-1 is expressed by the injected cardiac progenitors and is associated with CPC proliferation and angiogenesis.
  • NF- ⁇ is a regulator of inflammation in the infarct area, but also growth and cell adhesion. Suppression of NF- ⁇ promotes an anti-inflammatory response and shrinks the size of the infarct, while its activation leads to inflammatory cytokine mRNA upregulation through TNF-a activation.
  • FIG. 49 relative GATA4 and NF-KB expression in the infarct zone did not change significantly. Islet- 1 transcript levels were elevated (up to 13 fold change) significantly.
  • Heme oxygenase- 1 is important for anti-inflammatory signaling because it protects against cardiomyocyte apoptosis by metabolizing cytotoxic heme. HMOX overexpression triggers SOD2 activation, Akt activation, and also anti-apoptosis signaling through mitochondrial biogenesis.
  • TNFAIP3 (A20) also has protective anti-inflammatory properties because it suppresses NF- ⁇ signaling. It is also known to improve cardiac function by inhibiting fibrosis and hypertrophy from TGF- ⁇ signaling.
  • BCL2 is an anti- apoptotic gene and it promotes VEGF secretion for angiogenesis in vitro.
  • HMOX transcript fold change in the infarct zone was elevated as much as 6.4 fold, but not in all sheep, therefore the average change was not found to be significant. Similar results were obtained for TNFAIP3 (elevated up to 22 in one animal), and for BCL2 (elevated up to 17 in one animal). When considered as an average trend, as shown in Figure 48, none of the prosurvival genes were elevated to an extent that was found to be significant.
  • Gap junction protein Connexin-43 is important for intercellular communication that affects wound healing, fibrosis, and remodeling after MI.
  • the average transcript levels of Cx43 were elevated in the infarct zone, as shown in Figure 52A.
  • Immunohistochemistry reveals that the CFSE-labeled Isl-1+ c-kit+ CPCs coexpressed the red connexin-43 stain as shown in Figure 52B.
  • Example 16 CPCs cultured on a biocompatible matrix
  • CSCs were isolated from biopsies of human neonates or adults under Institutional Review Board approval following a previous protocol as described in Example 1. Briefly, right atrial cardiac tissue ( ⁇ 1 mm ) was digested with collagenase (Roche Applied Science, Indianapolis, IN) for two hours at 37°C and then strained. Cells were cloned by limiting dilution (0.8 cells per well) to create the clonal populations that were used in this study. CSCs were isolated from sheep in accordance with the Animal Welfare Act, the NIH Guide for the Care and Use of Laboratory Animals, and the IACUC of Loma Linda University.
  • An 8x2-mm GroCell-3D polysaccharide scaffold (Molecular Matrix, Davis, CA) was placed in M199-supplemented media overnig ht. CSCs (2 x 10 6 ) in 20 ⁇ growth media were incubated on the scaffold for two hours at 37°C in 5% C0 2 before being placed in growth media in a T25 flask. Cells were seeded on both sides and maintained for three weeks. Scaffolds were dissolved for cell cycle, flow cytometry, and gene expression analysis using dissolution solution (Molecular Matrix, Davis, CA) following the manufacturer's protocol.
  • Flow cytometry was used to measure phosphorylated ERK and AKT.
  • Cells were fixed with 4% paraformaldehyde, permeabalized, and stained using antibodies to phosphorylated ERK 1/2 (Thr202/Tyr204, Cell Signaling Technology, Danvers, MA) at a 1/200 dilution, and p-AKT (Ser473, Cell Signaling Technology, Danvers, MA) at a 1/100 dilution.
  • FITC goat anti-rabbit IgG (BD Biosceinces San Jose, CA) at a dilution of 1/150 was used as a secondary antibody.
  • the geometric mean of each peak for each clone was used to calculate the staining of each cell.
  • Flow cytometry was used to measure the level of expression of the differentiation markers TropT and vWF..
  • the Lightning-Link Antibody Labeling Kit (Novus Biologicals, Littleton, CO) was used per the manufacturer's instructions to conjugate rabbit anti-human von-Willbrand factor antibody (Dako, Carpinteria, CA) to FITC and anti-cardiac troponin T antibody (Abeam, Cambridge, MA) to PE.
  • Cells were stained with FITC- conjugated vWF at a dilution of 1/10 and PE-conjugated TropT at a dilution 1/250. Isotype controls were used to define populations.
  • RNA 500ng was isolated and used to prepare cDNA with the Superscript III (Invitrogen, Carlsbad, CA) protocol.
  • RT-PCR was performed on the IQ5 machine (Bio-rad, Hercules, CA) with ⁇ -actin as a housekeeping gene. The PCR conditions were: 94°C for 10 minutes, 94°C for 15 seconds, 52°C for 60 seconds, and 72°C for 30 seconds for 40 cycles.
  • the primers were (forward) 5 ' -TTTGAATGATGAGCCTTCGTCCCC-3 ' and (reverse) 5'- GGTCTC AAGTCAGTGTACAGGTAAGC-3 ' for ⁇ -Actin; (forward) 5'- CAGAGCAGATAGAGCCTGCG-3 ' and (reverse) 5 ' -C AGGTAACTCGTGC AGAGC A-3 ' for IGF-1; (forward) 5 ' -C ACG AACAC AGCTTTTTGCC-3 ' and (reverse) 5'- TGATCCC AGCGCTGACAAAT-3 ' for HGF; and (forward) 5'- CTACAGATGCCC ATGCCGAT-3 ' and 5 ' -GTGGGTCTAGCGGAAAGTCC-3 ' for SDF- la (Integrated DNA Technologies, Coralville, IA).
  • mice anti-Ki-67 (Biolegend, San Diego, CA) at a 1: 100 dilution
  • mouse anti-Isll Abeam, Cambridge, MA
  • rabbit anti-vWF rabbit anti-vWF
  • mouse anti-TropT (Abeam, Cambdridge, MA) at a 1: 100 dilution. Incubation was performed at 4°C. Each section was washed with PBS/Tween20 and incubated with a secondary antibody for one hour at room temperature.
  • the secondary antibodies were either goat anti-mouse PE (Southern Biotech, Birmingham, AL) at a 1: 100 or 1:250 dilution or donkey anti-rabbit Alexa Fluor 647 (Life Technologies, Carlsbad, CA) at a 1:200 dilution.
  • Slides were mounted in Prolong Gold with DAPI (Life Technologies, Carlsbad, CA). Sections were imaged (20x) using a LSM 710 NLO laser- scanning, confocal microscope (Carl Zeiss Microscopy Gmblt, Jena, Germany). Images were processed using ImageJ (v.1.49, NIH, http://imagej.nih.gov/ij).
  • Ovine-derived, scaffold-cultured CSCs express increased levels of Isll and Ki-67 and a pro- stem cell shift in cell cycling
  • ovine-derived CSCs that are cultured using a molecular scaffold can both proliferate and differentiate.
  • RT-PCR was used to assess the expression of IGF-1, HGF, and SDF- ⁇ . It was found that the expression of HGF and SDF- ⁇ was significantly higher in the scaffold- cultured CSCs as shown in Figure 32A, HGF: 48-fold increase, P ⁇ 0.01; SDF- ⁇ : 77-fold increase, P ⁇ 0.05. While an increase was observed for IGF-1, this change was not significant.
  • the amplified cDNA products of the statistically significant growth factors were visualized on a 1% agarose gel, and an increase in expression and a conformation of the appropriate product length can be qualitatively observed in Figure 32B.
  • Example 17 CPCs exhibit in vivo retention, division, differentiation, and recruitment of endogenous endothelial cells
  • Tissue was harvested from the right atrium of neonatal sheep and cut into
  • a 40 mm cell strainer was used to isolate the CPCs.
  • the solution was diluted to 0.8 cells per well and the isolated clones were expanded and characterized.
  • Flow cytometry was used to identify both isl- 1 and ckit expression, and PCR was used to verify the expression of isl- 1 and c-kit on these undifferentiated isl 1+ c-Kit+ CPCs.
  • the cells were expanded in culture prior to transplantation.
  • LAD ligation was used to induce a myocardial infarction in four sheep. EKG and echocardiograms were done prior to infarction, on the day of cardiovascular progenitor cell transplantation in these animals, and at sacrifice of the animals two months after cardiovascular progenitor cell injection.
  • the CSC clones to be administered Prior to cardiovascular progenitor cell administration, the CSC clones to be administered (106) were suspended in 5 ⁇ of CFSE and incubated for 10 minutes at 37 °C to label them with a green fluorescent tag for tracking. This reaction was quenched by 10% sheep serum (S2263, Sigma Aldrich, St Louis, MO)-supplemented M-199 (Life Technologies, Carlsbad, CA) media. Labeling of the cells was verified by flow cytometry prior to cardiovascular stem cell administration in vivo. CFSE labeled allogenic CPCs were injected 28-38 days post infarction. Histology was performed by immunostaining of frozen sections. At 55-60 days after cell injection, the animals were sacrificed to examine the effect of CSC administration on cardiovascular regeneration and repair.
  • Tissue pieces were frozen for later sectioning and immunostaining. Additional pieces were kept in RNAlater for preparation of RNA for gene expression analysis. Immunostaining was done on frozen sections using antibodies to identify proliferation, differentiation into cardiomyocytes and endothelial cells and cell-cell interaction. The concentration of the antibodies was established in preliminary experiments performed to optimize conditions for staining sections using these reagents. The reagents used included anti-ki67, anti-troponin I, anti-von-Willebrand factor and anti-connexin 43 antibodies along with the appropriate secondary antibodies. Two color staining of CFSE (green) and the red Alexa Fluor 647 antibodies allowed identification of cardiovascular progenitor cell differentiation and cell division.
  • Sheep A ( Figure 54E): 12-13% of cells are CFSE positive, from a total of 1410 cells imaged.
  • Sheep B ( Figure 54F): 10-31% of the cells are CFSE positive, from a total of 2593 cells imaged.
  • Sheep C ( Figure 54G): 11- 19% of the cells are CFSE positive, from a total of 2007 cells imaged.
  • Sheep D (Figure 54H) 11-43% of the cells are CFSE positive, from a total of 3624 cells imaged.
  • Sheep A Average 78%, range 46-95% CFSE+ CPCs expressing Ki-67, from a total of 904 cells imaged.
  • Sheep B Average 26%, range 9-32% CFSE+ CPCs expressing Ki-67, from a total of 798 cells imaged.
  • Sheep C Average 68%, range 56-89% CFSE+ CPCs expressing Ki-67, from a total of 1 103 cell imaged.
  • Sheep D Average 9%, range 5-14% CFSE+ CPCs expressing Ki-67, from a total of 1164 cells imaged. CFSE-labeled allogenic neonatal CPCs differentiated into cardiac and endothelia cells
  • Infarct zone staining with anti-Troponin I illustrated co-localization (yellow) of anti-Troponin I (red) labeling and the CFSE (green) labeling of the introduced allogenic CPCs ( Figure 57B).
  • the non-infarct area labeled with anti-Troponin I is shown in Figure 57A.
  • Figure 57C the introduced CPCs in the infarct zone exhibited differentiation into cardiomyocytes in all four sheep models
  • Sheep A Average 31%, Range 28-42% CFSE+ CPCs expressing Trop I from a total of 1508 cells imaged.
  • Sheep B Average 43%, Range 29-61% CFSE+ CPCs expressing Trop I from a total of 760 cells imaged
  • Sheep C Average 40% Range 39-41% CFSE+ CPCs expressing Trop I, from a total of 1069 cells imaged
  • Sheep D Average 27% Range 12-40% CFSE+ CPCs expressing Trop I, from a total of 889 cells imaged
  • non-infarct section of the left ventricle exhibits von Willebrand Factor (vWF) staining (red, Figure 57D) indicative of endothelial cells
  • vWF von Willebrand Factor
  • Figure 57E shows von Willebrand Factor labeling (red) and introduced CPCs which have differentiated into endothelial cells (colocalized- yellow, indicated by arrows, Figure 57E).
  • the introduced CPCs exhibited endothelial cell differentiation in three of the four sheep studied.
  • Sheep A Average 36%, Range 27-48% CFSE+ CPCs expressing vWF, from a total of 1360 cells imaged.
  • Sheep B Average 14%, Range 0-21% CFSE+ CPCs expressing vWF, from a total of 706 cells imaged
  • Sheep C Average 4% Range 0-9% CFSE+ CPCs expressing vWF, from a total of 1841 cells imaged
  • Sheep D Average 0% Range 0-0% CFSE+ CPCs expressing vWF, from a total of 1209 cells imaged Endothelial cells are recruited into the infarct zone after CPC injection.
  • Figure 58A shows a representative section of the non-infarct area of the left ventricle, showing vascularization. Endothelial cells are stained with anti-vWF (red).
  • Figure 58B shows a representative section of the infarct area of the left ventricle, showing vascularization. Endothelial cells are stained with anti-vWF (red). CFSE labeled CPCs are green.
  • Figure 58C shows that the infarct area contains a large number of vWF positive endothelial cells (red). An increase in the total percentage of cells which are vWF+ in the infarct zone was found in all four sheep, illustrating recruitment of endogenous endothelial cells as shown in Figure 58D.
  • Non-infarct Average 6%, range 1-13% cells expressing vWF, with a total of 1701 cells imaged.
  • Infarct Average 21%, range 12-34% cells expressing vWF, with a total of
  • Sheep B Non-infarct: Average 8%, range 7-11 % cells expressing vWF, with a total of 903 cells imaged.
  • Infarct Average 14%, range 10-19% cells expressing vWF, with a total of 706 cells imaged.
  • Non-infarct Average 7%, range 2-11 % cells expressing vWF, with a total of 2147 cells imaged.
  • Infarct Average 19%, range 14-24% cells expressing vWF, with a total of 1841 cells imaged.
  • Non-infarct Average 7%, range 7-11 % cells expressing vWF, with a total of 626 cells imaged.
  • Infarct Average 12%, range 7-17% cells expressing vWF, with a total of 1209 cells imaged.

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Abstract

Compositions and methods of using isl-l+c-kit+ cardiac progenitor cells are disclosed for promoting repair of cardiac tissue and improve cardiac function in post-myocardial infarction patients.

Description

COMPOSITIONS AND USE OF ISL-1+C-KIT+ CELLS FOR CARDIOVASCULAR
REPAIR
BACKGROUND
Field
[0001] The present disclosure relates to compositions and use of a cloned population of isl-l+c-kit+ progenitor cells for cardiovascular repair and regeneration in treating cardiac tissue damage.
