WO2020097440A1 - Procédés de prédiction de récupération fonctionnelle de tissu à l'aide d'exosomes circulants dérivés de cellules transplantées - Google Patents

Procédés de prédiction de récupération fonctionnelle de tissu à l'aide d'exosomes circulants dérivés de cellules transplantées Download PDF

Info

Publication number
WO2020097440A1
WO2020097440A1 PCT/US2019/060436 US2019060436W WO2020097440A1 WO 2020097440 A1 WO2020097440 A1 WO 2020097440A1 US 2019060436 W US2019060436 W US 2019060436W WO 2020097440 A1 WO2020097440 A1 WO 2020097440A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
exosomes
cells
transplanted
subject
Prior art date
Application number
PCT/US2019/060436
Other languages
English (en)
Inventor
Sunjay Kaushal
Progyaparamita SAHA
Sudhish SHARMA
Prashanth VALLABHAJOSYULA
Original Assignee
University Of Maryland, Baltimore
The Trustees Of The University Of Pennsylvania
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Maryland, Baltimore, The Trustees Of The University Of Pennsylvania filed Critical University Of Maryland, Baltimore
Publication of WO2020097440A1 publication Critical patent/WO2020097440A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • CPCs cardiac progenitor cells
  • CDCs cardiosphere-derived cells
  • MI myocardial infarction
  • the components of the stem cell secretome promote myocardial recovery through donor age-dependent pathways [5, 7-13]
  • an in-depth proteomic analysis of the CPCs secretome was performed, demonstrating that a single intramyocardial injection of the exosomes derived from neonatal CPCs promoted myocardial recovery at a level similar to that observed following neonatal CPCs injection [5]
  • Exosomes are extracellular nanovesicles released by many tissue types into body fluids, including blood, urine, and bronchoalveolar secretions [18-21]
  • Other studies have identified the presence of circulating tissue-specific exosomes derived from transplanted solid organs in recipient plasma [22, 23]
  • exosomes contain specific proteomic and RNA signatures that reflect the conditional and functional status of their cells of origin [19, 24] Given these attributes, exosomes are being actively investigated for their functional and diagnostic potential in many medical fields.
  • circulating transplant tissue- specific exosome characterization enables noninvasive surveillance of transplanted solid organs in a time-sensitive, condition-specific manner [21, 25, 26]
  • the present invention has been realized via the work of the inventors reported herein, demonstrating for the first time that progenitor cell-specific exosomes are present in the circulation of subjects into which such cells have been transplanted.
  • Provided in the Examples are the results of a head-to-head comparison in a xenogeneic model of rodent myocardial infarction (MI) that studied the cardiac regenerative potential of two well-studied progenitor cells, CDCs and CPCs, derived from the same human heart biopsy.
  • MI rodent myocardial infarction
  • the results show the monitoring potential of a stem cell-specific exosome platform, and demonstrate that intra- exosomal microRNA cargoes reflect the functional myocardial recovery achieved by the transplanted stem cells. Data from initial studies conducted in humans is provided as well.
  • the present invention is directed to methods of monitoring cells transplanted into a subject.
  • the method comprises screening a biological sample obtained from a subject into whom cells have been transplanted for the presence of transplanted cell-derived exosomes.
  • the presence of exosomes in the biological sample indicates the presence of the transplanted cells in the subject.
  • An increase/decrease in the number of transplanted cell-derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells in the subject over time.
  • monitoring of cells transplanted into the subject can be achieved.
  • the present invention is directed to methods of monitoring a subject receiving cell-based therapy.
  • the method comprises screening a biological sample obtained from a subject receiving cell therapy for the presence of transplanted cell-derived exosomes.
  • the presence of exosomes in the biological sample indicates the presence of the transplanted cells in the subject.
  • An increase/decrease in the number of transplanted cell-derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells in the subject over time.
  • monitoring a subject receiving cell-based therapy can be achieved.
  • the present invention is directed to methods for predicting functional recovery of ischemic myocardium in a subject into which cells have been
  • the method comprises screening a biological sample obtained from a subject that has ischemic myocardium and into which cells have been transplanted for the presence of transplanted cell-derived exosomes.
  • the presence of exosomes in the biological sample indicates functional recovery of the ischemic myocardium in the subject is more likely than a
  • An increase/decrease in the number of transplanted cell-derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells, which may aid in the functional recovery of the ischemic myocardium, in the subject over time.
  • a prediction of functional recovery of ischemic myocardium in the subject can be achieved.
  • the methods may further comprise enumerating the number of exosomes present in the biological samples.
  • the methods may further comprise repeating the method at one or more additional time points and enumerating the number of exosomes present in the biological samples from each time point to determine whether there is a change in the number of exosomes over time.
  • the transplanted cells may be allogeneic, that is, the transplanted cells may be obtained from an individual that is different from the subject into which the cells are transplanted. When the transplanted cells are allogeneic, exosome produced by the cells can be more easily identified in the biological sample.
  • the transplanted cells include, but are not limited to, stem cells and progenitor cells.
  • the cells may be one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).
  • the subject is one into whom cells have been transplanted.
  • the cells will have been transplanted into a target organ or into a target organ system in the subject.
  • Suitable target organs include, but are not limited to, heart, lungs, kidneys, liver, pancreas, spleen, brain, bladder, or lymph nodes.
  • Suitable target organ systems include, but are not limited to, cardiovascular system, digestive system, endocrine system, excretory system, lymphatic system, muscular system, nervous system, reproductive system, and respiratory system.
  • the biological sample may be screened within 6, 12,
  • the biological sample may be screened within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after completion of cell therapy or transplantation of the cells into the subject.
  • the biological sample may be, but is not limited to, sputum/oral fluid, amniotic fluid, blood, a blood fraction, or fine needle biopsy samples (e.g., surgical biopsy, fine needle biopsy, etc.) urine, peritoneal fluid, and pleural fluid.
  • the biological sample is blood plasma.
  • the biological samples may be screened using antibodies having binding specificity for molecules displayed by the exosomes.
  • the molecules may be, for example, leukocyte antigen surface molecules, including human leukocyte antigen (ELLA) surface molecules and human mismatch ELLA surface molecules.
  • ELLA human leukocyte antigen
  • the subject may be a mammal, including, but not limited to, a human.
  • the methods of the invention include profiling the contents of exosomes collected from the biological samples. For example, polynucleotides and polypeptides within the exosomes can be isolated and characterized.
  • the invention is directed to methods of profiling exosomes derived from cells transplanted into a subject.
  • the method comprises (i) collecting, from a biological sample obtained from a subject into whom cells have been transplanted, exosomes derived from the transplanted cells and (ii) characterizing cargo of the collected exosomes.
  • profiling of exosomes derived from cells transplanted into a subject can be achieved.
  • the cargo may be, but is not limited to, one or more of polynucleotides, polypeptides, and lipids.
  • the cargo is polynucleotides, such as, but not limited to, microRNA.
  • the invention is directed to methods of profiling exosomes derived from cells transplanted into a subject.
  • the method comprises (i) collecting, from a biological sample obtained from a subject into whom cells have been transplanted, exosomes derived from the transplanted cells and (ii) characterizing microRNA cargo of the collected exosomes.
  • profiling of exosomes derived from cells transplanted into a subject can be achieved.
  • characterizing microRNA cargo may comprise sequencing one or more species of the microRNA present in the collected exosomes.
  • Characterizing microRNA cargo may alternatively comprise otherwise identifying one or more species of microRNA present in the collected exosomes. Characterizing microRNA cargo may also comprise screening the contents of the exosomes for the presence of one or more specific species of microRNA; in some aspects of the invention, the specific species of microRNA are known to be associated with one or more activities performed by the target organ.
  • the cargo may screened for the presence of one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27- 5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR- 623, and miR-362-5p.
  • the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, miR-623, miR-94l, miR- 1224, and miR-l256.
  • the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224.
  • Suitable means for identifying one or more species of microRNA present in the collected exosomes include, but are not limited to, sequencing and hybridization to labeled probes.
  • the methods may further comprise repeating the method at one or more additional time points and characterizing cargo from each additional time point to determine whether there is a change in the cargo over time.
  • the cells may be allogeneic, that is, the cells may be obtained from an individual that is different from the subject into whom the cells are
  • exosome produced by the cells can be more easily identified in the biological sample.
  • the transplanted cells include, but are not limited to, stem cells and progenitor cells.
  • the cells may be one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).
  • CPCs cardiac progenitor cells
  • CDCs cardiosphere-derived cells
  • MSCs mesenchymal stem cells
  • BMCs bone marrow cells
  • ESCs embryonic stem cells
  • the subject is one into which cells have been transplanted.
  • the cells will have been transplanted into a target organ or into a target organ system in the subject.