Description of the Related Art
[0002] In the United States alone, 1.2 million heart attacks occur annually. One out of four people die of heart failure. Cardiovascular disease and stroke continue to be the nation's first and third most frequent causes of death respectively, and are responsible for over 870,000 deaths each year. Moreover, approximately 35,000 newborns are diagnosed with a congenital heart defect annually. Pharmacological treatments do not regenerate the heart, but stem cell-based therapies offer new hope that cardiovascular regeneration can be achieved in the future. For the millions of Americans with heart disease, and children born with congenital abnormalities of the heart, stem cell research offers the hope of identifying new ways of regenerating heart tissue. Clinical trials testing whether autologous bone marrow-derived stem cells could improve heart function have not been as beneficial as initially hoped. Although these autologous stem cells were shown to be safe, the modest improvement in ejection fraction appears to have been the result of paracrine effects, and a lack of adequate controls render the clinical significance of this data questionable.
SUMMARY
[0003] Methods, compositions, and systems for treating cardiac damage and/or for cardiac repair are disclosed.
[0004] In some embodiments, a method for treating a patient having damage within a region of cardiac tissue, comprising transplanting into the region or into cardiac tissue surrounding the region, a clonal population of isl-l+c-kit+ cardiac progenitor cells. In some embodiments, the clonal population of isl-l+c-kit+ progenitor cells is preconditioned under hypoxic conditions prior to transplantation.
[0005] In some embodiments, a composition comprising a cell type for cell therapy treatment for treating cardiac damage is disclosed. The composition comprises: clonal population of isl-l+c-kit+ progenitor cells configured to differentiate into one or more cardiovascular lineages. In some embodiments, the isl-l+c-kit+ progenitor cells comprise autologous or allogeneic cells.
[0006] In some embodiments, a method is disclosed for providing a therapeutic cell matrix for use in regenerative cardiovascular medicine. In some embodiments, the method for providing a therapeutic cell matrix for use in regenerative cardiovascular medicine comprises obtaining a cloned population of isl-l+c-kit+ progenitor cells; and culturing the isl-l+c-kit+ progenitor cells on a support structure which may be removed either prior to or after implantation of the cells into the patient. In some embodiments, the support structure is an implantable biodegradable matrix configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the matrix into an infarct zone, wherein the isl-l+c-kit+ progenitor cells are capable of regenerating damaged heart tissue. In some embodiments, a method is disclosed for providing a therapeutic cell suspension for use in regenerative cardiovascular medicine. In some embodiments, the method for providing a therapeutic cell suspension for use in regenerative cardiovascular medicine comprises obtaining a cloned population of isl-l+c-kit+ progenitor cells; and suspending the isl-l+c-kit+ progenitor cells in a pharmaceutically acceptable medium configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the suspension into an infarct zone, wherein the isl-l+c-kit+ progenitor cells are capable of regenerating damaged heart tissue. In some embodiments, the cloned population of isl-l+c-kit+ progenitor cells are obtained from the atrial appendage, epicardium, or ventricle of one or more human tissue donors. In some embodiments, the cloned population of isl-l+c-kit+ progenitor cells are configured to differentiate into one or more cardiovascular lineages. In some embodiments, the cloned population of isl-l+c-kit+ progenitor cells comprises autologous or allogeneic cells. In some embodiments, the cloned population of isl-l+c-kit+ progenitor cells is preconditioned under hypoxic conditions.
[0007] In some embodiments, a composition for treating cardiac damage is disclosed. In some embodiments, the composition for treating cardiac damage comprises a cloned population of isl-l+c-kit+ progenitor cells; wherein the isl-l+c-kit+ progenitor cells are adapted to promote repair of damaged heart tissue when transplanted into a region of cardiovascular tissue comprising damaged heart tissue. In some embodiments, the cloned population of isl-l+c-kit+ progenitor cells are obtained from the atrial appendage, epicardium, or ventricle of one or more human tissue donors. In some embodiments, the cloned population of isl-l+c-kit+ progenitor cells are configured to differentiate into one or more cardiovascular lineages. In some embodiments, the clonal population of isl-l+c-kit+ progenitor cells comprises autologous or allogeneic cells. In some embodiments, the composition further comprises one or more cytokines, growth factors or other agents that promote survival, proliferation and/or differentiation of the isl-l+c-kit+ progenitor cells. In some embodiments, the composition further comprises a support structure which may be removed either prior to or after implantation of the cells into the patient. In some embodiments, the support structure is an implantable biodegradable matrix associated with the isl-l+c-kit+ progenitor cells, wherein the biodegradable matrix promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c-kit+ progenitor cells. In some embodiments, the composition further comprises a biocompatible nutrient medium that promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c- kit+ progenitor cells.
[0008] In some embodiments, a use of a composition for treating damaged cardiac tissue in a patient in need thereof is disclosed. In some embodiments, the composition comprises a cloned population of isl-l+c-kit+ progenitor cells. In some embodiments, the composition is configured for transplantation into a region of cardiovascular tissue comprising damaged cardiac tissue. In some embodiments, the cloned population of isl-l+c- kit+ progenitor cells are obtained from the atrial appendage, epicardium, or ventricle of one or more human tissue donors. In some embodiments, the cloned population of isl-l+c-kit+ progenitor cells are configured to differentiate into one or more cardiovascular lineages. In some embodiments, the clonal population of isl-l+c-kit+ progenitor cells comprises autologous or allogeneic cells. In some embodiments, the composition further comprises one or more cytokines, growth factors or other agents that promote survival, proliferation and/or differentiation of the isl-l+c-kit+ progenitor cells. In some embodiments, the composition further comprises a support structure which may be removed either prior to or after implantation of the cells into the patient. In some embodiments, the support structure is an implantable biodegradable matrix associated with the isl-l+c-kit+ progenitor cells, wherein the biodegradable matrix promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c-kit+ progenitor cells. In some embodiments, the composition further comprises a biocompatible nutrient medium that promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c-kit+ progenitor cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figures 1A-B show the isl-l+ckit+ cardiovascular progenitor cells uniquely express markers of early cardiogenic mesoderm, thereby indicating multipotency to differentiate into a variety of cardiovascular derivatives. Figure IB shows the results of flow cytometry used to show expression of markers of early cardiogenic mesoderm.
[0010] Figures 2A-B illustrate steps of selecting (FIG. 2A) and isolating (FIG. 2B) cardiac progenitor cells.
[0011] Figures 3A-C show characterization of the CPCs. Figure 3D shows the results of gel electrophoresis to demonstrate that c-kit+isl-l+ cells express Mesp-1 the early cardiovascular progenitor cell marker, in addition to low levels of Brachyury and PDGF receptor alpha.
[0012] Figure 4 shows flow cytometry used to identify phenotypes of neonatal and adult progenitor cells.
[0013] Figure 5 shows histocompatibility antigens on cardiac progenitors.
[0014] Figure 6 shows a cell cycle analysis comparing neonatal and adult CPCs.
[0015] Figure 7 shows a higher frequency of adult progenitors are in the Gl phase, while a higher frequency of neonatal progenitors are in the S and G2 phases.
[0016] Figure 8 illustrates the Transwell invasion assay. [0017] Figure 9A shows a higher number of neonatal CPCs invade in response to
SDF.
[0018] Figure 9B shows that SDF-1 receptors CXCR4 and CXCR7 were adequately expressed on both neonatal and adult CPCs.
[0019] Figure 10 shows activation of the AKT pathway is higher during differentiation of neonatal CPCs.
[0020] Figure 1 1 shows transcripts SDF- la and IGF-1 are expressed at higher levels in neonatal CPCs.
[0021] Figures 12A-B illustrates that AKT signaling is increased in neonatal CPCs in response to SDF- la.
[0022] Figures 13A-B shows that AKT signaling was increased in neonatal CPCs with IGF- 1 treatment and AKT signaling was unchanged in adult CPCs with IGF- 1 treatment.
[0023] Figures 14 and 15 identify isl-1 derivatives in the heart.
[0024] Figure 16 shows that endogenous CPCs are heterogeneous.
[0025] Figure 17 shows that isl-1 + CPC clones express KDR, PDGFR and CXCR4.
[0026] Figure 18 illustrates isl-1 +c-kit+ PDGFRa +IGF1R +CPC clones.
[0027] Figure 19 illustrates the time to transplant, for a representative neonatal clone, from 1 cell to 1x10 cells.
[0028] Figure 20 shows the distribution of markers for cell types illustrating that both neonatal adult CPCs differentiate into all three cardiac lineages. This data was captured using the dexamethasone protocol.
[0029] Figure 21 illustrates the differentiation into cardiomyocytes and the timeline of the differentiation procedure.
[0030] Figure 22 shows the differentiation of progenitor clones.
[0031] Figures 23A-C illustrates that the CPCs form endothelial cells in a tube formation assay.
[0032] Figures 24A-B shows that neonatal and adult CPCs form capillary-like networks on Matrigel. [0033] Figure 25 shows that 42 significant microRNA expression differences were identified when comparing neonatal and adult cardiac progenitors.
[0034] Figure 26 shows that microRNAs that promote proliferation are highly expressed in neonatal CPCs.
[0035] Figure 27A shows that microRNAs that prevent senescence are more highly expressed in adult CPCs. Figure 27B shows that microRNAs associated with senescence are more highly expressed in adult CPCs.
[0036] Figure 28A shows isl- 1 expression was significantly elevated when cultured ovine isl- l+c-kit+ CPC were cultured on a scaffold. Figure 28B shows the results of cell cycle analysis performed on scaffold cultured CPC where an elevated number of cells are in G2 after scaffold culture.
[0037] Figures 28C-H show that cardiac stem cells show elevated expression of isl- 1 after culture on a scaffold and continue to proliferate actively as shown by Ki67 labeling of sections and quantitation using Image Pro software.
[0038] Figure 29A-F shows representative histograms of TropT-positive and vWF-positive cells, demonstrating that scaffold culturing induces differentiation of cardiac stem cells.
[0039] Figure 29G-L shows the results of confocal microscopy, which demonstrates that TropT and vWF were induced by differentiation of scaffold-cultured cells.
[0040] Figure 30 A shows fewer scaffold cultured cardiac stem cells are in the Gl phase as shown by flow cytometry. Figure 30B shows the expression of TropT and vWF was significantly increased in scaffold cultured cardiac stem cells.
[0041] Figure 31 A shows a significant decrease in ERK phosphorylation as verified by flow cytometry. Figure 3 IB shows elevated AKT phosphorylation shown by flow cytometry.
[0042] Figure 32A shows a significant increase HGF, and SDF-Ια expression, and an increase in IGF-1 expression in scaffold cultured cells. Figure 32B shows the amplified cDNA products of the statistically significant growth factors on an agarose gel. [0043] Figure 33 shows microRNAs associated with enhanced capacity to invade are elevated in neonatal CPCs. MicroRNAs that inhibit invasion are more highly expressed in adult CPCs.
[0044] Figure 34 shows alterations in CPC after short term hypoxia treatment. Gene expression changes associated with the AKT pathway were induced in neonatal CPC (FIG. 34A) and adult CPC (FIG. 34B). Quantification of seven independent hCPCs revealed significantly higher (15.4% increase, p = 0.013) Akt phosphorylation in neonatal hypoxia- pretreated CPC. Akt phosphorylation measurements taken by flow cytometry analyzed as a function of age reveals that hypoxia-mediated Akt activation occurs more readily in neonatal hCPCs (FIG. 34C). Elevated levels of phosphorylated AKT after short term hypoxia in adult CPC identified by Western blot are shown in (FIG. 34D), elevated transcripts encoding prosurvival genes after short term hypoxia (FIG. 34E), and elevated phosphorylation of AKT after short term hypoxia are shown by flow cytometry (FIG. 34F). Heat shock proteins 40 and 90 are elevated after short term hypoxia (FIG. 34G), apoptosis (FIG. 34H) and differentiation (FIG. 341) are not induced by short term hypoxia.
[0045] Figure 35 shows hypoxia-pretreated hCPCs exhibit significantly improved invasion as measured by Transwell invasion assay.
[0046] Figure 36 shows the progenitor cell distribution in sheep.
[0047] Figure 37 shows the subpopulations of sheep CPC that include progenitors which are phenotypically comparable to those isolated from humans.
[0048] Figure 38 shows CPC clone used in cardiovascular stem cell transplantation.
[0049] Figure 39 shows CFSE labeling of CPC prior to transplantation.
[0050] Figure 40 shows the heart of a sheep harvested 2 months after CPC injection.
[0051] Figure 41 shows sectioning of frozen tissues harvested from the sheep heart.
[0052] Figure 42 shows the procedure for preparation and fluorescent IHC staining of frozen tissue sections. [0053] Figure 43 shows the results of gel electrophoresis used to compare gene expression of CXCR4, CXCL12, and IGF1 at non-infarcted and infarcted sites.
[0054] Figure 44 shows the results of real-time PCR analysis used to compare transcripts of genes associated with paracrine effects after transplantation of isl-l+c-kit cells at infarcted and non-infarcted sites.
[0055] Figure 45 shows the results of real time PCR used to show transcripts of genes in the Akt pathway were elevated at the site of infarction after transplantation with isl- l+c-kit+ progenitors.
[0056] Figure 46 shows the results of real time PCR used to show Notch transcripts were elevated at the site of infarction after transplantation with isl-l+c-kit+ progenitors.
[0057] Figure 47A-E show the results of real time PCR used to show heat shock protein transcripts were elevated at the site of infarction after transplantation with isl-l+c- kit+ progenitors.
[0058] Figure 48 shows the results of real time PCR used to show pro survival transcripts were elevated at the site of infarction after transplantation with isl-l+c-kit+ progenitors.
[0059] Figure 49 shows the results of real time PCR used to show transcription factors were elevated at the site of infarction after transplantation with isl-l+c-kit+ progenitors.
[0060] Figure 50A shows the results of real time PCR used to show superoxide dismutase 2 transcripts were elevated at the site of infarction after transplantation with isl- l+c-kit+ progenitors, and Figure 50B shows the results were verified using gel electrophoresis.
[0061] Figure 51A shows the results of real time PCR used to show c-Kit transcripts were elevated at the site of infarction after transplantation with isl-l+c-kit+ progenitors, and Figure 5 IB shows the results were verified using gel electrophoresis.
[0062] Figure 52A shows the results of real time PCR used to show Connexin-43 transcripts were elevated at the site of infarction after transplantation with isl-l+c-kit+ progenitors, and Figure 52B shows a Connexin-43 stained image showing connexin staining between the newly introduced cardiovascular cells in the myocardium.
[0063] Figure 53A-D shows the results of real-time PCR used to identify gene expression changes in the infarct zone of four sheep after transplantation with isl-l+c-kit+ progenitors. Figure 53E shows the pooled real-time PCR data.
[0064] Figures 54A-H show the retention of transplanted CFSE-labeled allogeneic neonatal cardiac progenitor cells in the sheep ventricle at 2 months post-injection.
[0065] Figures 55A-B show transplanted CSFE-labeled CPCs remain in the infarct zone 2 months post-injection.
[0066] Figures 56A-D show that transplanted CPCs are actively dividing in the infarct zone 60 days post-injection.
[0067] Figures 57A-F show introduced sheep cardiac progenitor cells are capable of differentiation into cardiac and endothelial cells.
[0068] Figures 58A-D show endothelial cells are recruited into the infarct zone after CPC injection.
[0069] Figures 59A-E show that CPCs form capillary-like networks on Matrigel.
DETAILED DESCRIPTION
[0070] Cardiovascular disease is the number 1 cause of death and disability worldwide. Numerous clinical trials are underway to study the efficacy of stem cell-based cardiac therapy. Initial human clinical trials have shown that endogenous cardiac progenitor cells, isolated from the heart itself, show promise to repair cardiovascular damage, including e.g., myocardial infarction.
[0071] Cardiovascular progenitor cells (CPCs) were examined for myocardial repair, and were found to be capable of safe and effective regeneration of myocardial tissue in vivo. Key features required for effective myocardial repair were studied including: retention at the site of injection; differentiation into endothelial cells in vivo and differentiation into cardiac myocytes in vivo; recruitment of endogenous progenitors to the site of injury; and examination of arrhythmias was conducted. In vivo, progenitor cells are retained, differentiate and proliferate at the infarct site. There is evidence for recruitment of endogenous endothelial cells into the infarct zone.
[0072] An isolated, clonal population of isl-l+c-kit+ progenitor cells which can differentiate into all cardiovascular lineages in vitro and in vivo have been obtained. Evidence for regeneration following administration of neonatal, allogenic CPCs in vivo has been obtained in the sheep model of myocardial infarction. For instance, Figure 36 shows the progenitor cell distribution and Figure 35 shows the subpopulations of sheep CPC. Figure 38 shows CPC clone used in cardiovascular stem cell transplantation. In addition, the isl-l+c- kit+ cells recruit endogenous cells to the infarct site in vivo to participate in the repair process. Therefore, cloned isl-l+c-kit+ endogenous cardiovascular progenitors represent a cell product that may benefit patients after cell transplantation for the repair of any cardiovascular damage, including e.g., myocardial infarction. The cell product may be provided for regenerative cardiovascular medical treatment as: (1) cells cultured on a support structure which may be removed either prior to or after implantation of the cells into the patient, or (2) a therapeutic cell suspension comprising a cloned population of isl-l+c-kit+ progenitor cells in a biocompatible nutrient medium, where the suspension is configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the suspension into an infarct zone. In some embodiments, the support structure is an implantable biodegradable matrix comprising a cloned population of isl-l+c-kit+ progenitor cells, where the matrix is configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the matrix into an infarct zone. At the present time, no other cloned comparable cardiovascular progenitors have been described, nor are there published reports describing the use of isl-l+c-kit+ endogenous cardiovascular stem cell clones for cardiac repair in any models in vivo.
[0073] Isl- 1+ are a renewable, rare subset of master heart progenitor cells. These multipotent cells give rise to over two thirds of the heart as well as the three major cell types of the heart including: cardiac muscle; smooth muscle; and endothelium cells. Figures 14 and 15 display colored dots to illustrate the contribution of progenitors that express isl-1 to the coronary vasculature, valves, pulmonary artery/aorta, endothelial/ smooth muscle cell layers of the proximal area of the great vessels, atrioventricular myocardium, conduction system, and cardiac ganglia. The use of these cells requires: 1) migration & retention; 2) proliferation; and 3) differentiation including cardiomyocytes and endothelial cells. The capacity of isl- 1 + progenitors to repair the adult heart was studied. Functional outcomes are promising including the safety, decrease in scar size, and increase in viable myocardium. Long-term studies can be conducted in additional patients to assess outcome relative to controls and clarify roles of paracrine effects in cardiovascular regeneration. Nonetheless, current evidence indicates that CPCs are able to effectively regenerate damaged cardiac tissue.
[0074] Evidence has been obtained showing that CPCs are retained in vivo, and actively divide at the site of infarction. For instance, Figures 54A-H show that CPCs, labeled with CFSE and injected into four sheep approximately one month after infarction, were retained for at least 55 days post-injection. Figure 55A displays a representative section showing CFSE+ CPCs retained in the infarct zone, and Figure 55B displays a graph of the percentage of total cells in each sheep that are retained CFSE+ CPCs. Furthermore, Figures 56A-D show that transplanted CPCs are actively dividing in the heart. Figures 56A-B display representative sections from the infarct zone of two sheep, stained with anti-Ki-67 to identify the dividing cells. The CFSE+ cells, shown in green, are the introduced CPCs. The sections were stained with anti-Ki-67 to identify dividing cells shown in red. The dividing CPCs are co-localized and image yellow. Figure 56C shows a graph of the percentage of CFSE+ CPCs which are actively dividing in each animal. Figure 56D illustrates a compiled percentage of the CFSE+CPCs which were actively dividing among all four sheep.
[0075] Evidence has been obtained showing that CPCs are able to differentiate into all cardiovascular lineages. Figure 57A shows that non-infarct sections exhibit Troponin I staining, while Figure 57B shows infarct zone staining with anti-Troponin I, illustrating co- localization (yellow) of anti-Troponin I (red) labeling and the CFSE (green) labeling of the introduced cells. Figure 57C shows that the introduced CPCs exhibited cardiac differentiation in all four sheep studied. Figure 57D shows the non-infarct section of the left ventricle exhibits von Willebrand Factor (vWF) staining (red) indicative of endothelial cells. Figure 57E shows that the infarct zone section exhibits CFSE labeled introduced CPCs (green), von Willebrand Factor labeling (red) and introduced CPCs which have differentiated into endothelial cells (colocalized - yellow, and indicated by arrows). Figure 57F shows that the introduced CPCs exhibited endothelial cell differentiation in three of the four sheep studied.
[0076] Evidence has been obtained showing that endothelial cells are recruited into the infarct zone after CPC injection. Figure 58A shows a representative section of the non-infarct area of the left ventricle, showing vascularization. Endothelial cells are stained with anti-vWF (red). Figure 58B shows a representative section of the infarct area of the left ventricle, showing vascularization. Endothelial cells are stained with anti-vWF (red). The CPCs are labeled with CFSE (green). Figure 58C shows the infarct area contains a large number of vWF positive endothelial cells (red). Figure 58D shows that there is an increase in the total percentage of cells which are vWF+ in the infarct zone in all four sheep, illustrating recruitment of endogenous endothelial cells.
[0077] Furthermore, these cells can be rapidly expanded. Time to transplant, from 1 cell to 1x10 cells, is illustrated in Figure 19. Cardiac progenitor cells were isolated from a 1-month-old patient. Begin with 0.8 cells/well in a 96 well plate. One clone expanded to determine time from single cell to enough for a patient. At day 0, about one cell can be present. At day 24, about 1.38x10 total cells can be present. Therefore, the time to transplant can be about 3.5 weeks. In some embodiments, the time to transplant can be about 2.5 to 4.5 weeks, for example, the time to transplant can be about 2.5 weeks, about 3 weeks, about 3.5 weeks, about 4 weeks, about 4.5 weeks, about 5 weeks, about 5.5 weeks, about 6 weeks, or greater than 6 weeks.
[0078] Thus understanding endogenous cardiac progenitor cells is important because they represent a heart's endogenous regenerative potential. These cells have been isolated as isl-l+c-kit+ cells which are clonogenic and can also be grown as cardiospheres. These cells are HLA Class I positive and HLA Class II negative as shown in Figure 5. They have been shown to express various growth factors such as IGF-1, SDF-Ια, and HGF. The cells also express Mesp- 1 and low levels of Brachyury as shown in Figures 1A-C. They are therefore very early cardiovascular progenitors that have the capability to proliferate and produce additional early cardiovascular progenitor cells as well as differentiate in vivo into all lineages necessary for cardiovascular repair in vivo. Furthermore, they have been noted to be heterogeneous in phenotype and regenerative capability. Figure 16 shows that endogenous CPCs are heterogeneous. The results depicted in Figure 17 shows that isl-l+ CPC clones express KDR, PDGFR and CXCR4. Figure 18 illustrates isl-1 +c-kit+ PDGFRa +IGF1R +CPC clones. PDGFR and IGF1R are present on subpopulations of c-kit+ progenitors with superior regenerative capacity as shown in Figure 18. In particular, neonatal CPCs have been seen to regenerate the heart more effectively than adult CPCs. The mechanism of how this heterogeneity and the aging process impact the regenerative capacity of cardiac progenitors has been studied. It was found that microRNAs that promote proliferation (mir-17, mir-20a, mir-106b, and mir-93) were highly expressed in neonatal cardiac progenitors. Cell cycle analysis shows that a higher percentage of neonatal vs. adult CPC were actively dividing. MicroRNAs that promote senescence were highly expressed in adult CPC. There are significant (p<0.05) microRNA differences between neonatal and adult cardiac progenitors which impact their capacity to invade. Neonatal CPCs further have increased AKT autocrine/paracrine signaling as shown by enhanced levels of AKT activation.
[0079] In summary, isl- 1 +c-kit+ endogenous CPC represent a promising new population of cardiovascular progenitors that can be expanded rapidly and studied using single cell, clonal populations isolated from human neonates and adults. Furthermore, these cells are suitable for a repairing a wide variety of cardiovascular damage, including myocardial infarction among other types of damage.
Isl- l+c-kit cardiac progenitor cells are useful for cardiac repair
[0080] Cardiac progenitor cells suitable for cardiac repair can be obtained from a variety of sources. Cardiovascular progenitor cells residing within the heart co-express isl-1 and c-kit. In some embodiments, endogenous stem cell-derived cardiomyocytes are isolated from humans. In some embodiments, CPCs are isolated from the atrial appendage, epicardium or ventricle of human patients. Isolation and selection of cardiac stem cells may be conducted as depicted in Figures 2A-B. Further characterization of the CPCs, as shown in Figures 3A-B, shows that some clones co-express the markers isl-1 and c-kit. As can be seen in Figure 3 A, flow cytometry was used to verify the cells positively express isl-1. This was confirmed by RT-PCR where the indicated bands correspond to isl-1 expression and c-kit. These CPCs coexpressing isl-1 and c-kit were identified in clones from both neonatal and adult sources and they represent a new cardiovascular progenitor cell population.
[0081] In some embodiments, autologous stem cells can be used for cardiovascular repair. These can include bone marrow-derived cells and mesenchymal stem cells as are used in early clinical trials. These cells can be safe but have modest, if any, functional improvement. In some embodiments, resident cardiac progenitor cells can be used for cardiac repair. Resident cardiac progenitor cells exhibit heterogeneity, including phenotype and regenerative capacity cell type differences and age-related differences. There is evidence that aging plays a role in the quality of stem cells that are isolated from the heart. Cells isolated from cardiac biopsies and expanded as c-kit+ cells or cardiosphere-derived cells can improve cardiac function in animal models.
[0082] In some embodiments, human embryonic stem cell-derived cardiovascular cells can be used for cell-based therapy. These are available "off the shelf and it has been shown that these cells can improve cardiac function after a myocardial infarction in animals. Cell retention and survival is limited and the long-term efficacy may be improved. Additionally, safety concerns that allogeneic cells may initiate an immune response may be addressed.
[0083] Cardiac progenitor cells can be isolated using various markers. In some embodiments, these progenitors are c-kit positive cells or cardiosphere-derived cells. The cells are safe and have been reported to reduce scar size and increase cardiac muscle mass. The newly introduced cells can be used to effectively regenerate the damaged heart tissue. While there is a wide range of expression of cell markers in CPCs, some markers were found to be expressed in all the clones such as ckit, pdgfr, kdr. Some markers were found to be selectively expressed, such as SSEA-4, where some clones were positive and some negative. Flow cytometry may be used to identify phenotypes of neonatal and adult progenitor cells, and to identify phenotypically similar clones as shown in Figure 4. In Figure 4, each column represents one clone and three representative neonatal and adult clones are shown. Using phenotype data, clones that were most similar when comparing neonatal and adults CPCs were selected for further differentiation and functional studies. [0084] The isl-l+ckit+ cardiovascular progenitor cells uniquely express markers of early cardiogenic mesoderm, thereby indicating multipotency to differentiate into a variety of cardiovascular derivatives. As shown in Figure 1A, mesoderm differentiates into cardiogenic mesoderm, which has the capacity to become several types of cardiac cells, including cardiomyocytes, endothelium, smooth muscle cells, and sinoatrial nodal cells. While cardiogenic mesoderm and more committed cells express Isll and c-kit, cardiovascular progenitors that precede the cardiogenic mesoderm stage express Mesp-1 , Brachyury, KDR/Flk-1, and PDGFRa. The expression of markers of early cardiogenic mesoderm was assessed by visualizing amplicons of genes of interest using gel electrophoresis (Figure IB) and by flow cytometry (Figure 1C). Gene expression analysis indicates the presence of Mesp- 1 and a low level of Brachyury as well as of the robust co-expression of Isl 1 and c-kit, which suggests a transition from primordial cardiovascular progenitor to early cardiogenic mesoderm. Protein expression confirms the expression of committed first and second heart field cells, as indicated by the co-expression of Isll and c-kit, while also demonstrating the expression of nascent cardiac progenitor markers, including PDGFRa and KDR. Ultimately, the cardiac stem cells used herein are well positioned to develop into the myriad of cardiac derivatives. Further, Figure 20 shows the distribution of markers for cell types illustrating that endogenous CPCs differentiate into all cardiovascular lineages. This data was captured using the dexamethasone protocol. There are several theories of the cellular hierarchy during cardiovascular commitment at day 0 through after day 6. One theory is described in Bondue A et al. J Cell Biol 201 1 ; 192:751-765, which is hereby incorporated by reference in its entirety and should be considered a part of this specification.
[0085] Several procedures can be used for differentiation. In one embodiment, the differentiation protocol used can be similar to the differentiation protocol described in Smits AM, van Vliet P, Metz CH, Korfage T, Sluijter JP, et al. (2009) Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology. Nat Protoc 4: 232-243, which is hereby incorporated by reference in its entirety and should be considered a part of this specification. Gene expression during differentiation can be identified. In one experiment, RT-PCR was used to identify gene expression changes during differentiation of these clones. In this experiment, Trop T was acquired and other markers such as Oct 4 were lost. This along with other gene expression data demonstrates that these clones can be differentiated into cardiomyocytes. Both neonates and adults can be differentiated in adult cardiomyocytes. Their differentiation markers are the same or similar.