  • Suitable target organs include, but are not limited to, heart, lungs, kidneys, liver, pancreas, spleen, brain, bladder, or lymph nodes.
  • Suitable target organ systems include, but are not limited to, cardiovascular system, digestive system, endocrine system, excretory system, lymphatic system, muscular system, nervous system, reproductive system, and respiratory system.
  • the exosomes may be collected within 6, 12, 18, 24,
  • the exosomes may be collected within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after completion of cell therapy or transplantation of the cells into the subject.
  • the biological sample may be, but is not limited to, sputum/oral fluid, amniotic fluid, blood, a blood fraction, or fine needle biopsy samples (e.g., surgical biopsy, fine needle biopsy, etc.) urine, peritoneal fluid, and pleural fluid.
  • the biological sample is blood plasma.
  • the exosomes may be collected using antibodies having binding specificity for molecules displayed by the exosomes.
  • the molecules may be, for example, leukocyte antigen surface molecules, including human leukocyte antigen (HLA) surface molecules and human mismatch HLA surface molecules.
  • HLA human leukocyte antigen
  • the subject may be a mammal, including, but not limited to, a human.
  • the methods of the invention include predicting functional recovery of ischemic myocardium in a subject based on the miRNA cargo of exosomes collected from the biological samples.
  • the invention is directed to methods of predicting functional recovery of a target organ in a subject.
  • the method comprises (i) collecting, from a biological sample obtained from a subject having a target organ into which cells have been transplanted, exosomes derived from the transplanted cells, (ii) characterizing miRNA cargo of the collected exosomes, and (iii) predicting functional recovery of the target organ in the subject based on characteristics of the miRNA cargo, wherein the target organ is diseased, damaged, ischemic, or in some other manner malfunctioning.
  • predicting functional recovery of target organ in a subject can be achieved.
  • the invention is directed to methods of predicting functional recovery of ischemic myocardium in a subject.
  • the method comprises (i) collecting, from a biological sample obtained from a subject having ischemic myocardium and into which progenitor cells have been transplanted, exosomes derived from the transplanted progenitor cells, (ii) characterizing miRNA cargo of the collected exosomes, and (iii) predicting functional recovery of the ischemic myocardium in the subject based on characteristics of the miRNA cargo.
  • predicting functional recovery of ischemic myocardium in a subject can be achieved.
  • characterizing microRNA cargo may comprise sequencing one or more species of the microRNA present in the collected exosomes. Characterizing microRNA cargo may alternatively comprise otherwise identifying one or more species of microRNA present in the collected exosomes. Characterizing microRNA cargo may also comprise screening the contents of the exosomes for the presence of one or more specific species of microRNA; in some aspects of the invention, the specific species of microRNA are known to be associated with one or more activities performed by the target organ.
  • the cargo may screened for the presence of one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27- 5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR- 623, and miR-362-5p.
  • the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, miR-623, miR-94l, miR- 1224, and miR-l256. In further aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224.
  • miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27-5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR-623, and miR-362-5p is identified as being present in the cargo of the collected exosomes
  • functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.
  • miR-378b, miR-384, miR- 515-5p, miR-525-3p, miR-623, miR-94l, miR-l224, and miR-l256 when one or more of miR-378b, miR-384, miR- 515-5p, miR-525-3p, miR-623, miR-94l, miR-l224, and miR-l256 is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.
  • miR-378b, miR-384, miR- 515-5p, miR-525-3p, and miR-l224 when one or more of miR-378b, miR-384, miR- 515-5p, miR-525-3p, and miR-l224 is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.
  • the methods may further comprise repeating the method at one or more additional time points and characterizing miRNA cargo from each additional time point to determine whether there is a change in the miRNA cargo over time.
  • the cells may be allogeneic, that is, the cells may be obtained from an individual that is different from the subject into which the cells are transplanted.
  • the cells are allogeneic, exosome produced by the cells can be more easily identified in the biological sample.
  • the transplanted cells include, but are not limited to, stem cells and progenitor cells.
  • the cells may be one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).
  • CPCs cardiac progenitor cells
  • CDCs cardiosphere-derived cells
  • MSCs mesenchymal stem cells
  • BMCs bone marrow cells
  • ESCs embryonic stem cells
  • the subject is one into which cells have been transplanted.
  • the cells will have been transplanted into a target organ or into a target organ system in the subject.
  • Suitable target organs include, but are not limited to, heart, lungs, kidneys, liver, pancreas, spleen, brain, bladder, or lymph nodes.
  • Suitable target organ systems include, but are not limited to, cardiovascular system, digestive system, endocrine system, excretory system, lymphatic system, muscular system, nervous system, reproductive system, and respiratory system.
  • the exosomes may be collected within 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72 or more hours after completion of cell therapy or transplantation of the cells into the subject.
  • the exosomes may be collected within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after completion of cell therapy or transplantation of the cells into the subject.
  • the biological sample may be, but is not limited to, sputum/oral fluid, amniotic fluid, blood, a blood fraction, or fine needle biopsy samples (e.g., surgical biopsy, fine needle biopsy, etc.) urine, peritoneal fluid, and pleural fluid.
  • the biological sample is blood plasma.
  • the exosomes may be collected using antibodies having binding specificity for leukocyte antigen surface molecules displayed by the exosomes.
  • the leukocyte antigen surface molecules are human leukocyte antigen (ELLA) surface molecules and human mismatch ELLA surface molecules.
  • the subject may be a mammal, including, but not limited to, a human.
  • Figure 1 Phenotypic characterization and cardiac functional assessment in rat myocardial infarction (MI) model after adult CPCs and CDCs transplantation.
  • MI myocardial infarction
  • FIG. S2A Schematic diagram of the isolation of adult CPC and CDCs.
  • FIG. 2 Functional assessment of medium (TCM) derived from CPCs, CDCs and mixed cell population in vitro and in vivo.
  • TCM medium
  • D Effect of growth medium on growth properties of CPCs and CDCs as assessed by onset of senescence (E) and cellular proliferation by Alamar blue (see also Fig.
  • Figure 3 Characterization of exosomes obtained from CPCs and CDCs both in vitro and from rat plasma after injection of CPCs or CDCs respectively.
  • A Transmission electron microscope (TEM) visualization of exosomes obtained in vitro from CPCs and CDCs labeled with exosome marker CD63 using immunogold.
  • HLA-A goat secondary Qdot 605 by NTA (Nanosight NS300)
  • NTA Sonight NS300
  • H Exosomes from rat plasma were analyzed on NanoSight nanoparticle detector on light scatter (total exosomes) and fluorescence modes (HLA Qdot 605) for transplanted CPCs and CDCs derived exosomes using anti-HLA-A.
  • Human exosomes were isolated from rat plasma obtained after transplanted myocardial injections in rat MI model (See also schematic diagram at Fig. S7) (n 8).
  • Figure 4 Computational model of covariant microRNA using the cue-signal response paradigm. Computational model and the prediction of cardiac functions of exosomes miRNA cargo.
  • PCA Principal component analysis
  • C-E The predicted plasma (red bars) and in vitro (blue bars) CPCs and CDCs functional outcomes were compared with the observed functional data (green bars) for EF (C) and angiogenesis (D) in comparison with fibrosis function (E).
  • F Partial least squares regression (PLSR) and miRNA target analysis. Top microRNAs with known validated targets were identified among the 60 matched miRNAs using miRTarBase and plotted in PC space. Thirty-one miRNAs with validated targets were identified by miRTarBase. Clusters of miRNAs are formed based on the functional outcome.
  • CPCs cardiac progenitor cells
  • CDCs cardiosphere-derived cells
  • VIP variable importance for projection
  • EF ejection fraction.
  • FIG. 5 Verification of functional role of miRs as identified by computational analysis.
  • A Quantitative PCR depicting the enrichment of individual miRs in HMECs after transfection with miR mimics. Cells were transfected with individual miRs. (See also Fig. Sll).
  • B Cell proliferation assay using Alamar blue of the transfected cells with scrambled, miR 378, miR 384, miR 515, miR 525 and miR 1224.
  • C-D Trans well migration assay of the transfected cells with scrambled, miR 378, miR 384, miR 515, miR 525 and miR 1224.
  • E-F Wound healing assay of the transfected cells with scrambled, miR 378, miR 384, miR 515, miR 525 and miR 1224. * ⁇ 0.05, ** ⁇ 0.01, ***_p ⁇ 0.00l, and ****_p ⁇ 0.000l. Data are analyzed using one- way ANOVA followed by Mann- Whitney’s analysis (B, D, F).
  • Figure 6 Schematic of donor exosome purification and identification in rat plasma.
  • Figure 7 Computational modeling of EXOs miRNA cargo.
  • PCA Principal component analysis
  • PC principal component
  • B Predictability measurements of angiogenesis functional outcome. PLSR model was created with the top 300 genes of only patients (neonate, infant, child) and this model determined the predictability of CPCs and CDCs functions.
  • C cardiac progenitor cells
  • CDCs cardiosphere-derived cells
  • mRNA extracted from serum combined with exosomal pediatric hypoxic and normoxic CPCs [57] using hypoxic/normoxic cardiac functional data. Predictability rates were high for EF and angiogenesis and slightly less for fibrosis.
  • D Canonical pathway analysis. Ingenuity pathways analysis (IP A) was used to determine top canonical pathways for these genes. Cardiac and immune response related pathways are specified by orange and brown colors respectively.
  • FIG. 8 Transplanted MSCs release donor-specific exosomes into the recipient circulation of the HLHS patients post-operative 2 and 7, but not seen preoperatively (pre).
  • B Total HLA-A expressing plasma exosome numbers were quantified on the NanoSight and expressed as number of nanoparticles per milliter per microgram of exosome protein at different times postoperatively (day 2 and 7) and preoperatively (pre).
  • “a” or“an” may mean one or more.
  • the words“a” or“an” may mean one or more than one.
  • “another” may mean at least a second or more.
  • “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
  • the term“about” generally refers to a range of numerical values (e.g., +/- 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
  • the term“about” may include numerical values that are rounded to the nearest significant figure.
  • transplanted stem cell/progenitor cell-derived exosome characterization was investigated as a noninvasive tool to monitor their cellular counterpart’s presence and function in vivo.
  • the present invention provides methods directed to screening biological samples from subjects into which cells have been transplanted or otherwise transferred for the presence of exosomes, or for a change in the number of exosomes over time. Such methods provide a clear indication that transplanted cells have survived transfer into recipient tissue and that the cells are present in the recipient.
  • the invention is directed to methods of monitoring cells transplanted into a subject.
  • the method comprises screening a biological sample obtained from a subject into which cells have been transplanted for the presence of transplanted cell- derived exosomes.
  • the presence of exosomes in the biological sample indicates the presence of the transplanted cells in the subject.
  • An increase/decrease in the number of transplanted cell- derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells in the subject over time.
  • monitoring of cells transplanted into the subject can be achieved.
  • the invention is directed to methods of monitoring a subject receiving cell-based therapy.
  • the method comprises screening a biological sample obtained from a subject receiving cell therapy for the presence of transplanted cell-derived exosomes.
  • the presence of exosomes in the biological sample indicates the presence of the transplanted cells in the subject.
  • An increase/decrease in the number of transplanted cell-derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells in the subject over time.
  • the invention is directed to methods for predicting functional recovery of ischemic myocardium in a subject into which cells have been
  • the method comprises screening a biological sample obtained from a subject that has ischemic myocardium and into which cells have been transplanted for the presence of transplanted cell-derived exosomes.
  • the presence of exosomes in the biological sample indicates functional recovery of the ischemic myocardium in the subject is more likely than a
  • An increase/decrease in the number of transplanted cell-derived exosomes over time may indicate a corresponding increase/decrease in the number of transplanted cells, which may aid in the functional recovery of the ischemic myocardium, in the subject over time.
  • a prediction of functional recovery of ischemic myocardium in the subject can be achieved.
  • the methods may further a step of enumerating the number of exosomes present in the biological samples, whether based on the specific number of exosomes in a biological sample or based on the weight or volume of cells in the sample.
  • the methods may further comprise repeating the method at one or more additional time points and enumerating the number of exosomes present in the biological samples from each time point to determine whether there is a change in the number of exosomes over time.
  • the method can be conducted on a subject within a couple of days of receiving progenitor cells, and then a week, month, etc. later to determine whether the transplanted cells continue to survive in the subject.
  • the present invention includes profiling the contents of exosomes collected from the biological samples.
  • the methods of the invention include profiling the contents of exosomes collected from the biological samples. For example, polynucleotides and polypeptides within the exosomes can be isolated and characterized.
  • the invention is directed to methods of profiling exosomes derived from cells transplanted into a subject.
  • the method comprises (i) collecting, from a biological sample obtained from a subject into which cells have been transplanted, exosomes derived from the transplanted cells and (ii) characterizing cargo of the collected exosomes.
  • profiling of exosomes derived from cells transplanted into a subject can be achieved.
  • the cargo may be, but is not limited to, one or more of polynucleotides, polypeptides, and lipids.
  • the cargo is polynucleotides, such as, but not limited to, microRNA.
  • the invention is directed to methods of profiling exosomes derived from cells transplanted into a subject.
  • the method comprises (i) collecting, from a biological sample obtained from a subject into which cells have been transplanted, exosomes derived from the transplanted cells and (ii) characterizing microRNA cargo of the collected exosomes.
  • profiling of exosomes derived from cells transplanted into a subject can be achieved.
  • characterizing the cargo may be via means that include, but are not limited to, identifying specific types of cargo contained in the exosomes, e.g. the polynucleotide, polypeptide, or lipid species within the exosomes, and identifying specific species of polynucleotide, polypeptide, or lipid within the exosomes.
  • identifying specific types of cargo contained in the exosomes e.g. the polynucleotide, polypeptide, or lipid species within the exosomes
  • identifying specific species of polynucleotide, polypeptide, or lipid within the exosomes e.g. the polynucleotide, polypeptide, or lipid species within the exosomes.
  • the skilled artisan will readily understand that a variety of means are available for performing such characterizations that include, but are not limited to, Northern, Southern and Western blots, sequencing, NMR analysis, HPLC analysis, immunologic analysis, etc.
  • the polynucleotide may be sequenced in order to determine its identity or subject to hybridization with an labeled probe, to name only two of the multitude of means for identifying a polynucleotide molecule.
  • characterizing the microRNAs may comprise sequencing one or more species of the microRNA present in the collected exosomes. Characterizing microRNA may alternatively comprise otherwise identifying one or more species of microRNA present in the collected exosomes. Characterizing microRNA cargo may also comprise screening the contents of the exosomes for the presence of one or more specific species of microRNA; in some aspects of the invention, the specific species of microRNA are known to be associated with one or more activities performed by the target organ.
  • the cargo may screened for the presence of one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27- 5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR- 623, and miR-362-5p.
  • the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, miR-623, miR-94l, miR- 1224, and miR-l256. In further aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224.
  • the methods may further comprise repeating the method at one or more additional time points and characterizing cargo from each additional time point to determine whether there is a change in the cargo over time.
  • the method can be conducted on a subject within a couple of days of receiving progenitor cells, and then a week, month, etc. later to determine whether the contents of the cargo have changed over time.
  • the present invention is extended to include predicting functional recovery of diseased, damaged, or ischemic organs or tissues in a subject into which cells have been transplanted based on the miRNA cargo of exosomes collected from biological samples obtained from the subject.
  • miRNA microRNA
  • miRNA cargo of the collected exosomes for example determining whether specific miRNAs known to be associated with improve cardiac function are present, predictions as to whether functional recovery of ischemic myocardium in a subject will be achieved can be made.
  • exemplary miRNAs are those associated with enhanced angiogenesis, cellular proliferation, cellular migration, and wound healing.
  • the invention is directed to methods of predicting functional recovery of a target organ in a subject.
  • the method comprises (i) collecting, from a biological sample obtained from a subject having a target organ into which cells have been transplanted, exosomes derived from the transplanted cells, (ii) characterizing miRNA cargo of the collected exosomes, and (iii) predicting functional recovery of the target organ in the subject based on characteristics of the miRNA cargo, wherein the target organ is diseased, damaged, ischemic, or in some other manner malfunctioning.
  • predicting functional recovery of a target organ in a subject can be achieved.
  • the methods of the invention include predicting functional recovery of ischemic myocardium in a subject based on the miRNA cargo of exosomes collected from the biological samples.
  • the invention is directed to methods of predicting functional recovery of ischemic myocardium in a subject.
  • the method comprises (i) collecting, from a biological sample obtained from a subject having ischemic myocardium and into which cells have been
  • characterizing microRNA cargo may thus comprise sequencing one or more species of the microRNA present in the collected exosomes. Characterizing microRNA may alternatively comprise otherwise identifying one or more species of microRNA present in the collected exosomes. Characterizing microRNA cargo may also comprise screening the contents of the exosomes for the presence of one or more specific species of microRNA; in some aspects of the invention, the specific species of microRNA are known to be associated with one or more activities performed by the target organ.
  • the cargo may screened for the presence of one or more of miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27- 5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR- 623, and miR-362-5p.
  • the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, miR-623, miR-94l, miR- 1224, and miR-l256. In further aspects of the invention, the cargo may screened for the presence of one or more of miR-378b, miR-384, miR-5l5-5p, miR-525-3p, and miR-l224.