[0086] There are various procedures for differentiation, for example the procedure described in D'Amario D, Fiorini C, Campbell PM, Goichberg P, Sanada F, et al. (201 1) Functionally competent cardiac stem cells can be isolated from endomyocardial biopsies of patients with advanced cardiomyopathies. Circ Res 108: 857-861, which is hereby incorporated by reference in its entirety and should be considered a part of this specification. Figure 21 illustrates the differentiation into cardiomyocytes and the timeline of the differentiation procedure as described in the differentiation protocol in Smits AM, van Vliet P, Metz CH, Korfage T, Sluijter JP, et al. (2009) Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology. Nat Protoc 4: 232-243, incorporated by reference herein. Figure 22 shows the differentiation of progenitor clones. Figures 23A-C illustrates that the CPCs form endothelial cells in a tube formation assay. Figure 23A illustrates the 96 well plate with plating 10,000 cells in medium containing VEGF and coat with Matrigel. Figure 23B shows an image tube formation 5 hours post-plating. Quantifying using Wimasis software is illustrated with Figure 23C including: branching points; tube length, numbers, and statistics; loop numbers, areas, and perimeters; and cell-covered area. The tube assay is described in detail in Arnaoutova I, George J, Kleinman HK Benton G. The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art. Angiogenesis (2009) 12:267-274, which is hereby incorporated by reference in its entirety and should be considered a part of this specification. Figure 20 shows that both neonatal and adult CPCs differentiate into all three cardiac lineages. Figures 59A-D show that CPCs form capillary-like networks on Matrigel. Similarly, 24A-B shows that neonatal and adult CPCs form capillary-like networks on Matrigel. Figure 24A shows neonatal CPCs at about 5 hours. Figure 24B shows adult CPCs at about 5 hours.
Neonatal cells provide superior regenerative capacity [0087] Functional differences were observed between neonatal and adult CPCs, including cell cycling and proliferation as well as motility and invasion. Therefore, the regulatory mechanisms that govern the differences in functional ability between neonatal and adult CPCs was explored. For example, differences in cell cycle and invasion, signaling in response to cytokines and growth factors, gene expression, and microRNA were examined.
[0088] The cell cycle includes the Gl , S, G2 and M phases. The cell cycle can be analyzed using ethanol fixation, RNAse (Remove Cellular RNA), propidium iodide (intercalates into DNA), and run on Flow Cytometry. As illustrated in Figure 6, it has been shown that a greater percentage of adult cardiac progenitors are in Gl. Conversely, as shown in Figure 7, a greater percentage of neonatal cardiac progenitors are actively dividing, as shown by the significantly higher frequency of neonatal CPC in the S and G2 phase.
[0089] Differences in microRNA expression and intracellular signaling in response to growth factors impact the function of CPCs isolated from older adults as compared to neonates. MicroRNAs regulate gene expression. MicroRNAs can be small 20 nucleotide molecules. They can regulate cardiac development. MicroRNA is conserved across species and gene expression is controlled through microRNA degradation or inhibition of translation. MicroRNA can target multiple genes. MicroRNA profiling was studied. 42 significant microRNA expression differences were identified when comparing neonatal and adult cardiac progenitors as illustrated in Figure 25.
[0090] The microRNA expression pattern of neonatal cardiac progenitors was more similar to that of human embryonic stem cells. Figure 26 shows that microRNAs that promote proliferation are highly expressed in neonatal CPCs. Figures 27A-B shows the microRNA expression and senescence. Figure 27A shows that microRNAs that prevent senescence are elevated in neonatal CPC. Additionally, Figure 27B shows that miR -371-3p is upregulated in senescent stem cells and is highly expressed in adult CPC. Further, as shown in Figure 33, microRNA expression is associated with enhanced capacity to invade in neonatal CPCs. Neonatal CPCs have higher levels of expression of microRNAs that promote invasion and lower levels of microRNAs that inhibit invasion.
[0091] Paracrine effects are largely responsible for the improvements in cardiac function that have been achieved with stem cell-based therapies. Insulin-like growth factor- 1 (IGF-1) and stromal cell-derived factor 1 (SDF- 1) have been well documented for their ability to promote cardiovascular cell growth, migration and regeneration. The beneficial effects of these and other growth factors when administered without a cell type capable of cardiovascular regeneration are transient. Stem cell-based therapy may thus be improved by understanding how growth factors promote cellular expansion. Autocrine and paracrine actions of the transplanted stem cells may promote better cardiac regeneration. Likewise, exogenous growth factors, including IGF- 1 and SDF-1 , administered together with the stem cells or in conjunction with (before or after) administration of the stem cells may promote better stem cell proliferation and cardiac regeneration.
[0092] However, signaling is impaired in cardiovascular progenitor cells isolated from the aged adult population, rendering them less responsive to growth factors that stimulate migration to the site of injury. Neonatal CPCs produce and are activated by IGF-1 and SDF-1, factors that play a key role in cardiovascular repair, and exist as a heterogeneous population. As shown in Figure 11 , transcripts for SDF-1 a and IGF-1 were expressed at higher levels in neonatal CPCs compared to adult CPCs. These findings suggest elevated autocrine and paracrine signaling and elaboration of growth factors in neonatal CPCs, as compared to adult CPCs, may aid in progenitor cell survival, proliferation, and migration.
[0093] Neonatal clones could be further distinguished by their differential response to SDF- 1 and IGF-1. The response of isl- l+ c-kit+ co-expressing neonatal and adult CPCs to IGF- 1 and SDF- 1 was compared. Cardiomyocytes from neonates responded to IGF- 1 treatment with a nearly two-fold increase in AKT phosphorylation while cells from adults showed no response. Flow cytometry revealed that IGF-1 receptor levels were present on both neonatal and adult CPCs. Accordingly, age impacts cardiovascular progenitor cell signaling and may account for the inability of cardiovascular progenitor cells to mobilize to the site of injury and repair the heart in older adults.
[0094] Neonatal clones could be further distinguished by their differential response to SDF- 1. The adult cells were not stimulated to invade in response to SDF- 1 and there was no evidence of AKT activation, although the receptors were present on the cells as identified by flow cytometry. A higher number of neonatal CPCs invade in response to SDF as shown in Figure 9A. The possibility that this finding was due to a lack of SDF- 1 receptors on the adult progenitors was ruled out; flow cytometry was used to show SDF-1 receptors CXCR4 and CXCR7 were adequately expressed on both neonatal and adult CPCs as shown in Figure 9B. Figure 11 shows transcripts SDF-1 a and IGF-1 are expressed at higher levels in neonatal CPCs when compared to adults. These findings suggest elevated autocrine and paracrine signaling in neonatal CPCs which aid in cell survival, proliferation, and migration.
[0095] AKT is well documented to increase cell survival and induce antiapoptotic effects, to promote cardiomyocyte cycling and expansion, and also increase cell motility and migration. Accordingly, the AKT pathway was studied to look at the differences between neonatal and adult CPCs.
[0096] Activation of the AKT pathway is higher during differentiation of neonatal CPCs as shown in Figure 10. Using RT-PCR, it was found that genes associated with AKT pathway are induced at higher levels during differentiation of neonatal CPCs than adult. Figure 12A-B illustrates that AKT signaling is increased in neonatal CPCs in response to SDF-1 a. Figure 12 shows that AKT signaling is increased in neonatal CPCs and not in adult CPCs in response to SDF-1 a. AKT signaling was increased in neonatal CPCs with SDF-1 a treatment. AKT was not phosphorylated in adult CPCs after SDF- 1 a treatment.
[0097] Similarly, with IGF-1, AKT signaling was also found to be increased in neonatal CPCs. Figures 13A-B shows that AKT signaling was increased in response to IGF- 1 treatment in neonatal CPCs, but not in adult CPCs. This indicates a possible mechanism for the observed differences noted between neonatal and adult CPCs.
[0098] Additionally, as described with reference to Figure 10, the activation of the AKT pathway were examined for neonatal vs. adult CPCs. Activation of the AKT pathway is higher during differentiation of neonatal CPCs as shown in Figure 10. Using RT-PCR, it was found that genes associated with AKT pathway are induced at higher levels during differentiation of neonatal CPCs than adult.
[0099] Hypoxic pretreatment can be beneficial to these cells and may benefit cardiac regeneration in vivo, as evidence suggests that short-term hypoxia activates the AKT signaling pathway as shown in Figures 34A-D and 34F. Accordingly, hypoxic pretreatment significantly elevates AKT phosphorylation and upregulates the expression of genes that promote proliferation and survival, as shown in Figures 34E and 34G. The additional benefits of hypoxia preconditioning include anti-apoptotic effects and improved invasion capabilities in vitro. Furthermore, hypoxic pretreatment has no significant effect on the differentiation process or cell cycle progression in isl-l+ hCPCs. In some embodiments, the cells may undergo hypoxic pretreatment prior to administration. In several embodiments, the hypoxic pretreatment comprises subjecting the CPCs to reduced levels of oxygen, for a period of time. For instance, the reduced level of oxygen may range from about 0.1% to about 10%, such as for example, about 0.5%, about 1%, about 2%, about 4%, about 6%, about 8%, about 10% and overlapping ranges thereof. The dwell time for hypoxic pretreatment is variable depending on the embodiment. For example, in several embodiments, the reduced oxygen levels are maintained for a period of time ranging from about 10 minutes to about 10 hours, such as for example, about 15 minutes, about 30 minutes, about 1 hour, about 2 hour, about 4 hours, about 6 hours, about 8 hours, about 10 hours, and overlapping ranges thereof.
[0100] In summary, neonatal CPCs have an increased ability to proliferate and expand compared to CPCs isolated from an adult source. Neonatal CPCs were more invasive than adult CPCs in response to SDF-Ια in vitro despite similar levels of expression of SDF-1 receptors CXCR4 and CXCR7. Neonatal CPCs have increased AKT autocrine/paracrine signaling and an increased intracellular response to growth factor as seen by enhanced levels of AKT activation. The impact of these differences on the ability of adult cardiac progenitors to repair the heart in vivo was determined in further studies.
Definitions
[0101] A "patient" as used herein may refer to a human or a non-human mammal, e.g. ., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.
[0102] An "effective amount" or a "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent that is effective to relieve, to some extent, or to reduce the likelihood of onset of, one or more of the symptoms of a disease or condition, and includes curing a disease or condition. [0103] "Treat," "treatment," or "treating," as used herein refers to administering a compound or pharmaceutical composition to a subject for prophylactic and/or therapeutic purposes. The term "prophylactic treatment", "preventing" or "prevent" refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term "therapeutic treatment" refers to administering treatment to a subject already suffering from a disease or condition.
[0104] Administration of the therapeutic agents disclosed herein or the pharmaceutically acceptable salts thereof can be via any of the accepted modes of administration for agents that serve similar utilities. Such modes include, but are not limited to, administration via a therapeutic cell matrix, a therapeutic cell suspension, and transplantation of a clonal population of cells. In some embodiments, a therapeutic cell matrix may be an implantable biodegradable matrix configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the matrix into any region of cardiovascular damage, such as an infarct zone, wherein the isl-l+c-kit+ progenitor cells are capable of regenerating damaged heart tissue. In some embodiments, a therapeutic cell suspension may be administered intramuscularly into cardiovascular tissue.
[0105] In embodiments that include administering a combination of a cell or compound described herein and another agent, the combination may be provided to caregivers as a mixture, or the caregivers may mix the two agents prior to administration, or the two agents may be administered separately.
[0106] The actual dose of the stem cells or active compounds described herein depends on the specific cells or compounds, and on the condition to be treated; the selection of the appropriate dose is well within the knowledge of the skilled artisan. For instance, the number of cells to be administered may range from less than 10 3 to at least 1010. In some embodiments, the number of cells to be administered may be 10 3J, 106°, 108°, 108°, 1010, and overlapping ranges thereof. In some embodiments, the number of cells to be administered may be 10 . [0107] Some embodiments include the combination of compounds, therapeutic agents, and/or pharmaceutical compositions described herein. In such embodiments, the two or more agents may be administered at the same time or substantially the same time. In other embodiments, the two or more agents are administered sequentially. In some embodiments, the agents are administered through the same route (e.g. orally) and in yet other embodiments, the agents are administered through different routes (e.g. one agent is administered orally while another agent is administered intravenously).
[0108] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
[0109] Various numerical examples, tables, graphs, and data are presented herein. These numerical examples, tables, graphs, and data are intended to illustrate certain example embodiments and not intended to limit the scope of the disclosed methods and kits.
[0110] The various features, kits, and methods described above may be used independently of one another, or may be combined in various ways. All possible combinations and subcombinations are intended to fall within the scope of this disclosure. In addition, certain method or process blocks may be omitted in some implementations. The methods and processes described herein are also not limited to any particular sequence or order, and the blocks or operations relating thereto can be performed in other sequences or orders that are appropriate. For example, described blocks or operations may be performed in an order other than that specifically disclosed, or multiple blocks or operations may be combined in a single block or operation. The example blocks or operations may be performed in serial, in parallel, or in some other manner. Blocks or operations may be added to, removed from, or rearranged compared to the disclosed example embodiments. The example kits and components described herein may be configured differently than described. For example, elements may be added to, removed from, or rearranged compared to the disclosed example embodiments.
[0111] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to volume of wastewater can be received in the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.
[0112] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., " a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to "at least one of A, B, or C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., " a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."
[0113] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
[0114] Conditional language used herein, such as, among others, "can," "could," "might," "may," "e.g.," and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and/or steps. Thus, such conditional language is not generally intended to imply that features, elements and/or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and/or steps are included or are to be performed in any particular embodiment. The terms "comprising," "including," "having," and the like are synonymous and are used inclusively, in an open-ended fashion, and do not exclude additional elements, features, acts, operations, and so forth. Also, the term "or" is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term "or" means one, some, or all of the elements in the list.
EXAMPLES
[0115] The following Examples are presented for the purposes of illustration and should not be construed to limit the invention to the particular features or embodiments described.
Example 1: Isolation and culture of Isll+ human cardiac progenitor cells
[0116] The Institutional Review Board of Loma Linda University approved the protocol for use of tissue that was discarded during cardiovascular surgery, without identifiable private information, for this study with a waiver of informed consent. Human Isll+ cardiac progenitor cells (hCPCs) were isolated from discarded cardiac tissue as illustrated in Figures 2A-B. Briefly, discarded atrial tissue was cut into small pieces then enzymatically digested using collagenase. The resulting solution was then passed through a 40μπι cell strainer. Cells were diluted and plated at a concentration of 0.8 cells per well to create clonal populations for expansion. Human CPC cultures were supplemented with growth media comprised of 12.0% fetal bovine serum (Thermo Scientific, Waltham, MA), 1.0% Penicillin-Streptomycin (Life Technologies, Carlsbad, CA) and 20.0% Endothelial Cell Growth Media (Lonza, Basel, Switzerland) in M199 (Life Technologies, Carlsbad, CA). Expression of Isll and cKit was quantified using Flow Cytometry.