  • miR-4649-3p, miR-548d-5p, miR-l256, miR-l270, miR-384, miR-2355-3p, miR-3 l27-5p, miR-7l8, miR-378b, miR-92l, miR-l224-5p, miR-337-5p, miR-5l5-3p, miR-767-5p, miR-623, and miR-362-5p is identified as being present in the cargo of the collected exosomes
  • functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.
  • miR-378b, miR-384, miR- 515-5p, miR-525-3p, miR-623, miR-94l, miR-l224, and miR-l256 when one or more of miR-378b, miR-384, miR- 515-5p, miR-525-3p, miR-623, miR-94l, miR-l224, and miR-l256 is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.
  • miR-378b, miR-384, miR- 515-5p, miR-525-3p, and miR-l224 when one or more of miR-378b, miR-384, miR- 515-5p, miR-525-3p, and miR-l224 is identified as being present in the cargo of the collected exosomes, functional recovery of the ischemic myocardium in the subject is predicted to be more likely than functional recovery of ischemic myocardium in a subject in which the miRNAs are not identified as being present in the collected exosomes.
  • the methods may further comprise repeating the method at one or more additional time points and characterizing miRNA cargo from each additional time point to determine whether there is a change in the miRNA cargo over time.
  • the transplanted cells may be autologous, syngeneic, allogeneic or xenogeneic cells, when consider in the context of the subject receiving the cells.
  • the transplanted cells will commonly be allogeneic.
  • exosome produced by the cells can be more easily identified in the biological sample.
  • the transplanted cells include, but are not limited to, stem cells and progenitor cells.
  • stem cells refers to biological cells having the potential to differentiate into other types of cells and that retain the ability to divide to produce more of the same type of stem cell.
  • Stem cells include embryonic stem cells and adult stem cells.
  • progenitor cells refers to biological cells that are descendants of stems cells that differentiate into a specific type of cell. Progenitor cells are more limited than stem cells in their ability to divided.
  • Suitable cells for use in the methods of the invention include, but are not limited to, one or more of cardiac progenitor cells (CPCs), cardiosphere-derived cells (CDCs), mesenchymal stem cells (MSCs), bone marrow cells (BMCs) and embryonic stem cells (ESCs).
  • CPCs cardiac progenitor cells
  • CDCs cardiosphere-derived cells
  • MSCs mesenchymal stem cells
  • BMCs bone marrow cells
  • ESCs embryonic stem cells
  • the term“biological sample” refers to a sample of biological tissue, cells, or fluid that may comprise exosomes and that can be obtained from a subject and screened for the presence of exosomes.
  • suitable biological samples include, but are not limited to, sputum/oral fluid, amniotic fluid, blood, a blood fraction, or fine needle biopsy samples (e.g., surgical biopsy, fine needle biopsy, etc.) urine, peritoneal fluid, pleural fluid, and the like.
  • the biological sample is blood plasma. The sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample.
  • such pretreatment may include preparing plasma from blood, diluting viscous fluids and so forth.
  • Methods of pretreatment may also involve, but are not limited to, filtration, precipitation, dilution, distillation, mixing, centrifugation, freezing, lyophilization, concentration, inactivation of interfering components, the addition of reagents, lysing, etc.
  • the term“transplanted cells” refers to one or more individual cells (e.g. a stem cell or progenitor cell, such as a cardiac progenitor cell (CPC), cardiosphere-derived cell (CDC), mesenchymal stem cell (MSC), bone marrow cell (BMC) or embryonic stem cell (ESC)) that has been isolated from its endogenous tissue or organ before being introduced into a subject in need thereof.
  • a stem cell or progenitor cell such as a cardiac progenitor cell (CPC), cardiosphere-derived cell (CDC), mesenchymal stem cell (MSC), bone marrow cell (BMC) or embryonic stem cell (ESC)
  • CPC cardiac progenitor cell
  • CDC cardiosphere-derived cell
  • MSC mesenchymal stem cell
  • BMC bone marrow cell
  • ESC embryonic stem cell
  • transplanted cell-derived exosomes as meaning exosomes derived from one or more cells that have transplanted into a subject in need of such cell transplantation.
  • the biological sample may be screened within 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72 or more hours after completion of cell therapy or transplantation of the cells into the subject.
  • the biological sample may be screened within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after completion of cell therapy or transplantation of the cells into the subject.
  • the exosomes may be collected within 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72 or more hours after completion of transplanted cell therapy or transplantation of the cells into the subject.
  • the exosomes may be collected within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days after completion of cell therapy or transplantation of the cells into the subject.
  • the biological samples may be screened for the presence of exosomes using antibodies having binding specificity for molecules displayed by the exosomes using a variety of means, including, for example, flow cytometry, enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay, magnetic immunoassay, radioimmunoassay, fluorescent immunosassay, Western immunoblot assay, dot immunoblot assay, slot immunoblot assay, and a particle analyzer (e.g., Nanosight) which detects nano-particles, etc.
  • ELISA enzyme-linked immunosorbent assay
  • lateral flow immunoassay magnetic immunoassay
  • radioimmunoassay radioimmunoassay
  • fluorescent immunosassay e.g., fluorescent immunosassay
  • Western immunoblot assay e.g., dot immunoblot assay
  • slot immunoblot assay e.g., slot immunoblot assay
  • a particle analyzer e.
  • the exosomes may be isolated from a biological sample using well-known techniques such as flow cytometry, immunosorbent plates and columns, ultracentrifugation, immune precipitation, etc.
  • the molecules displayed by the exosomes may be, for example, leukocyte antigen surface molecules, including human leukocyte antigen (HLA) surface molecules and human mismatch HLA surface molecule.
  • HLA human leukocyte antigen
  • the subject is one into which progenitor cells have been transplanted.
  • the progenitor cells will have been transplanted into a target organ or into a target organ system in the subject.
  • Suitable target organs include, but are not limited to, heart, lungs, kidneys, liver, pancreas, spleen, brain, bladder, or lymph nodes.
  • Suitable target organ systems include, but are not limited to, cardiovascular system, digestive system, endocrine system, excretory system, lymphatic system, muscular system, nervous system, reproductive system, and respiratory system.
  • the subject is a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.
  • c-kit + /CD45 CPCs were isolated from RAA biopsies of adult myocardium using a previously described protocol [6,28] Briefly, samples were minced and digested in Ham’s F12 (Lonza # 12-615F) basal medium containing 1-2 mg/ml of collagenase type II (Worthington # 4177) on an orbital shaker for 45 min at 37°C.
  • CPCs cardiac progenitor cells
  • phase-bright cells originating from explants were removed by mild trypsinization and plated on fibronectin coated flasks at low density (1.5 to 3 x lO 4 cells/mL) in cardiospheres-growing medium (CGM: FBS (3.5%,), IMDM (35%), Pen/Strep (1%), Glutamine (1%), B27 Serum substitute (2%), Cardiotroponin I (4 ng/ml), Epidermal Growth Factor (25 ng/ml), human basic Fibroblast Growth Factor (80 ng/ml,
  • cardiospheres were collected by centrifugation and expanded on fibronectin coated flasks in CDCs complete growth medium to obtain cardiosphere derived cells (CDCs).
  • c-kit + /CD45 cells derived from adult patients at passage 4 for both CPCs and CDCs were labeled with fluorochrome-conjugated primary antibodies: mesenchymal stem cell marker CD 105 or CD90, cardiac-specific transcription factors NKX2.5, GATA4, cardiac stem cell marker c-kit + , endothelial cell marker CD31, mast cell marker tryptase, hematopoietic cell lineage markers CD45 and CD34.
  • Conjugated isotype antibodies were used as negative controls.
  • the labeled cells were evaluated by flow cytometry with a Becton -Dickinson FACS Calibur (San Jose, CA), with 25,000 events/sample collected. Cell transplantation and echocardiography
  • Myocardial infarction was induced by permanent ligation of the left anterior descending (LAD) coronary artery in athymic nude male rats (weight, 250-300 g). The heart was exposed via a left thoracotomy, and the proximal LAD was ligated. Subsequently, 1 million aCPCs or aCDCs suspended in 100 pL of vehicle (IMDM) were injected into the myocardium at four sites adjacent to the infarct. Transthoracic echocardiograms were acquired 1 day, 7 days, and 28 days after myocardial infarct surgery.