[0117] This clonal model of CPCs has the benefit of being pure and thus the cells can be well characterized. They can be examined for efficacy and safety in large animal models, and are capable of becoming "available off-the-shelf." A total of 240 cardiovascular clones were isolated from a number of patients.
Example 2: Flow Cytometry
[0118] Human cardiac progenitor cells were fluorescently labeled with antibodies as recommended by their respective manufacturers. Progenitor cell populations were analyzed using a MACSquant analyzer (Miltenyi Biotec, Auburn, CA). Quantification of data was performed using FlowJo software (Ashland, OR). Small particulate matter, dead cells, and gas-bubbles were excluded from final analysis using forward-scatter and side- scatter data. Antibodies used in this study include: Phospho-Akt (S473) (D9E) Rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA), FITC goat anti-rabbit IgG polyclonal antibody (BD Biosciences, San Jose, CA) and Flourescein-anti-BrdU (PRB-1) monoclonal antibody (Phoenix Flow Systems, San Diego, CA).
Example 3: Quantitative Real-Time PCR
[0119] Human cardiac progenitors were washed with DPBS, then lysed using TRIzol reagent (Life Technologies, Carlsbad, CA). Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, and Limburg) then used as a template for cDNA synthesis using superscript III (Life Technologies, Carlsbad, CA). Quantitative RT-PCR was performed using Go-Taq® qPCR Mastermix (Promega, Madison, WI). Measurements were recorded using the iCycler iQ5 PCR Thermal Cycler (Bio-Rad, Hercules, CA). Cycler settings were set to 94°C for 10 minutes, 94°C for 15 seconds, 52-56°C (depending on the primer) for 60 seconds, and 72°C for 30 seconds for a total of 45 cycles. Human primers were created using NCBI primer blast as listed in Table 1. Relative gene expression data was analyzed using the comparative d method.
Table 1. Primer List
GENE FORWARD PRiMER REVERSE PRiMER
β-Aciin TTT GAA TGA GCC TTC GTC CGC GGT CTC AAG TCA GTG TAC AGG TAA GC c-Jun GTC CGC ACT GAT CCG CTC CG GGG CTG CGC GCA CAA GTT TC hRe!A GCG AGA GGA GCA GAG ATA CC GGG GTT GTT GTT GGT CTG GA c-Myc AAG AGA GCG GCA GCC CGA AC TGG GCG AGC TGC TGT CGT TG
CCMD1 TGC TGG AGT CAA GCC TGC GC AGG ACA TGC ACA CGG GCA CG
PiKSCA AACAAT GCC TCC ACG ACC AT TCA CGG TTG CCT ACT GGT TC
A20 CTG GGA CCA TGG CAC AAC TC CGG AAG GTT CCA TGG GATTC
Bd2 TGC ACC TGA CGC CCT TCA C AGA CAG CCA GGA GAA ATC AAA CAG
HiT!O l CTC TCG AGC GTC CTC A ACT ATC AGA CAA TGT TGT
HSP4G TTT ICG GAG GGT CCA ACC CCT TCT TGT TTG AGG CGG GAT GGC C
HSP70 TGA CCA AGA TGA AG G AGA TCG GTC AAA GAT GAG CAC GTT GC
HSP72 GGT CCG GAT AAC GGC TAG CCT CCA CCG GGT CGC CGA ACT TG
HSPSO GGA TTT GAG GGG AAG A TGA GCT TTC ATG ATT C
HSP105 TTATCA GCC AGC CGC CGC TG CCT GCC TGC TTC TCC TGC CG
Brachyury ACT GGA TGA AGG CTC CCG TCT CCT T CCA AGG CTG GAC CAA TTG TCA TGG G
Oct4 AAC CTG GAG TTT GTG CCA GGG TTT TGA ACT TCA CCT TCC CTC CAA CCA
TropT GTG GGA AGA GGC A.GA CTG AG ATA GAT GCT CTG CCA CAG C
MLC2V TAT TGG AAC ATG GCC TCT GGA T GGT GCT GAA GGC TGA TTA CGT T
CGC TAT ATC GGC CAC CTG TC GGC ATC CAG GTC TCC AAC AG
Example 4: TUNEL Assay
[0120] Human cardiac progenitor cells were washed with DPBS and treated with a solution of 0.5mM hydrogen peroxide in culture media for 21 hours. TUNEL assay was performed following the manufacturers recommendations. Briefly, cardiac progenitors were counted using trypan blue and concentrated to 106cells/ml. The concentrated progenitor cell solution was then re-suspended in 1.0ml of 1% paraformaldehyde in DPBS, placed on ice for 30 minutes, then reconstituted into 1.0ml of 70% ethanol for overnight incubation at -20°C. The next day, the cardiac progenitor cells were labeled with Br-dUTP (Phoenix Flow Systems, San Diego, CA) and re-suspended in Antibody Solution containing the Flourescein anti-BrdU antibody (Phoenix Flow Systems, San Diego, CA). Population analysis was performed using flow cytometry.
Example 5: Trans well Invasion Assay
[0121] Cultrex® basement membrane extract (Trevigen, Gaithersburg, MD) was applied to the upper chamber of a Corning HTS Transwell® plate (8.0μπι pore size, Venlo, Limburg). Human cardiac progenitor cells were suspended in starvation media composed of 98.5% IMDM with GlutaMAX™ (Life Technologies, Carlsbad, CA), 1.0% Insulin- Transferrin-Selenium (Life Technologies, Carlsbad, CA), and 0.5% fetal bovine serum (Thermo Scientific, Waltham, MA) then plated onto the coated wells at a density of 50,000 cells per well. Stromal cell-derived factor 1-a (SDF-la, Life Technologies, Carlsbad, CA), a chemoattractant, was diluted with growth media to a final concentration of lOOng/ml and administered to the lower chamber. After forty-eight hours of incubation at 37°C, the cells in the lower chamber were dissociated, stained with calcein AM (BD Biosciences, San Jose, CA), and analyzed using an FLx800 Microplate Fluorescence Reader (BioTek Instruments, Winooski, VT).
Example 6: Cell Cycle Analysis
[0122] Human Isll+ cardiac progenitor cells at 80% confluency were trypsinized, counted, and concentrated to 105 cells/0.3 ml DPBS. Ice-cold 70% ethanol (0.7ml) was added drop-wise to fix the cells then stored at -20°C overnight. Human CPCs were washed then incubated at 37°C for one hour with RNase A (0.5 mg/ml, Life Technologies, Carlsbad, CA). Propidium Iodide solution (10 μg/ml) was added, and the resulting cell solution was analyzed using a MACSquant analyzer (Miltenyi Biotec, Auburn, CA). Cytometer data was quantified using Flow Jo software (Ashland, OR).
Example 7: Hypoxic Preconditioning
[0123] A population of hCPCs was subjected to hypoxic preconditioning to determine the effect of short-term hypoxia on Akt phosphorylation. A sample of hCPCs was obtained through the procedure detailed in Example 1. The day before hypoxic pretreatment, the hCPC samples fresh culture media, and if necessary, passaged to achieve 80% confluency within 24 hours. Experimental hCPCs were then placed in a Heracell 150 tri-gas incubator (Thermo Scientific, Waltham, MA) set to 1.0% 02, 5.0% C02, and 94% N2 for 6 hours at 37°C. Control CPC conditions were 37°C and 5.0%CO2. The effects of short term hypoxic preconditioning on hCPCs were analyzed with Flow Cytometry, Quatitative Real-Time PCR, Transwell Invasion Assay, TUNEL Assay, and Cell Cycle analysis. Flow cytometry analysis
[0124] The effect of short-term hypoxia on Akt phosphorylation in CPCs was analyzed with flow cytometry according to the procedure detailed in Example 2. Briefly, hypoxia-pretreated hCPCs and non-treated hCPCs were labeled with an antibody specific for
Ser 473 -phosphorylated Akt. Flow cytometry data revealed markedly higher fluorescence intensity readings in preconditioned hCPCs, as shown by Figure 34E. Subsequent trials demonstrated a significant increase in Akt activation in hCPCs that received hypoxic pretreatment as compared to hCPCs kept under control conditions, with a more significant increase observed in neonatal hCPCs as shown by Figure 34C. qRT-PCR analysis
[0125] The effect of short-term hypoxia on the expression of genes that are known to play a role in the Akt pathway was analyzed with Quantitative RT-PCR according to the procedure detailed in Example 3. The data shows that pretreated, hypoxic hCPCs express significantly higher levels of nearly all Akt-related genes when compared to non- treated, normoxic hCPCs, including phosphoinositide 3-kinase, which is known to activate Akt, lending further credence to the link between hypoxia and Akt activation, as shown in Figure 34A. Figure 34B shows a greater change was observed in neonatal hCPCs.
[0126] The effect of short-term hypoxia on the expression of several heat-shock proteins was also analyzed with Quantitative RT-PCR according to the procedure detailed in Example 3. The expression profiles of several heat- shock proteins in control and experimental populations using qRT-PCR. Data analysis confirms that the pretreated hCPCs indeed express higher amounts of heat-shock protein RNA transcripts, as shown by Figure 34G. Heat-shock proteins 40 and 90 were significantly upregulated, 2 and 3-fold respectively, in preconditioned hCPCs. Further gene expression analysis revealed that short- term hypoxia not only induces a stress response but also significantly upregulates the transcription of genes associated with cell survival, as shown by Figure 34E. Pretreated hCPCs displayed a 6.5-fold overall increase of B-cell lymphoma 2 (Bcl-2) and a 5-fold increase of Hemoxygenase 1 (Hmoxl), two genes known to regulate apoptosis. [0127] Finally, the effects of short-term hypoxia on differentiation in Isll+ human cardiac progenitors was analyzed using qRT-PCR to measure the expression of several differentiation markers in control and experimental hCPCs. Quantification of data demonstrates that there is no significant difference between pretreated and non-treated hCPCs in four out of five markers examined, as shown by Figure 341. MLC2v, a marker expressed in differentiated cardiomyocytes, was significantly downregulated by hypoxia. However, none of the other surface antigens associated with differentiation were significantly different in pretreated hCPCs. Thus, short-term hypoxia does not impact the differentiation process in Isir hCPCs.
TUNEL Assay
[0128] To ascertain the functional benefits associated with the changes in heat- shock protein expression, a TUNEL assay was performed on both control and experimental hCPCs according to the procedure detailed in Example 4. The data reveals a downward trend in total apoptotic cells with hypoxia preconditioning, as shown in Figure 34H. However, statistical analysis deems this result non-significant.
Transwell Invasion Assay
[0129] To determine the effect of short term hypoxia on cellular motility, the chemotaxis in response to SDF-la amongst non-treated and pretreated hCPCs was compared using a Transwell Invasion Assay according to the procedure detailed in Example 5. A statistically significant difference was noted between non-treated hCPCs and pretreated hCPCs. The human cardiac progenitors preconditioned with hypoxia invaded through the basement membrane extract and Transwell pores in significantly greater numbers than the non-treated hCPCs as shown by Figure 35.
Cell Cycle Analysis
[0130] Cell Cycle Analysis was performed on hypoxia pre-treated CPCs and non- treated CPCs according to the procedure detailed in Example 6. No significant difference was identified between the two populations, indicating that short-term hypoxia does not inhibit normal cell cycle progression.
Example 8: Neonatal CPC are more responsive to growth factors than adult CPCs
[0131] 264 clones of human neonatal and adult cardiovascular progenitor cells (CPCs) were isolated from patients undergoing cardiothoracic surgery, using the procedure described in Example 1. Forty-seven clones were selected for surface phenotype characterization using flow cytometry. Real time PCR was used to further distinguish the clones during differentiation into cardiac myocytes based on the expression of isl- 1 , MESP- 1 , c-kit and TropT. Differences in AKT cell signaling were identified in CPC clones in response to SDF- 1 and IGF- 1 by Western blotting. Invasion assays were also performed.
[0132] Results indicated that cardiovascular progenitor cell clones can produce both IGF-1 and SDF-1, factors that play a key role in cardiovascular repair. Functional differences were identified in the response of CPC populations to various growth factors. The response of isl-l+ c-kit+ co-expressing neonatal and adult CPCs to IGF-1 and SDF-1 was compared. Cardiomyocytes from neonates responded to IGF-1 treatment with a nearly two-fold increase in AKT phosphorylation while cells from the adult showed no response. Flow cytometry revealed that IGF-1 receptor levels were present on both neonatal and adult CPCs. Neonatal clones could be further distinguished by their differential response to SDF- 1. The adult cells were not stimulated to invade in response to SDF-1 and there was no evidence of AKT activation, although the receptors were present on the cells as identified by flow cytometry.
[0133] From these results, it was concluded that cardiovascular progenitor cells residing within the heart co-express isl- 1 and c-kit. CPCs produce and are activated by IGF- 1 and SDF-1 and exist as a heterogeneous population. Age impacts cardiovascular progenitor cell signaling and may account for the inability of cardiovascular progenitor cells to mobilize to the site of injury and repair the heart in older adults.
Example 9: A greater percentage of neonatal CPCs are actively dividing as compared to adult CPCs [0134] Propidium iodide based cell-cycle analysis and flow cytometry was used to define the number of cells in each stage of the cell cycle. A greater percentage of neonatal CPCs are actively dividing as shown in Figure 6. It was found that a higher frequency of adult progenitors were identified in the Gl phase while a higher frequency of neonatal progenitors were found in the S and G2 phase as shown in Figure 7. This revealed that a greater percentage of neonatal progenitors are actively dividing than compared to adult CPCs.
Example 10: Neonatal CPCs invade more readily in response to growth factors such as SDF-la
[0135] A Transwell invasion assay was also employed to see if the adults have a lesser capacity to respond to a site of injury. Figure 8 illustrates the Transwell invasion assay. The ability to respond and invade is important because cardiac regeneration in vivo requires CPCs to move from their stem cell niche and invade to the site of injury.
[0136] To measure this ability, a Transwell insert with 8 μπι pores was coated with a basement membrane extract. SDF-la, a growth factor known to be secreted in the heart to recruit CPCs to the site of injury, was selected as the chemoattractant. After 24 hours, the number of cells that migrated through the membrane by flow cytometry was quantified. Components included: Starved media 24 hours; SDF-la lOOng/mL of M199 + EGM-2; Coated with: Cultrex basement extract from Trevigen.
[0137] Neonatal CPCs invade more readily in response to SDF-la. What was found was that a higher number of neonatal CPCs invade in response to SDF as shown in Figure 9 A. The possibility that this finding was due to a lack of SDF- 1 receptors on the adult progenitors was ruled out. By flow cytometry, it was found that SDF-1 receptors CXCR4 and CXCR7 were adequately expressed on both neonatal and adult CPCs as shown in Figure 9B.
Example 11: Neonatal CPCs exhibit increased phosphorylation of the AKT pathway as compared to adult CPCs, leading to increased proliferation and motility.
[0138] In studying the signaling pathways and looking at the differences between neonatal and adult CPCs, the AKT pathway is studied. AKT is well documented to increase cell survival and induce antiapoptotic effects, to promote cardiomyocyte cycling and expansion, and also increase cell motility and migration. The observed functional differences between neonatal and adult CPCs include cell cycling and proliferation as well as motility and invasion.
[0139] Activation of the AKT pathway is higher during differentiation of neonatal CPCs as shown in Figure 10. Using RT-PCR, it was found that genes associated with AKT pathway are induced at higher levels during differentiation of neonatal CPCs than adult.
[0140] Figure 1 1 shows transcripts SDF-Ια and IGF-1 are expressed at higher levels in neonatal CPCs. Furthermore, transcripts for SDF-la and IGF1 were expressed at higher levels in neonatal CPCs when compared to adults. These findings suggest elevated autocrine and paracrine signaling in neonatal CPCs which aid in cell survival, proliferation, and migration.
[0141] Figures 12A-B illustrates that AKT signaling is increased in neonatal CPCs in response to SDF-la. To directly characterize the differences in AKT signaling, AKT protein phosphorylation in CPCs when exposed to growth factor was measured. In this assay, cells were treated with growth factors SDF-la or IGF-1 for 30 minutes. The samples were lysed and western blots were performed to measure AKT activation or phosphorylation. AKT signaling was found to be increased in neonatal CPCs in response to both SDF-la while signaling was found to be reduced in adult CPCs. Figures 12A-B show that AKT signaling was increased in neonatal CPCs with SDF-la treatment and AKT signaling was reduced in adult CPCs with SDF-la treatment. Included: AKT phos on ser473; lOOng/mL SDF-la; +some neonates also have a decreased or no AKT phosphorylation.
[0142] Similarly with IGF-1, AKT signaling was also found to be increased in neonatal CPCs. Figures 13A-B shows that AKT signaling was increased in neonatal CPCs with IGF- 1 treatment and AKT signaling was unchanged in adult CPCs with IGF- 1 treatment. This indicates a possible mechanism for the observed differences noted between neonatal and adult CPCs. Included: lOOng/mL IGF-1.
[0143] In summary, neonatal CPCs have an increased ability to proliferate and expand compared to CPCs isolated from an adult source. Neonatal CPCs were more invasive than adult CPCs in response to SDF-la in vitro despite similar levels of expression of SDF-1 receptors CXCR4 and CXCR7. Neonatal CPCs have increased AKT autocrine/paracrine signaling and an increased intracellular response to growth factor as seen by enhanced levels of AKT activation. The impact of these differences on the ability of adult cardiac progenitors to repair the heart in vivo was determined in further studies.
Example 12: The mechanism responsible for differences in regenerative capacity of adult vs neonatal CPCs
[0144] The mechanisms responsible for differences in regenerative capacity in human neonatal and adult CPC were explored. For example, microRNA and gene expression, cell cycle and invasion, and signaling in response to cytokines/growth factors were examined.
[0145] Differences in microRNA expression and intracellular signaling in response to growth factors impact function in CPCs isolated from older adults vs. neonates. This was observed by using cardiovascular progenitor cell clones isolated from the heart of human patients. Pure, well-characterized CPC populations were used. Progenitors capable of regeneration were identified and safety and effectiveness in large animal models was observed.
[0146] Isolation and selection of cardiac stem cells was conducted as described above with reference to Figures 2A-B. Further, these results take into account the histocompatibility of antigens on cardiac progenitors, flow cytometry of the phenotype of CPC populations, and the co-expression of isl-1 and c-kit in neonatal and adult cardiovascular progenitors as described above with reference to Figures 3A-B, 4, and 5.
[0147] MicroRNAs regulate gene expression. MicroRNA profiling was studied. 42 significant microRNA expression differences were identified when comparing neonatal and adult cardiac progenitors as illustrated in Figure 25. The microRNA expression pattern of neonatal cardiac progenitors was more similar to that of human embryonic stem cells. Figure 26 shows that microRNAs that promote proliferation are highly expressed in neonatal CPCs. Figures 27A-B shows the microRNA expression and senescence. Figure 27A shows that microRNAs that prevent senescence are elevated in neonatal CPC. Additionally, Figure 27B shows that miR-371-3p is upregulated in senescent stem cells and is highly expressed in adult CPC.
[0148] MicroRNAs that promote proliferation (mir-17, mir-20a, mir-106b, and mir-93) were highly expressed in neonatal cardiac progenitors. Cell cycle analysis shows that a higher percentage of neonatal CPCs were actively dividing as compared to adult CPCs. MicroRNAs that promote senescence were highly expressed in adult CPC. There are significant (p<0.05) differences between neonatal and adult cardiac progenitors in microRNAs impacting their capacity to invade. Neonatal CPCs have increased AKT autocrine/paracrine signaling as shown by enhanced levels of AKT activation.
Example 13: Flow cytometry and RT-PCR indicates CPCs co-express isl-1 and c-kit
[0149] It has been shown that isl-1 and c-kit are co-expressed in neonatal and adult cardiovascular progenitors. Further characterization of the CPCs, as shown in Figures 3A-B, shows that certain clones co-express the markers isl-1 and ckit. As can be seen in Figure 3 A by flow cytometry, the cells positively express isl- 1. This was confirmed by RT- PCR where these bands corresponds to isl- 1 expression and these bands corresponds to ckit. These CPCs coexpressing isl-1 and ckit were identified in clones from both neonatal and adult sources and they represent a new cardiovascular progenitor cell population.
[0150] Further, a wide range of expression of cell markers was found. Some markers were found to be expressed in all the clones such as ckit, pdgfr, kdr. Some markers were found to be selectively expressed, such as SSEA-4, where some clones were positive and some negative.
Example 14: Flow Cytometry indicates CPCs are HLA Class I positive and HLA Class II negative
[0151] Figure 5 shows histocompatibility antigens on cardiac progenitors. Flow cytometry characterization was used to identify similar phenotypic populations of CPC for analysis of microRNA expression and functional analysis. It was found that CPCs are HLA Class I positive and HLA Class II negative as shown in Figure 5. Example 15: Effects of isl-l+ c-kit+ CPCs at site of infarction in sheep model
CPC Isolation, Culture and Administration Post- Infarction
[0152] Tissue was harvested from the right atrium of neonatal sheep and cut into 1 mm cubed pieces. The pieces were then digested with collagenase for about 2 hours at 37°C. A 40 mm cell strainer was used to isolate the CPCs. The solution was diluted to 0.8 cells per well and single cell clones were expanded and confirmed to be isll+ c-Kit+ CPCs by flow cytometry and PCR. The neonatal clones selected for cardiac repair were expanded to ten million cells which was the number of cells administered into the infarct zone of each animal. The CPC were labeled with CFSE on the day of injection. Four allogeneic adult sheep were treated with ten million isl-l+c-kit+ neonatal cardiovascular stem cells at 3-4 weeks post-infarction which was induced by ligation of the left anterior descending coronary artery. Cells were administered in each animal as ten separate injections of one million cells each, directly into the heart in the area surrounding the infarction.
Cardiac Tissue Extraction
[0153] Three months after the infarction, the sheep were sacrificed and the hearts were harvested. Figure 40 shows a harvested sheep heart. The left ventricle (LV) was cut into 1.5 cm pieces, and each piece saved for further study was labeled according to the location. The pieces intended for IHC were wrapped in aluminum foil and frozen with dry ice and liquid nitrogen. The pieces were stored in a -80°C freezer until sectioned. The pieces intended for RNA extraction were placed in RNAlater® solution and stored in a 4°C refrigerator.
Immunohistochemistr and Connexin-43 Quantification
[0154] Tissue Tek® O.C.T. compound was added to surround the LV tissue in a Leica® CM1900 cryostat set to -20°C to ensure clean 6 μηι microtome cuts. The smoothest tissue slices were placed against charged Surgipath® X-tra® slides. Extra tissue in O.C.T. compound from cutting was saved in 15 mL tubes for later RNA extraction. The frozen slides were warmed to room temperature for 30 minutes then put into cold PBS for 10 minutes to rinse off the O.C.T. compound. The slide were then were fixed with cold acetone/methanol 1 : 1 solution for 20 minutes on ice, then washed three times with cold PBS. The slides were left to dry enough to circle the sections with a PAP pen. The slides were blocked for 30 minutes in 5% goat serum in PBS and dilutions of the primary antibody (mouse monoclonal 79010 to Connexin 43 from Abeam®) were prepared in aliquots of the same blocking solution. The blocking solution was aspirated off from the slides and the diluted primary antibody ( 1 :50 to 1 : 1000 concentration) was added. The slides were incubated overnight with wet Kimwipes® at 4°C. The next day the primary antibody was aspirated off carefully and then washed in cold PBS three times for 10 minutes each. The slides were then washed in cold blocking buffer twice for 10 minutes each. 1 :500 dilutions were made of the secondary antibody in (Alexa Fluor® 633 goat anti-mouse IgG (H+L)) in blocking buffer in the dark. After aspirating the blocking solution, the diluted secondary antibody was added in the dark to incubate for an hour. The secondary antibody was aspirated and then washed three times with cold PBS for 10 minutes each. The slides were dried for 10 minutes and then mounted with Prolong Gold with DAPI and a coverslip in the dark. The next day they were sealed with nail polish and viewed under the microscope. The slides stained for connexin 43 were quantified for DAPI, CFSE (injected cells), and connexin 43 with Image Pro software. This procedure is illustrated in Figure 42.
RNA Extraction and Purification
[0155] 30 mg of heart tissue was removed from the RNAlater® solution and put in a round bottom tube. 300 μL· of RLT buffer with b-ME was quickly added to the tube. A Tekmar® homogenizer was washed with 70% EtOH and let dry. The tissue was then lysed. 500 μL· of RNase-free water was added to the lysate. The lysate was then transferred to a 1.5 μL· microfuge tube. 10 of Proteinase K was added and mixed via pipetting then incubated at 55°C for 10 minutes on a heat block. Afterwards the tube was centrifuged at 23 °C for 3 minutes at 10,000g. The supernatant was pipetted into a new sterile microfuge tube and diluted 2: 1 in 100% EtOH and mixed with pipetting. ^00μL· of the supernatant in EtOH was transferred to an RNeasy Mini-spin column in a 2 mL collection tube. It was then centrifuged at 23 °C for 15 seconds at 9,000g. Flow through was discarded. The remainder of the supernatant in EtOH solution was transferred to an RNeasy Mini-spin column in a 2 mL collection tube. It was then centrifuged at 23 °C for 15 seconds at 9,000g. Flow through was discarded. 350 μL· of RWl Buffer was added to the column and centrifuged for 15 seconds at 9,000g. Flow through was discarded. 10 μL· DNase 1 stock solution was added to 70 μL· RDD Buffer, mixed with inversion, then briefly centrifuged. This S0μL· solution was then added to the column's membrane and incubated for 15 minutes on the bench. 350 RWl Buffer was added to the column and centrifuged at 9,000g for 15 seconds. Flow through was discarded. 500 μL· RPE Buffer was added to the column and centrifuged at 9,000g for 15 seconds. Flow through was discarded. 500 μL· RPE Buffer was added to the column and centrifuged for 2 minutes at 9,000g. The column was put into a new 1.5 mL centrifuge tube and 21 of RNase-free water was added to it. The column was then centrifuged for 1 minute at 9,000g at room temperature to elute the RNA. The concentration of the RNA was checked with a NanoDrop spectrophotometer.
First-Strand DNA Synthesis
[0156] 1 μL· of oligo (dT)20 (50μΜ) was added to a nuclease-free microfuge tube. 2μg of RNA in solution up to 11 μL· was added. 1 μL· of lOmM dNTP Mix was added and then nuclease free water was added to bring the total volume to 13 μί. The mixture was heated to 65 °C for 5 minutes with a heat block then put on ice for at least one minute. The contents of the tube were collected via centrifugation then 4 μL· 5X First-Strand Buffer and Ιμί 0.1 M DTT were added. Then I μL· RNaseOUT Recombinant RNase inhibitor and I μL· Superscript III RT were added and mixed gently via pipetting. The tube was incubated at 50°C for 60 minutes in a water bath then the reaction was inactivated at 70°C on a heat block for 15 minutes.
Real-time PCR
[0157] Mastermix was prepared so that each used well of a 96 well plate contained 12.5 μL· SYBR Green, 10.3 μL· nuclease-free water, and (20/96) μL· cDNA. An extra 10% mastermix was included to account for pipetting error. Each primer set was run in triplicate. 3 μL· of each forward and reverse primer was used. After loading the plate, the wells were covered with 8-strip caps and spun with a plate spinner. After placing the plate into a iQ5 real-time PCR cycler the camera was allowed to warm up while the temperature rose to 94°C for 10 minutes. The denaturation temperature was set to 94°C for 15 seconds, the annealing temperature was set to 52°C for 60 seconds, and the elongation temperature was set to 72°C for 30 seconds. This cycle would be repeated 45 times. When the cycles completed the cycle threshold for each well was then recorded.
Gel Electrophoresis
[0158] The agarose gels were composed of 1% or 2% Bio-Rad agarose in 60 mL of IX TBE and 6μL· SYBR Safe from Invitrogen. 5 μL· of a plate well of interest was run with loading dye and a low mass ladder for 45 minutes at 70V. The gel was imaged with a Bio-Rad Gel Doc XR+ with Image Lab software. The noninfarct and infarct zone bands were then compared for each gene of interest. Figure 43 shows exemplary gel electrophoresis results used to compare expression of CXCR4, CXCL12 and IGF-1 at infarcted and non- infarcted sites.
Table 2. Primer List
Figure imgf000041_0001
Results
Transcripts Encoding. Paracrine Factors are Elevated at the Site of Infarction in the Sheep Model
[0159] Paracrine factors are the cell-to-cell mechanism for promoting regeneration after infarction. IGF1, CXCL12, HGF, VEGF, CXCR4, IL10, and TGFB 1 were measured at infarct and non-infarct cites in four animals as shown in Figures 53A-D. VEGF and HGF were important to examine because they promote angiogenesis to restore blood flow to the damaged region. PCR was used to detect significantly elevated levels of VEGF in three of four animals. HGF transcripts were also elevated to a significant extent (8-26 fold) in two animals when comparing the non-infarct zone and the infarct zone in four animals.
[0160] IGF-1 was examined in the context of the regenerating myocardium because it activates Akt signaling for cell survival. IGF-1 levels in the infarct zone were elevated to a significant extent in one animal. CXCL12 and its receptor CXCR4 recruit progenitors to promote angiogenesis and CXCL12 was significantly elevated in two animals while CXCR4 was significantly elevated in three of four animals (up to 4 fold). Elevated transcripts for IL-10 noted in two of four animals could potentially contribute to preventing immune-mediated rejection of allogeneic cells.