  • IMDM vehicle
  • Tissues were processed as previously described [5,6,28] Briefly, rat hearts were excised under anesthesia after collection of echocardiographic data and perfused with 4% paraformaldehyde. Tissues were cryo-preserved using 30% sucrose and embedded in OCT (TissueTek). Sections were cut to 7 pm using a cryostat and immunostained for isolectin B4 (Invitrogen; Carlsbad, CA), a-SMA (Sigma; St. Louis, MO), sarcomeric a-actin (Sigma), human nuclear antigen (HNA, Millipore; Billerica, MA), human mitochondrial antigen (HMA,
  • DAPI 4,6-diamidino-2-phenylindole nuclear stain
  • infarct size Masson trichrome-stained sections at various levels along the long axis were analyzed for collagen deposition. The midline technique for infarct size determination was used as previously described [27] The stained sections were analyzed by Image-Pro software [27] To calculate the amounts of viable and non-viable tissue, the number of pink pixels (viable tissue) and blue pixels (non-viable tissue) were measured and the ratio of non-viable tissue/overall number of the pixels was presented. 6 sections per animal and at least 15 animals per group were analyzed.
  • CPCs and CDCs are mixed together in the proportion of 20%, 40% and 80% of CPCs in the CDCs population for in vivo and in vitro studies.
  • CDCs populations inherently contain 10% c-kit + cells, which was factored into the proportions presented below, and which is why artificial mixtures started from 20% CPCs in CDCs.
  • IMDM vehicle
  • ELISA was performed for human VEGFA, SDF-la, PDGFB, IGF-l, ANG-l, bFGF, and HGF in the core facility at the University of Maryland School of Medicine using human-specific ELISA kits (Millipore and R&D systems), according to the manufacturers’ protocols.
  • CPCs and CDCs were placed on the upper layer of a cell culture insert with permeable fluorescence block (8.0 um pore size, Cat # 351152) membrane and the media with serum and without serum are placed below the cell permeable membrane in a 24 well cell culture plate (Cat # 353504). Following an incubation period (6-7hours) at 37°C, the cells that migrated through the membrane were stained with Calcein (Calcein AM C3100MP, Thermo Fisher;
  • Cell proliferation was assessed using Alamar blue as per manufacturer’s instructions (Alamar Blue 10% of the total volume of the medium). Briefly, 5000 cells/well were seeded in 96 well plates in their respective medium. After overnight incubation at 37°C, 10 ul of Alamar- blue cell viability reagent (Invitrogen cat # 1933424) was added per well and absorbance was taken immediately (basal absorbance) and after 3 hours (proliferation absorbance) of incubation at 37°C. To obtain the actual absorbance, basal absorbance was subtracted from proliferation absorbance.
  • MCDB 131 basal medium containing exosomes derived from CPCs or CDCs. Cells were fixed in their wells after 16 hours; migrated distances were calculated using image Pro software.
  • Exosome proteins were separated using NuPAGE 4-12% Bis-Tris Gels and transferred onto nitrocellulose membranes (Life Technologies, CA, EISA). The blots were blocked with 5% non-fat dry milk at room temperature for 1 hour and incubated overnight at 40°C with desired primary antibodies at concentration per manufacturer’s protocol, followed by incubation with HRP-conjugated secondary antibodies (Santa Cruz Biotechnologies Inc.) at room temperature for 1 hour. The membrane blots were developed with ECL detection reagent (Luminata Forte, Millipore Corporation, Billerica, MA) per manufacturer’s protocol and detected through Chemiluminescence using Image quant LAS 400 Phospho-Imager (GE Health, USA).
  • ECL detection reagent Luminata Forte, Millipore Corporation, Billerica, MA
  • Thermo Scientific PageRuler Plus Prestained protein Ladder (# 26619) was used.
  • Antibodies specific to c-kit (Cat# 18696-1-AP, Protein Tech Labs), Troponin-I (ab56357, Abeam), HLA-A (ab52922, Abeam), Flotilin-l (3253, Cell Signaling Technology), HLA-A1 (BIH0331, One Lambda Inc), NKX2.5 (SC-376565) (according to company’s data sheet this antibody recognizes two bands in some cell lines), CD-63 (Sc-7080), and Cytochrome- C (Sc-l3 l56) were purchased from Santa Cruz Biotechnology, Inc.
  • HLA-A specific antibodies were covalently conjugated to N-Hydroxy Succinamide magnetic beads (NHS beads, Pierce Inc.) as per manufacturer’s suggestions. 50 pg protein equivalent of exosomes were incubated with antibody -magnetic-beads complex for overnight at 4°C on a rocker platform. The bead bound exosomes were washed using PBS and eluted using manufacturer’s protocol and utilized for downstream analysis. Isolation procedure of exosomes and exosomal micro RNA
  • Exosomes were isolated from CPC and CDC conditioned media (48 hours) by size exclusion chromatography and micro RNA immediately isolated from the exosomes using Exo RNeasy kit (Cat# 77023, Qiagen Inc.) as per manufacturer instruction. Total RNA was quantified on a NanoDrop ND-1000 spectrophotometer followed by RNA quality assessment on an Agilent TapeStation. Micro RNA labeling was performed by FlashTag Biotin HSR RNA Labeling Kit (Applied Biosystems). GeneChip miRNA Arrays 4.0 Arrays were hybridized with Flash Tag Biotin Labeled total RNA (100 ng) from experimental and control samples in 100 pl
  • Target denaturation was performed at 99°C for 5 min. and then 45°C for 5 min. followed by hybridization for 18 hrs at 48°C.
  • Arrays were washed and stained using Genechip Fluidic Station 450 according to protocol. Chips were scanned on an Affymetrix Gene Chip Scanner 3000, using Command Console Software. These studies were performed at Cancer Genomics Laboratory of the Thomas Jefferson University.
  • Exosomes from CDCs and CPCs were isolated and analyzed as previously described [22] Briefly, exosomes were isolated using by size exclusion chromatography using a Sepharose 2B column (Sigma-Aldrich) and eluted fractions were analyzed using nanosight NS300 (405 nm laser diode) for the presence of 40-120 nm diameter vesicles. For cell based in vitro assays, exosomes from CDCs and CPCs were used at the constant number 0.5 x 10 9 /100 ul, equivalent to 10 ng/lOO ul proteins.
  • HLA class I The surface expression of HLA class I was analyzed using exosomes (2 x 10 8 ) incubated with anti-HLA class I (0.5 ug, Cat #311402, Bio Legend) for 2 hrs. Thereafter, goat anti-mouse Qdot 605 (1 :50 dilution, Q-11001MP, Thermo Fisher) was added as fluorescent secondary antibody and incubated for 2 hours. The unbound primary and secondary antibodies were removed using Exosquick plus (EQPL10A-1, System Biosciences) exosome isolation kit according to the manufacturer protocol. Total exosomes were counted in bright field emission and the HLA class I labeled exosomes were counted using fluorescent emission in Nanosight.
  • HLA-specific exosome signal was quantified using following formula: (HLA Flourescence/HLA light scatter) - (POD 0 Flourescence/POD 0 light scatter) - (IgG isotype Flourescence/IgG isotype light scatter). miRNAs mimic transfections
  • human miRIDIAN mimics (miRNA 378, miRNA 384, miRNA 515-5p, miRNA 525-3p and miRNA 1224) along with the transfection control -Dy547 (cat # CP-004500-01-05), positive control (cat # CP-001000-02-05) and scrambled (non-targeting) miR, were procured from Dharmacon.
  • Cells were transfected with 50 nM of each miR mimic using reverse transfection protocol of lipofectamine RNAiMAX® (cat # P/N 100014472).
  • Exosomes were negatively stained after absorption onto carbon-coated copper grids for 2 minutes. Grids were washed twice for 1 minute each in dELO and stained for 1 minute with 1% aqueous uranyl acetate (Ted Pella; Redding, CA). Samples were viewed on a JEOL 1200EX transmission electron microscopy (JEOL USA; Peabody, MA) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques; Woburn, MA).
  • grids were incubated with mouse anti-CD63 antibody (AB193349, Abeam), for 30 minutes, followed by secondary goat anti-mouse IgG Ab conjugated to colloidal gold (Jackson Immuno Research Laboratories; West Grove, PA) for 30 minutes. Grids were washed and stained with uranyl acetate and viewed by transmission EM as described above.
  • Fig. IB characterized for cell surface markers and had similar cellular morphologies as previously described for each cell type.
  • Both cell types expressed mesenchymal stem cell markers (CD 105 and CD90) and the cardiomyocyte lineage-specific markers, transcription factor NKX2.5, and Troponin I. They did not express hematopoietic markers (CD34 and CD45), mast cell marker (tryptase), or cardiomyocyte lineage-specific transcription factor GATA4, however, CPCs were 85% c-kit + positive (CD117; Fig. IB).
  • progenitor cells Another key functional property of the progenitor cells is their intrinsic migration ability after transplantation into the infarcted myocardium. To recapitulate this functional activity in vitro , a trans-well migration assay was performed to measure the migrator ⁇ ' response of both progenitor cell types. Derived from same RAA of four biological replicates, CPCs migration was significantly more as compared to CDCs (Fig. I E) after 6 hours of incubation in presence of serum.
  • IMDM Modified Dulbecco’s Medium
  • LV functional improvement was significantly greater with CPCs as compared to CDCs (Fig. 1F-1I).
  • the functional improvement was apparent 1 week after MI and sustained for the entire 4 weeks of follow-up.
  • Structural changes in the LV were further evaluated by histologic analysis at 28 days post-MI, focusing on fibrosis (Masson trichrome), arteriolar density (smooth muscle actin), and total vascular density (Isolectin IB4). Representative images of myocardial fibrosis and quantification of the three different treatment groups are shown in Fig. 1 J. At 4 weeks post-MI, infarct size was analyzed by measuring the area of fibrosis relative to total stained myocardial area.
  • HMECs keeping TCM proteins concentration constant (50 ng/ul proteins concentration).
  • Exosomes were purified from the conditioned medium using size exclusion chromatography [5,38] Transmission electron microscopy (TEM) confirmed that the isolated extracellular vesicles were in the size range of exosomes and expressed canonical exosome marker CD63, as identified by immunogold staining (Fig. 3A). Flow cytometry using CD63 -conjugated magnetic beads demonstrated higher CD63 expression on CDCs derived exosomes as compared to CPCs derived exosomes (Fig. 3B).
  • TEM Transmission electron microscopy
  • CDCs-derived exosomes were not only larger in size (average size 165 nm) than CPCs-derived exosomes (average size 124 nm), but also existed at a higher concentration as compared to CPCs-derived exosomes (Fig. 3C).
  • CPCs-derived exosomes showed more proliferation of HMEC and exhibited more angiogenic potential when compared to CDCs- derived exosomes in transwell migration assay and wound healing assay, when keeping exosome numbers constant and equal (Fig. 3D-E. It was concluded that despite being fewer in number, CPCs-derived exosomes maybe more potent for myocardial repair as compared to CDCs derived exosomes.
  • Fig. IB The exosomes derived from the CPCs and CDCs contained NKX2.5, HLA, Troponin I, c-kit + , and exosomes markers flotillin-l and CD63 (Fig. 3F). Importantly, cytochrome C, a marker for apoptotic bodies, could not be detected showing the homogenous population of exosomes.
  • HLA-A is selectively present on the surface of human stem/progenitor cell exosomes. Therefore, the MHC class I specificity was utilized to quantify and purify the human exosome subpopulation (Fig. 6) from the rat plasma after cellular transplantations.
  • This platform was tested in vitro on exosomes derived from CDCs or CPCs in culture. Purified exosomes were analyzed on the NanoSight in fluorescence mode (Qdot 605) for HLA-detection [39,40] HLA-A specific signal using goat secondary Qdot 605, was detected on the exosomes derived from CPCs or CDCs (Fig. 3G).
  • progenitor cells specific exosome signal in the recipient rat plasma from days 2 and 7 post- progenitor cell transplantation was quantified in the total exosomes using anti-HLA-A
  • HLA-A and HLA-A1 enriched progenitor cell specific exosome subpopulations expressed HLA molecules
  • flotillin 1 exosome marker
  • c-kit progenitor cell marker
  • cardiomyocyte marker troponin I Fig. 3J
  • PCA principal component analysis
  • PLSR partial least squares regression
  • IP A Ingenuity Pathway Analysis
  • miRNAs were identified associated with improvement in ejection fraction (miRs 378b, 623, and 941), reduction in fibrosis (1256 and 384), and induction of angiogenesis (525- 3p, 515-5p, and 1224).
  • Table 4 List of canonical signaling pathways affected by VIP miRs
  • Bioinformatics tools facilitate the study of miRNAs by providing a list of potential functions, however, due to multi-targeted approach of miRNAs, it is important to validate the predicted functions of miRNA.
  • miRNAs 378, 384, 515, 525, and 1224 [10,9,15,11,14, respectively] were identified and predicted to improve cardiac function after MI by enhancing angiogenesis. These miRNAs were not detected in the in vitro cultured CPCs exosomes but were enriched in the circulating CPCs exosome subset purified from rat plasma in the MI model.
  • HMECs were transfected with the mimics of these miRNAs and their angiogenic potential was assessed by three well -accepted angiogenic assays: a) endothelial cell proliferation, b) transwell migration, and c) wound healing assay [45,46] Transfection with specific miRNA mimics resulted in multifold enrichment of that specific miRNA in the transfected cells (Fig. 5A). All the shortlisted miRNAs, as predicted, significantly induced cellular proliferation (Fig. 5B) as compared to non-specific miR transfection control. Next, transwell migration assay (Fig.
  • the ELPIS Phase I study is an open-label study to primarily determine the safety and feasibility of injecting allogeneic human mesenchymal stem cells (MSCs) into the right ventricle (RV) of human patients having hypoplastic left heart syndrome (HLHS) and undergoing the Stage II operation.
  • the secondary objective is to determine the efficacy of MSC treatment from baseline to 12 month follow-up in all MSC-treated subjects by serial cardiac magnetic resonance (CMR).
  • CMR serial cardiac magnetic resonance
  • Efficacy endpoints included RV regional and global cardiac function, ventricular volume, heart failure status, size of fibrosis, and somatic growth.
  • TGF i p superfamily plays a key role in attenuation of cardiac hypertrophy, cardioprotection, and remodeling after myocardial infarction (MI) as an autocrine/paracrine factor [67-69] Similar to the technique described for isolating tissue-specific exosomes for monitoring immunologic rejection for solid organ transplantation, mismatched anti-HLA antibody were used to isolate and quantify MSC-specific donor exosomes in the serum of ELPIS HLHS patients.
  • the miRNAs enriched in the circulating progenitor cell exosomes may be
  • Computational modeling provides an insightful avenue for determining the mechanistic pathways driving progenitor cell mediated remodeling of the MI myocardium. Similar to a previous validation study for the computational modeling of transplanted pediatric progenitor cells [42,55], the initial validation of the model was performed by examining miRNAs involved in angiogenesis. The angiogenesis endpoint was selected from the model because its high significance for cardiac function recovery and well-established in vitro angiogenesis assays. Although miRNAs 378 and 525 failed to promote cell migration (Fig. 5), all the shortlisted miRNAs induced cellular proliferation, suggesting that the methodology adopted by the computational analyses presented herein is accurate and the exosomes secreted after cellular transplantation are more effective in predicting miRNAs involved in angiogenesis.
  • CPCs By direct head-to-head comparison of miRNA in vitro functionality, functional superiority of CPCs as compared to CDCs was demonstrated. Since both cell types were derived from the same heart biopsy sample, the approach presented herein eliminated patient variability, strengthening the observed comparative data. CPCs were superior in terms of paracrine factor secretion, angiogenesis, myocardial tissue preservation, and functional improvement. As expected, increasing the c-kit+ cell concentration in the CDCs increased myocardial recovery, supporting the critical function of the c-kit+ cell population. Despite increased exosome secretion by CDCs in vitro , CPCs demonstrated increased exosome secretion in vivo in this model. Further, the cardioprotective pathways identified by the miRNA-driven pathways potentially mediated by exosome transfer gives an unparalleled insight into the mechanisms of cellular recovery following CPCs and CDCs administration.
  • VEGF nanoparticles repair the heart after myocardial infarction.
  • MiR-384 inhibits human colorectal cancer metastasis by targeting KRAS and CDC42. Oncotarget 7, 84826-84838 (2016).
  • Hsa-miR-623 suppresses tumor progression in human lung
  • the transforming growth factor-beta superfamily member growth-differentiation factor- 15 protects the heart from ischemia/reperfusion injury. Circulation research, 98(3):351-60 (2006).
  • GDF15/MIC-1 functions as a protective and antihypertrophic factor released from the myocardium in association with SMAD protein activation. Circulation research , 98(3):342-50 (2006).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Endocrinology (AREA)
  • Biophysics (AREA)
  • Physiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Le domaine des cellules souches est gêné par l'incapacité à surveiller de manière non invasive des cellules transplantées dans un organe cible d'une manière reproductible, chronosensible et spécifique à la pathologie. On a avancé l'hypothèse que la quantification et la caractérisation de cargaison intraexosomale d'exosomes circulants spécifiques à une cellule transplantée permettraient qu'une plate-forme de surveillance fiable et non invasive reflète l'activité conditionnelle de leurs équivalents cellulaires. Pour tester cette hypothèse, on a eu recours à un modèle d'infarctus du myocarde xénogène humain-rat faisant intervenir deux types de cellules progénitrices bien étudiées : des cellules dérivées de la cardiosphère (CDC) et des cellules progénitrices cardiaques (CPC) c-kit+, dérivées du même appendice auriculaire droit d'êtres humains adultes. Pour surveiller de manière non invasive l'activité de CDC ou de CPC transplantées in vivo, des exosomes plasmatiques du receveur ont été purifiés à l'aide d'anticorps dirigés contre des molécules de surface d'antigènes des leucocytes humains (HLA) uniquement exprimées sur la surface d'exosomes humains. 7 jours après la transplantation, une augmentation de la concentration en exosomes plasmatiques spécifiques des CPC de 2,5 fois a été observée comparativement aux exosomes spécifiques des CDC. L'analyse de cheminement computationnelle n'est pas parvenue à établir une liaison entre l'ARNm cellulaire de CPC ou CDC et une récupération myocardique observée. Cependant, la récupération du myocarde était fortement liée à la cargaison en ARNmi des exosomes de CPC purifiés à partir du plasma du receveur. De plus, des cheminements mécanistes régissant la récupération du myocarde ont été identifiés vers des résultats spécifiques par les CPC transplantées. Collectivement, ces résultats démontrent le potentiel d'exosomes circulants spécifiques aux cellules progénitrices en tant que biopsie liquide qui fournit un aperçu de l'état des cellules transplantées.