[0161] In summary IGF-1 , HGF, VEGF, and CXCR4 were significantly more expressed in the infarct zone on average, as shown in Figure 53E. TGF-βΙ (0.84-0.96), levels were not changed
[0162] PCR results were verified by agarose gel electrophoresis. CXCR4, CXCL12, and IGF-1 bands, for example, were shown to be the correct size in Figure43. GAPDH products taken from the matching PCR plates were run as loading controls, as shown in Figure 43.
Transcripts Encoding Akt and Wnt Pathway Proteins Show Elevation in the Site of Infarction in the Sheep Model
[0163] The importance of the AKT pathway is that it upregulates anti-apoptosis proteins to promote survival of cells in the infarct zone. Wnt signaling contributes to renewal and differentiation of Isll+ CPCs after their injection to continue regeneration of the infarct area. The average transcripts from the AKT pathway genes were increased significantly in the infarct zone except for c-Myc, as shown in Figure 45.
Transcripts Encoding Notchl and Notch! are Elevated in the Infarct Zone of the Sheep Model
[0164] Notch ligands are important for cardiac progenitor survival and differentiation to continue regeneration after myocardial infarction (MI). Notchl infarct fold changes were significantly elevated up to 12 fold. Notch2 expression was elevated, but the levels were not found to be significant due to animal-to-animal variation when the data was pooled from all animals, as shown in Figure 46.
Transcripts Encoding Heat Shock Proteins at the Site of Infarction in the Sheep Model
[0165] Heat shock protein 70 (HSP70) offers cytoprotection during MI, which compliments the process of regeneration. Noninfarct and infarct zone transcripts were recorded for each sheep, as shown in Figures 47A-D, and averaged as shown in Figure 47E. HSP40 transcripts were elevated in two of four animals and the fold change ranged from 0-5. HSP70 expression was significantly elevated in two animals but the change was not higher than two fold, as shown in. Figures 47A-D.
Transcripts Encoding Superoxide Dismutase 2 Show Increased Expression in the Infarct Zone in the Sheep Model
[0166] Regeneration after MI has been linked to increases in antioxidant activity because small changes in oxidation can have drastic effects on the survival and behavior of cells. Antioxidant enzyme superoxide dismutase (SOD) is important for oxidative balance in vivo and can act as a measure of oxidative stress. SOD2 transcripts affect the mitochondria. Transcript levels were increased in the infarct zone of every sheep. Minimum fold change in the infarct zone was 1.2 and the maximum was 1400. Transcripts Encoding C-kit are Elevated at the Site of Infarction in the Sheep Model
[0167] The receptor tyrosine kinase protein c-kit is a surface receptor for cytokines and it is also a surface marker. C-kit is critical for cell survival and proliferation in the myocardium and this is why cardiac progenitors that express c-kit a leading choice for regenerative therapy after MI. The transcript levels of c-kit were elevated in the infarct zone, as shown in Figures 51A-B. The infarct zone in individual sheep examined for this study saw a fold change of 23 fold at the maximum. One of the four sheep did not have a significant c- kit rise.
Transcripts Encoding Cardiac Transcription Factors
[0168] Transcription factors control the rate of gene transcription to RNA by binding to DNA. GATA4 is an important cardiac transcription factor that positively affects cell survival and growth factor secretion in an infarcted area to stimulate regeneration. Isl-1 is expressed by the injected cardiac progenitors and is associated with CPC proliferation and angiogenesis. NF-κΒ is a regulator of inflammation in the infarct area, but also growth and cell adhesion. Suppression of NF-κΒ promotes an anti-inflammatory response and shrinks the size of the infarct, while its activation leads to inflammatory cytokine mRNA upregulation through TNF-a activation. As shown in Figure 49, relative GATA4 and NF-KB expression in the infarct zone did not change significantly. Islet- 1 transcript levels were elevated (up to 13 fold change) significantly.
Transcripts Encoding Pro-Survival Proteins are Elevated in the Infarct Zone of the Sheep Model
[0169] Heme oxygenase- 1 (HMOX) is important for anti-inflammatory signaling because it protects against cardiomyocyte apoptosis by metabolizing cytotoxic heme. HMOX overexpression triggers SOD2 activation, Akt activation, and also anti-apoptosis signaling through mitochondrial biogenesis. TNFAIP3 (A20) also has protective anti-inflammatory properties because it suppresses NF-κΒ signaling. It is also known to improve cardiac function by inhibiting fibrosis and hypertrophy from TGF-β signaling. BCL2 is an anti- apoptotic gene and it promotes VEGF secretion for angiogenesis in vitro. HMOX transcript fold change in the infarct zone was elevated as much as 6.4 fold, but not in all sheep, therefore the average change was not found to be significant. Similar results were obtained for TNFAIP3 (elevated up to 22 in one animal), and for BCL2 (elevated up to 17 in one animal). When considered as an average trend, as shown in Figure 48, none of the prosurvival genes were elevated to an extent that was found to be significant.
Transcripts Encoding Connexin-43 Show Increased Expression in the Infarct Zone of the Sheep Model
[0170] Gap junction protein Connexin-43 is important for intercellular communication that affects wound healing, fibrosis, and remodeling after MI. The average transcript levels of Cx43 were elevated in the infarct zone, as shown in Figure 52A. Immunohistochemistry reveals that the CFSE-labeled Isl-1+ c-kit+ CPCs coexpressed the red connexin-43 stain as shown in Figure 52B.
Example 16: CPCs cultured on a biocompatible matrix
Cardiovascular progenitor cell isolation
[0171] CSCs were isolated from biopsies of human neonates or adults under Institutional Review Board approval following a previous protocol as described in Example 1. Briefly, right atrial cardiac tissue (~ 1 mm ) was digested with collagenase (Roche Applied Science, Indianapolis, IN) for two hours at 37°C and then strained. Cells were cloned by limiting dilution (0.8 cells per well) to create the clonal populations that were used in this study. CSCs were isolated from sheep in accordance with the Animal Welfare Act, the NIH Guide for the Care and Use of Laboratory Animals, and the IACUC of Loma Linda University.
Cell growth on the scaffold
[0172] An 8x2-mm GroCell-3D polysaccharide scaffold (Molecular Matrix, Davis, CA) was placed in M199-supplemented media overnig ht. CSCs (2 x 106) in 20μί growth media were incubated on the scaffold for two hours at 37°C in 5% C02 before being placed in growth media in a T25 flask. Cells were seeded on both sides and maintained for three weeks. Scaffolds were dissolved for cell cycle, flow cytometry, and gene expression analysis using dissolution solution (Molecular Matrix, Davis, CA) following the manufacturer's protocol.
Phosphorylation assay using flow cytometry
[0173] Flow cytometry was used to measure phosphorylated ERK and AKT. Cells were fixed with 4% paraformaldehyde, permeabalized, and stained using antibodies to phosphorylated ERK 1/2 (Thr202/Tyr204, Cell Signaling Technology, Danvers, MA) at a 1/200 dilution, and p-AKT (Ser473, Cell Signaling Technology, Danvers, MA) at a 1/100 dilution. FITC goat anti-rabbit IgG (BD Biosceinces San Jose, CA) at a dilution of 1/150 was used as a secondary antibody. The geometric mean of each peak for each clone was used to calculate the staining of each cell.
Differentiation marker detection using flow cytometry
[0174] Flow cytometry was used to measure the level of expression of the differentiation markers TropT and vWF.. The Lightning-Link Antibody Labeling Kit (Novus Biologicals, Littleton, CO) was used per the manufacturer's instructions to conjugate rabbit anti-human von-Willbrand factor antibody (Dako, Carpinteria, CA) to FITC and anti-cardiac troponin T antibody (Abeam, Cambridge, MA) to PE. Cells were stained with FITC- conjugated vWF at a dilution of 1/10 and PE-conjugated TropT at a dilution 1/250. Isotype controls were used to define populations.
Cell cycle analysis using flow cytometry
[0175] Cell cycle analysis was conducted using flow cytometry. Cells were fixed in 70% ethanol, stored overnight at -20°C, washed, and incubated at 37°C with RNase A (0.5 mg/ml Invitrogen, Carlsbad, CA) for one hour. Propidium iodide was added (10 μg/ml). Cells were analyzed using a MACSquant analyzer (Miltenyi Biotec, Auburn, CA) and Flow Jo software (Ashland, OR). Real time polymerase chain reaction (RT-PCR)
[0176] Cells were stored using Trizol reagent (Invitrogen, Carlsbad, CA). RNA (500ng) was isolated and used to prepare cDNA with the Superscript III (Invitrogen, Carlsbad, CA) protocol. RT-PCR was performed on the IQ5 machine (Bio-rad, Hercules, CA) with β-actin as a housekeeping gene. The PCR conditions were: 94°C for 10 minutes, 94°C for 15 seconds, 52°C for 60 seconds, and 72°C for 30 seconds for 40 cycles. The primers were (forward) 5 ' -TTTGAATGATGAGCCTTCGTCCCC-3 ' and (reverse) 5'- GGTCTC AAGTCAGTGTACAGGTAAGC-3 ' for β-Actin; (forward) 5'- CAGAGCAGATAGAGCCTGCG-3 ' and (reverse) 5 ' -C AGGTAACTCGTGC AGAGC A-3 ' for IGF-1; (forward) 5 ' -C ACG AACAC AGCTTTTTGCC-3 ' and (reverse) 5'- TGATCCC AGCGCTGACAAAT-3 ' for HGF; and (forward) 5'- CTACAGATGCCC ATGCCGAT-3 ' and 5 ' -GTGGGTCTAGCGGAAAGTCC-3 ' for SDF- la (Integrated DNA Technologies, Coralville, IA).
Immunohistochemistry
[0177] Scaffolds were fixed in 4% paraformaldehyde overnight, washed with serial dilutions of ethanol for thirty minutes, washed with xylene and embedded in paraffin at 58°C. The paraffin block was chilled on ice for 15 minutes and sectioned using a microtome (Model RM 2125, Leica Biosystems, Buffalo Grove, IL). Sections were deparaffinized and blocked using 10% species-appropriate serum in 3% glycine and PBS for 30 minutes. Primary antibodies included mouse anti-Ki-67 (Biolegend, San Diego, CA) at a 1: 100 dilution, mouse anti-Isll (Abeam, Cambridge, MA) at a 1:200 dilution, rabbit anti-vWF (Dako, Carpinteria, CA) at a 1:200 dilution, and mouse anti-TropT (Abeam, Cambdridge, MA) at a 1: 100 dilution. Incubation was performed at 4°C. Each section was washed with PBS/Tween20 and incubated with a secondary antibody for one hour at room temperature. The secondary antibodies were either goat anti-mouse PE (Southern Biotech, Birmingham, AL) at a 1: 100 or 1:250 dilution or donkey anti-rabbit Alexa Fluor 647 (Life Technologies, Carlsbad, CA) at a 1:200 dilution. Slides were mounted in Prolong Gold with DAPI (Life Technologies, Carlsbad, CA). Sections were imaged (20x) using a LSM 710 NLO laser- scanning, confocal microscope (Carl Zeiss Microscopy Gmblt, Jena, Germany). Images were processed using ImageJ (v.1.49, NIH, http://imagej.nih.gov/ij).
Cell proliferation and in vivo tracking.
[0178] Cells were stained with CFSE (Biolegend, San Diego) following the manufacturer's protocol. CSCs (106) were suspended in 5μΜ of CFSE and incubated for 10 minutes at 37°C. This reaction was quenched by 10% sheep serum (S2263, Sigma Aldrich, St Louis, MO)-supplemented M-199 (Life Technologies, Carlsbad, CA) media. Cells were seeded onto the scaffold and cultured prior to immunohistochemistry analysis.
Statistical Analyses
[0179] For all normally distributed data, a two-tailed, paired, student's t-test was performed using Microsoft Excel. Significance was p<0.05. Data is represented as the mean +/- standard error.
Results
Ovine-derived, scaffold-cultured CSCs express increased levels of Isll and Ki-67 and a pro- stem cell shift in cell cycling
[0180] To demonstrate the suitability of this scaffold for preclinical, large animal model transplantation, ovine-derived cells were cultured. Isll expression, a marker that identifies the clonal population of the CSCs that were seeded on the scaffold, was significantly elevated (5.6-fold, P<0.05) as shown in Figure 28A. A pro-stem cell shift in the cell cycle profile was observed using flow cytometry as shown in Figure 28B. Confocal microscopy was used to observe the expression of Isll and Ki-67, a marker of proliferation. CSCs were observed to stain more brilliantly for Isll following scaffold culturing, as shown in Figures 28C-E. A 1.91 -fold increase in the expression of Ki-67 was detected in scaffold- cultured cells Figures 28F-H, evidencing the proliferation of CSCs. Ovine-derived, scaffold-cultured CSCs express increased levels of the differentiation markers TropT and vWF
[0181] Scaffold culturing was observed to induce the differentiation of CSCs. Fluorophore-conjugated antibodies against the cardiomyocyte marker TropT and endothelium marker vWF were used to detect the presence of differentiated cells. Representative histograms of TropT-positive and vWF-positive cells are shown in Figures 29A-F. Unlike control-cultured CSCs depicted in Figures 29A and 29D, scaffold-cultured cells express increased levels of TropT and vWF, as shown in Figures 29B and 29E. This was confirmed by confocal microscopy, in which stains for TropT and vWF are intensely observed in scaffold-cultured cells, as shown in Figures 29G-L. When compared to the negative control, there was a 5.42-fold increase in TropT staining and an 1 1.70-fold increase in vWF staining in scaffold-cultured sections, which indicates that scaffold-cultured CSCs differentiate. Therefore, ovine-derived CSCs that are cultured using a molecular scaffold can both proliferate and differentiate.
Patient-derived CSCs differentiate and proliferate on the scaffold
[0182] Following scaffold-based culturing, changes in cell cycling and differentiation marker expression were detected using flow cytometry. We observed a significant decrease in the Gl phase of scaffold-cultured progenitors, as shown in Figure 30A. This shift in the cell cycling profile of scaffold-cultured CSCs (i.e., a punctuated Gl phase) is characteristic of stem cell cycling. Nevertheless, CSC-derivatives were observed in the scaffold-cultured cell populations. Fluorophore-conjugated antibodies against the cardiomyocyte marker TropT and endothelium marker vWF were used to detect the presence of differentiated cells using flow cytometry. As shown in Figure 30B, the expression of TropT and vWF were significantly increased in scaffold-cultured CSCs. Therefore, human- derived CSCs that are cultured using a molecular scaffold can both proliferate and differentiate. ERK signaling significantly decreases while AKT signaling is elevated in scaffold-cultured CSCs
[0183] To better understand the intracellular dynamics that are associated with the improved proliferation and differentiation capacity of scaffold-cultured CSCs, flow cytometry was used to measure phosphorylated ERK 1/2 (P-ERK) and phosphorylated AKT (P-AKT). A significant decrease in the number of P-ERK+ scaffold-cultured CSCs was observed, as well as a general increase in P-AKT+ scaffold-cultured CSCs, as shown in Figures 31A-B. This suggests that scaffold culturing decreased activity in the MEK/ERK pathway while increasing AKT signaling along the PI3K/AKT pathway. This is consistent with previous reports in which AKT signaling, through the PI3K/AKT pathway, and ERK suppression is evidenced to preserve sternness.