PCT/US2019/060436 2018-11-09 2019-11-08 Procédés de prédiction de récupération fonctionnelle de tissu à l'aide d'exosomes circulants dérivés de cellules transplantées WO2020097440A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862758209P 2018-11-09 2018-11-09
US62/758,209 2018-11-09

Publications (1)

Publication Number Publication Date
WO2020097440A1 true WO2020097440A1 (fr) 2020-05-14

Family

ID=68848360

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/060436 WO2020097440A1 (fr) 2018-11-09 2019-11-08 Procédés de prédiction de récupération fonctionnelle de tissu à l'aide d'exosomes circulants dérivés de cellules transplantées

Country Status (1)

Country Link
WO (1) WO2020097440A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210009935A1 (en) * 2019-07-12 2021-01-14 Hitachi, Ltd. Cell culture monitoring device and cell culture system
WO2022204045A1 (fr) * 2021-03-22 2022-09-29 Spiritus Therapeutics, Inc. Utilisations diagnostiques et thérapeutiques d'exosomes puissants purifiés contenant un cargo de signature basé sur une maladie et basé sur une thérapie

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014013258A1 (fr) * 2012-07-19 2014-01-23 Reneuron Limited Microparticules de cellules souches
WO2017066390A1 (fr) * 2015-10-13 2017-04-20 The Trustees Of The University Of Pennsylvania Méthodes d'utilisation d'exosomes enrichis comme plateforme pour surveiller l'état d'un organe

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014013258A1 (fr) * 2012-07-19 2014-01-23 Reneuron Limited Microparticules de cellules souches
WO2017066390A1 (fr) * 2015-10-13 2017-04-20 The Trustees Of The University Of Pennsylvania Méthodes d'utilisation d'exosomes enrichis comme plateforme pour surveiller l'état d'un organe

Non-Patent Citations (72)

* Cited by examiner, † Cited by third party
Title
"Molecular Biology and Biotechnology: a Comprehensive Desk Reference", 1995, WILEY, JOHN & SONS, INC.
"The Encyclopedia of Molecular Biology", 1994, BLACKWELL PUBLISHERS
A. G. IBRAHIMK. CHENGE. MARBAN: "Exosomes as critical agents of cardiac regeneration triggered by cell therapy", STEM CELL REPORTS, vol. 2, 2014, pages 606 - 619, XP055335389, DOI: 10.1016/j.stemcr.2014.04.006
A. HABERTHEUER ET AL.: "Donor tissue-specific exosome profiling enables noninvasive monitoring of acute rejection in mouse allogeneic heart transplantation", THE JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY
A. HABERTHEUER ET AL.: "Donor tissue-specific exosome profiling enables noninvasive monitoring of acute rejection in mouse allogeneic heart transplantation", THE JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY, vol. 155, 2018, pages 2479 - 2489, XP085396830, DOI: 10.1016/j.jtcvs.2017.12.125
A. IBRAHIME. MARBAN: "Exosomes: Fundamental Biology and Roles in Cardiovascular Physiology", ANNUAL REVIEW OF PHYSIOLOGY, vol. 78, 2016, pages 67 - 83
A. KRAEMER ET AL.: "Cell survival following radiation exposure requires miR-525-3p mediated suppression of ARRBl and TXN1", PLOS ONE, vol. 8, 2013, pages e77484
A. R. WILLIAMSJ. M. HARE: "Mesenchymal stem cells: biology, pathophysiology, translational findings, and therapeutic implications for cardiac disease", CIRCULATION RESEARCH, vol. 109, 2011, pages 923 - 940, XP055054593, DOI: 10.1161/CIRCRESAHA.111.243147
ANDERSON JDJOHANSSON HJGRAHAM CSVESTERLUND MPHAM MTBRAMLETT CSMONTGOMERY ENMELLEMA MSBARDINI RLCONTRERAS Z: "Comprehensive Proteomic Analysis of Mesenchymal Stem Cell Exosomes Reveals Modulation of Angiogenesis via Nuclear Factor-KappaB Signaling", STEM CELLS, vol. 34, no. 3, 2016, pages 601 - 13
B. WANGR. CHEHELTANIJ. ROSANOD. L. CRABBEM. F. KIANI: "Targeted delivery of VEGF to treat myocardial infarction", ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY, vol. 765, 2013, pages 307 - 314
BENJAMIN LEWIN: "Genes VII", 2000, OXFORD UNIVERSITY PRESS
C. GARDINERY. J. FERREIRAR. A. DRAGOVICC. W. REDMANI. L. SARGENT: "Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis", JOURNAL OF EXTRACELLULAR VESICLES, vol. 2, 2013, XP055365668, DOI: 10.3402/jev.v2i0.19671
C. LAWSONJ. M. VICENCIOD. M. YELLONS. M. DAVIDSON: "Microvesicles and exosomes: new players in metabolic and cardiovascular disease", THE JOURNAL OF ENDOCRINOLOGY, vol. 228, 2016, pages R57 - 71
C. P. HODGKINSONA. BAREJAJ. A. GOMEZV. J. DZAU: "Emerging Concepts in Paracrine Mechanisms in Regenerative Cardiovascular Medicine and Biology", CIRCULATION RESEARCH, vol. 118, 2016, pages 95 - 107
D. J. LOB. KAPLANA. D. KIRK: "Biomarkers for kidney transplant rejection", NATURE REVIEWS. NEPHROLOGY, vol. 10, 2014, pages 215 - 225
D. L. SIMPSON ET AL.: "A strong regenerative ability of cardiac stem cells derived from neonatal hearts", CIRCULATION, vol. 126, 2012, pages 46 - 53
E. CAMBRIA ET AL.: "Translational cardiac stem cell therapy: advancing from first-generation to next-generation cell types", NPJREGENERATIVE MEDICINE, vol. 2, 2017, pages 17
F. ARSLAN ET AL.: "Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury", STEM CELL RESEARCH, vol. 10, 2013, pages 301 - 312, XP055419473, DOI: 10.1016/j.scr.2013.01.002
G. M. ELLISON ET AL.: "Endogenous cardiac stem cell activation by insulin-like growth factor-1/hepatocyte growth factor intracoronary injection fosters survival and regeneration of the infarcted pig heart", JAM COLL CARDIOL, vol. 58, 2011, pages 977 - 986, XP028266059, DOI: 10.1016/j.jacc.2011.05.013
G. PHINNEY DONALDF. PITTENGER MARK: "Concise Review: MSC-Derived Exosomes for Cell-Free Therapy", STEM CELLS (DAYTON, OHIO), vol. 35, 2017, pages 851 - 858, XP055591076, DOI: 10.1002/stem.2575
H. JEONG ET AL.: "Mesenchymal Stem Cell Therapy for Ischemic Heart Disease: Systematic Review and Meta-analysis", INTERNATIONAL JOURNAL OF STEM CELLS, 2018
H. JULICHA. WILLMSV. LUKACS-KORNEKM. KORNEK: "Extracellular vesicle profiling and their use as potential disease specific biomarker", FRONTIERS IN IMMUNOLOGY, vol. 5, 2014, pages 413
H. VALADI ET AL.: "Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells", NATURE CELL BIOLOGY, vol. 9, 2007, pages 654 - 659
HABERTHEUER ANDREAS ET AL: "Donor tissue-specific exosome profiling enables noninvasive monitoring of acute rejection in mouse allogeneic heart transplantation", JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY, MOSBY-YEAR BOOK, INC., ST. LOUIS, MO, US, vol. 155, no. 6, 1 February 2018 (2018-02-01), pages 2479 - 2489, XP085396830, ISSN: 0022-5223, DOI: 10.1016/J.JTCVS.2017.12.125 *
I. S. VLACHOS ET AL.: "DIANA-miRPath v3.0: deciphering microRNA function with experimental support", NUCLEIC ACIDS RESEARCH, vol. 43, 2015, pages 460 - 466
I. S. VLACHOS ET AL.: "DIANA-miRPath v3.0: deciphering microRNA function with experimental support", NUCLEIC ACIDS RESEARCH, vol. 43, 2015, pages W460 - W466