Growth factor secretion significantly increases in human CSCs following scaffold culturing
[0184] Following the detection of a significantly altered intracellular signaling cascade, RT-PCR was used to assess the expression of IGF-1, HGF, and SDF-Ια. It was found that the expression of HGF and SDF-Ια was significantly higher in the scaffold- cultured CSCs as shown in Figure 32A, HGF: 48-fold increase, P<0.01; SDF-Ια: 77-fold increase, P<0.05. While an increase was observed for IGF-1, this change was not significant. The amplified cDNA products of the statistically significant growth factors were visualized on a 1% agarose gel, and an increase in expression and a conformation of the appropriate product length can be qualitatively observed in Figure 32B.
Example 17: CPCs exhibit in vivo retention, division, differentiation, and recruitment of endogenous endothelial cells
[0185] Tissue was harvested from the right atrium of neonatal sheep and cut into
1 mm cubed pieces. The pieces were then digested with collagenase for about 2 hours at
37°C. A 40 mm cell strainer was used to isolate the CPCs. The solution was diluted to 0.8 cells per well and the isolated clones were expanded and characterized. Flow cytometry was used to identify both isl- 1 and ckit expression, and PCR was used to verify the expression of isl- 1 and c-kit on these undifferentiated isl 1+ c-Kit+ CPCs. The cells were expanded in culture prior to transplantation. LAD ligation was used to induce a myocardial infarction in four sheep. EKG and echocardiograms were done prior to infarction, on the day of cardiovascular progenitor cell transplantation in these animals, and at sacrifice of the animals two months after cardiovascular progenitor cell injection. Prior to cardiovascular progenitor cell administration, the CSC clones to be administered (106) were suspended in 5μΜ of CFSE and incubated for 10 minutes at 37 °C to label them with a green fluorescent tag for tracking. This reaction was quenched by 10% sheep serum (S2263, Sigma Aldrich, St Louis, MO)-supplemented M-199 (Life Technologies, Carlsbad, CA) media. Labeling of the cells was verified by flow cytometry prior to cardiovascular stem cell administration in vivo. CFSE labeled allogenic CPCs were injected 28-38 days post infarction. Histology was performed by immunostaining of frozen sections. At 55-60 days after cell injection, the animals were sacrificed to examine the effect of CSC administration on cardiovascular regeneration and repair. Tissue pieces were frozen for later sectioning and immunostaining. Additional pieces were kept in RNAlater for preparation of RNA for gene expression analysis. Immunostaining was done on frozen sections using antibodies to identify proliferation, differentiation into cardiomyocytes and endothelial cells and cell-cell interaction. The concentration of the antibodies was established in preliminary experiments performed to optimize conditions for staining sections using these reagents. The reagents used included anti-ki67, anti-troponin I, anti-von-Willebrand factor and anti-connexin 43 antibodies along with the appropriate secondary antibodies. Two color staining of CFSE (green) and the red Alexa Fluor 647 antibodies allowed identification of cardiovascular progenitor cell differentiation and cell division. Slides were mounted in Prolong Gold with DAPI (Life Technologies, Carlsbad, CA). Sections were imaged (20x) using a LSM 710 NLO laser-scanning, confocal microscope (Carl Zeiss Microscopy Gmblt, Jena, Germany). Images were processed using ImageJ (v.1.49, NIH, http://im gej .nih. gov/ij ). The immunostaining verified cell retention, division, differentiation, and recruitment of endogenous endothelial cells to the site of infarction. Results
CFSE-labeled allogenic neonatal CPCs were retained 2 months post-injection
[0186] It was found that the CFSE-labeled allogenic CPCs were retained in the ventricle of all four sheep 2 months post injection (Figure 54).
Sheep A (Figure 54E): 12-13% of cells are CFSE positive, from a total of 1410 cells imaged.
Sheep B (Figure 54F): 10-31% of the cells are CFSE positive, from a total of 2593 cells imaged.
Sheep C (Figure 54G): 11- 19% of the cells are CFSE positive, from a total of 2007 cells imaged.
Sheep D (Figure 54H) 11-43% of the cells are CFSE positive, from a total of 3624 cells imaged.
On average, approximately 19% of cells analyzed were CFSE+ CPCs, as shown in Figure 55.
CFSE-labeled allogenic neonatal CPCs were actively dividins 2 months post-in jection
[0187] By staining the sections with Ki-67, the actively dividing cells were identified (Figure 56 A-C). It was found that approximately 45% of retained CFSE+ CPCs were actively dividing, as shown in Figure 56D.
Sheep A: Average 78%, range 46-95% CFSE+ CPCs expressing Ki-67, from a total of 904 cells imaged.
Sheep B: Average 26%, range 9-32% CFSE+ CPCs expressing Ki-67, from a total of 798 cells imaged.
Sheep C: Average 68%, range 56-89% CFSE+ CPCs expressing Ki-67, from a total of 1 103 cell imaged.
Sheep D: Average 9%, range 5-14% CFSE+ CPCs expressing Ki-67, from a total of 1164 cells imaged. CFSE-labeled allogenic neonatal CPCs differentiated into cardiac and endothelia cells
[0188] Infarct zone staining with anti-Troponin I illustrated co-localization (yellow) of anti-Troponin I (red) labeling and the CFSE (green) labeling of the introduced allogenic CPCs (Figure 57B). The non-infarct area labeled with anti-Troponin I is shown in Figure 57A. As shown in Figure 57C, the introduced CPCs in the infarct zone exhibited differentiation into cardiomyocytes in all four sheep models
Sheep A: Average 31%, Range 28-42% CFSE+ CPCs expressing Trop I from a total of 1508 cells imaged.
Sheep B: Average 43%, Range 29-61% CFSE+ CPCs expressing Trop I from a total of 760 cells imaged
Sheep C: Average 40% Range 39-41% CFSE+ CPCs expressing Trop I, from a total of 1069 cells imaged
Sheep D: Average 27% Range 12-40% CFSE+ CPCs expressing Trop I, from a total of 889 cells imaged
Likewise, non-infarct section of the left ventricle exhibits von Willebrand Factor (vWF) staining (red, Figure 57D) indicative of endothelial cells, and the infarct zone section exhibits CFSE labeled introduced CPCs (green, Figure 57E). Figure 57E shows von Willebrand Factor labeling (red) and introduced CPCs which have differentiated into endothelial cells (colocalized- yellow, indicated by arrows, Figure 57E). As shown in Figure 57F, the introduced CPCs exhibited endothelial cell differentiation in three of the four sheep studied.
Sheep A: Average 36%, Range 27-48% CFSE+ CPCs expressing vWF, from a total of 1360 cells imaged.
Sheep B: Average 14%, Range 0-21% CFSE+ CPCs expressing vWF, from a total of 706 cells imaged
Sheep C: Average 4% Range 0-9% CFSE+ CPCs expressing vWF, from a total of 1841 cells imaged
Sheep D: Average 0% Range 0-0% CFSE+ CPCs expressing vWF, from a total of 1209 cells imaged Endothelial cells are recruited into the infarct zone after CPC injection.
[0189] Figure 58A shows a representative section of the non-infarct area of the left ventricle, showing vascularization. Endothelial cells are stained with anti-vWF (red). Figure 58B shows a representative section of the infarct area of the left ventricle, showing vascularization. Endothelial cells are stained with anti-vWF (red). CFSE labeled CPCs are green. Figure 58C shows that the infarct area contains a large number of vWF positive endothelial cells (red). An increase in the total percentage of cells which are vWF+ in the infarct zone was found in all four sheep, illustrating recruitment of endogenous endothelial cells as shown in Figure 58D.
Sheep A: Non-infarct: Average 6%, range 1-13% cells expressing vWF, with a total of 1701 cells imaged.
Infarct: Average 21%, range 12-34% cells expressing vWF, with a total of
1360 cells imaged.
Sheep B: Non-infarct: Average 8%, range 7-11 % cells expressing vWF, with a total of 903 cells imaged.
Infarct: Average 14%, range 10-19% cells expressing vWF, with a total of 706 cells imaged.
Sheep C: Non-infarct: Average 7%, range 2-11 % cells expressing vWF, with a total of 2147 cells imaged.
Infarct: Average 19%, range 14-24% cells expressing vWF, with a total of 1841 cells imaged.
Sheep D: Non-infarct: Average 7%, range 7-11 % cells expressing vWF, with a total of 626 cells imaged.
Infarct: Average 12%, range 7-17% cells expressing vWF, with a total of 1209 cells imaged.

Claims

WHAT IS CLAIMED IS:
1. A method for treating a patient having damage within a region of cardiac tissue, comprising transplanting into the region or into cardiac tissue surrounding the region, a clonal population of isl-l+c-kit+ cardiac progenitor cells.
2. The method of claim 1, further comprising preconditioning the clonal population of isl-l+c-kit+ progenitor cells under hypoxic conditions prior to transplantation.
3. The composition of claims 1 or 2, wherein the clonal population of isl-l+c- kit+ progenitor cells are configured to differentiate into one or more cardiovascular lineages.
4. The composition of any one of claims 1-3, wherein the clonal population of isl-l+c-kit+ progenitor cells comprises autologous or allogeneic cells.
5. A method of providing a therapeutic cell matrix for use in regenerative cardiovascular medicine, the method comprising:
obtaining a cloned population of isl-l+c-kit+ progenitor cells; and culturing the isl-l+c-kit+ progenitor cells within an implantable biodegradable matrix configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the matrix into an infarct zone, wherein the isl-l+c-kit+ progenitor cells are capable of regenerating damaged heart tissue.
6. A method of providing a therapeutic cell suspension for use in regenerative cardiovascular medicine, the method comprising:
obtaining a cloned population of isl-l+c-kit+ progenitor cells; and suspending the isl-l+c-kit+ progenitor cells in a pharmaceutically acceptable medium configured to support at least one of viability, proliferation and differentiation of the isl-l+c-kit+ progenitor cells upon transplantation of the suspension into an infarct zone, wherein the isl-l+c-kit+ progenitor cells are capable of regenerating damaged heart tissue.
7. The method of claim 5 or 6, wherein the cloned population of isl-l+c-kit+ progenitor cells are obtained from the atrial appendage, epicardium, or ventricle of one or more human tissue donors.
8. The method of claim 5 or 6, wherein the cloned population of isl-l+c-kit+ progenitor cells are configured to differentiate into one or more cardiovascular lineages.
9. The method of claim 5 or 6, wherein the cloned population of isl-l+c-kit+ progenitor cells comprises autologous or allogeneic cells.
10. The method of any one of claims 5-9, further comprising preconditioning the cloned population of isl-l+c-kit+ progenitor cells under hypoxic conditions.
11. A composition for treating cardiac damage, the composition comprising: a cloned population of isl-l+c-kit+ progenitor cells;
wherein the isl-l+c-kit+ progenitor cells are adapted to promote repair of damaged heart tissue when transplanted into a region of cardiovascular tissue comprising damaged heart tissue.
12. The composition of claim 11, wherein the cloned population of isl-l+c-kit+ progenitor cells are obtained from the atrial appendage, epicardium, or ventricle of one or more human tissue donors.
13. The composition of claim 11 or 12, wherein the cloned population of isl-l+c- kit+ progenitor cells are configured to differentiate into one or more cardiovascular lineages.
14. The composition of any one of claims 11-13, wherein the clonal population of isl-l+c-kit+ progenitor cells comprises autologous or allogeneic cells.
15. The composition of any one of claims 11-14, further comprising one or more cytokines, growth factors or other agents that promote survival, proliferation and/or differentiation of the isl-l+c-kit+ progenitor cells.
16. The composition of any one of claims 11-15, further comprising an implantable biodegradable matrix associated with the isl-l+c-kit+ progenitor cells, wherein the biodegradable matrix promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c-kit+ progenitor cells.
17. The composition of any one of claims 11-15, further comprising a biocompatible nutrient medium that promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c-kit+ progenitor cells.
18. Use of a composition for treating damaged cardiac tissue in a patient in need thereof, the composition comprising a cloned population of isl-l+c-kit+ progenitor cells.
19. The use of claim 18, wherein the composition is configured for transplantation into a region of cardiovascular tissue comprising damaged cardiac tissue.
20. The use of claim 18 or 19, wherein the cloned population of isl-l+c-kit+ progenitor cells are obtained from the atrial appendage, epicardium, or ventricle of one or more human tissue donors.
21. The use of any one of claims 18-20, wherein the cloned population of isl-l+c- kit+ progenitor cells are configured to differentiate into one or more cardiovascular lineages.
22. The use of any one of claims 18-21, wherein the clonal population of isl-l+c- kit+ progenitor cells comprises autologous or allogeneic cells.
23. The use of any one of claims 18-22, wherein the composition further comprises one or more cytokines, growth factors or other agents that promote survival, proliferation and/or differentiation of the isl-l+c-kit+ progenitor cells.
24. The use of any one of claims 18-23, wherein the composition further comprises an implantable biodegradable matrix associated with the isl-l+c-kit+ progenitor cells, wherein the biodegradable matrix promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c-kit+ progenitor cells.
25. The use of any one of claims 18-23, wherein the composition further comprises a biocompatible nutrient medium that promotes survival, proliferation and/or differentiation of the clonal population of isl-l+c-kit+ progenitor cells.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080213230A1 (en) * 2006-11-07 2008-09-04 Keck Graduate Institute Enriched stem cell and progenitor cell populations, and methods of producing and using such populations
WO2014114465A1 (en) * 2013-01-24 2014-07-31 Bernardo Nadal-Ginard Modulation of cardiac stem-progenitor cell differentiation, assays and uses thereof
US20150328263A1 (en) * 2014-05-14 2015-11-19 University Of Maryland, Baltimore Cardiac stem cells for cardiac repair

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080213230A1 (en) * 2006-11-07 2008-09-04 Keck Graduate Institute Enriched stem cell and progenitor cell populations, and methods of producing and using such populations
WO2014114465A1 (en) * 2013-01-24 2014-07-31 Bernardo Nadal-Ginard Modulation of cardiac stem-progenitor cell differentiation, assays and uses thereof
US20150328263A1 (en) * 2014-05-14 2015-11-19 University Of Maryland, Baltimore Cardiac stem cells for cardiac repair

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