J. QIAN ET AL.: "MiR-1224-5p acts as a tumor suppressor by targeting CREB 1 in malignant gliomas", MOLECULAR AND CELLULAR BIOCHEMISTRY, vol. 403, 2015, pages 33 - 41, XP035445487, DOI: 10.1007/s11010-015-2334-1
K. M. BROUGHTON ET AL.: "Mechanisms of Cardiac Repair and Regeneration", CIRCULATION RESEARCH, vol. 122, 2018, pages 1151
K. U. HONG ET AL.: "c-kit+ Cardiac stem cells alleviate post-myocardial infarction left ventricular dysfunction despite poor engraftment and negligible retention in the recipient heart", PLOS ONE, vol. 9, 2014, pages e96725
KEMPF TEDEN MSTRELAU JNAGUIB MWILLENBOCKEL CTONGERS JHEINEKE JKOTLARZ DXU JMOLKENTIN JD: "The transforming growth factor-beta superfamily member growth-differentiation factor-15 protects the heart from ischemia/reperfusion injury", CIRCULATION RESEARCH, vol. 98, no. 3, 2006, pages 351 - 60, XP002399431, DOI: 10.1161/01.RES.0000202805.73038.48
L. BARILE ET AL.: "Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction", CARDIOVASCULAR RESEARCH, vol. 103, 2014, pages 530 - 541, XP055417520, DOI: 10.1093/cvr/cvu167
L. CHEN ET AL.: "Cardiac progenitor-derived exosomes protect ischemic myocardium from acute ischemia/reperfusion injury", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 431, 2013, pages 566 - 571, XP028980130, DOI: 10.1016/j.bbrc.2013.01.015
L. CHENJ. ZHANGX. HUK. D. PHILIPSONS. M. SCHARF: "The Na+/Ca2+ exchanger-1 mediates left ventricular dysfunction in mice with chronic intermittent hypoxia", JOURNAL OF APPLIED PHYSIOLOGY, vol. 109, 2010, pages 1675 - 1685
M. COLOMBOG. RAPOSOC. THERY: "Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles", ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, vol. 30, 2014, pages 255 - 289, XP055582304, DOI: 10.1146/annurev-cellbio-101512-122326
M. GNECCHI ET AL.: "Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells", NATURE MEDICINE, vol. 11, 2005, pages 367 - 368, XP002390236, DOI: 10.1038/nm0405-367
M. GYONGYOSI ET AL.: "Meta-Analysis of Cell-based CaRdiac stUdiEs (ACCRUE) in patients with acute myocardial infarction based on individual patient data", CIRCULATION RESEARCH, vol. 116, 2015, pages 1346 - 1360
M. KHAN ET AL: "Embryonic Stem Cell-Derived Exosomes Promote Endogenous Repair Mechanisms and Enhance Cardiac Function Following Myocardial Infarction", CIRCULATION RESEARCH, vol. 117, no. 1, 22 April 2015 (2015-04-22), US, pages 52 - 64, XP055433483, ISSN: 0009-7330, DOI: 10.1161/CIRCRESAHA.117.305990 *
M. MIROTSOUT. M. JAYAWARDENAJ. SCHMECKPEPERM. GNECCHIV. J. DZAU: "Paracrine mechanisms of stem cell reparative and regenerative actions in the heart", JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, vol. 50, 2011, pages 280 - 289, XP028132035, DOI: 10.1016/j.yjmcc.2010.08.005
M. ROTA ET AL.: "Local activation or implantation of cardiac progenitor cells rescues scarred infarcted myocardium improving cardiac function", CIRCULATION RESEARCH, vol. 103, 2008, pages 107 - 116
M. S. PENNJ. PASTORET. MILLERR. ARAS: "SDF-1 in myocardial repair", GENE THERAPY, vol. 19, 2012, pages 583 - 587, XP055312601, DOI: 10.1038/gt.2012.32
M. ZHANGS. MURALIMANOHARANA. C. WORTMANC. R. MENDELSON: "Primate-specific miR-515 family members inhibit key genes in human trophoblast differentiation and are upregulated in preeclampsia", PROC NATL ACAD SCI USA, vol. 113, no. 45, 2016, pages E7069 - E7076
P. MATHIYALAGANS. SAHOO: "Exosomes-Based Gene Therapy for MicroRNA Delivery", METHODS IN MOLECULAR BIOLOGY (CLIFTON, N.J.), vol. 1521, 2017, pages 139 - 152
P. NOWAK-SLIWINSKA ET AL.: "Consensus guidelines for the use and interpretation of angiogenesis assays", ANGIOGENESIS, 2018
P. P. ZHANG ET AL.: "DNA methylation-mediated repression of miR-941 enhances lysine (K)-specific demethylase 6B expression in hepatoma cells", JBIOL CHEM, vol. 289, 2014, pages 24724 - 24735
P. P. ZWETSLOOT ET AL.: "Cardiac Stem Cell Treatment in Myocardial Infarction: A Systematic Review and Meta-Analysis of Preclinical Studies", CIRCULATION RESEARCH, vol. 118, 2016, pages 1223 - 1232
P. V. JOHNSTON ET AL.: "Engraftment, differentiation, and functional benefits of autologous cardiosphere-derived cells in porcine ischemic cardiomyopathy", CIRCULATION, vol. 120, 2009, pages 1075 - 1083
P. VALLABHAJ OSYULA: "Ex Vivo Lung Perfusion Model to Study Pulmonary Tissue Extracellular Microvesicle Profiles", THE ANNALS OF THORACIC SURGERY, vol. 103, 2017, pages 1758 - 1766
P. VALLABHAJOSYULA ET AL.: "Tissue-specific exosome biomarkers for noninvasively monitoring immunologic rejection of transplanted tissue", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 127, 2017, pages 1375 - 1391
PROGYAPARAMITA SAHA ET AL: "Circulating exosomes derived from transplanted progenitor cells aid the functional recovery of ischemic myocardium", SCIENCE TRANSLATIONAL MEDICINE, vol. 11, no. 493, 22 May 2019 (2019-05-22), US, pages eaau1168, XP055666383, ISSN: 1946-6234, DOI: 10.1126/scitranslmed.aau1168 *
R. A. BOONS. DIMMELER: "MicroRNAs in myocardial infarction", NATURE REVIEWS. CARDIOLOGY, vol. 12, 2015, pages 135 - 142
R. A. DRAGOVIC ET AL.: "Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis", NANOMEDICINE : NANOTECHNOLOGY, BIOLOGY, AND MEDICINE, vol. 7, 2011, pages 780 - 788, XP055040874, DOI: 10.1016/j.nano.2011.04.003
R. BOLLI ET AL.: "Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial", LANCET (LONDON, ENGLAND), vol. 378, 2011, pages 1847 - 1857
R. GALLET ET AL.: "Exosomes secreted by cardiosphere-derived cells reduce scarring, attenuate adverse remodelling, and improve function in acute and chronic porcine myocardial infarction", EUROPEAN HEART JOURNAL, vol. 38, 2017, pages 201 - 211
R. J. LOBB ET AL., OPTIMIZED EXOSOME ISOLATION PROTOCOL FOR CELL CULTURE SUPERNATANT AND HUMAN PLASMA, vol. 2015
R. MISHRA ET AL.: "Characterization and functionality of cardiac progenitor cells in congenital heart patients", CIRCULATION, vol. 123, 2011, pages 364 - 373
R. R. MAKKAR ET AL.: "Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial", THE LANCET, vol. 379, 2012, pages 895 - 904, XP055245447, DOI: 10.1016/S0140-6736(12)60195-0
R. WUX. HUJ. A. WANG: "Concise Review: Optimized Strategies for Stem Cell-Based Therapy in Myocardial Repair: Clinical Translatability and Potential Limitation", STEM CELLS (DAYTON, OHIO), vol. 36, 2018, pages 482 - 500
S. BIAN ET AL.: "Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model", JOURNAL OF MOLECULAR MEDICINE, vol. 92, 2014, pages 387 - 397, XP055487476, DOI: 10.1007/s00109-013-1110-5
S. COSTANTINOF. PANENI: "Stem cell therapy in heart failure: Is the best yet to come?", INTERNATIONAL JOURNAL OF CARDIOLOGY, vol. 260, 2018, pages 135 - 136, XP085369989, DOI: 10.1016/j.ijcard.2018.03.001
S. M. LIUJ. LUH. C. LEEF. H. CHUNGN. MA: "miR-524-5p suppresses the growth of oncogenic BRAF melanoma by targeting BRAF and ERK2", ONCOTARGET, vol. 5, 2014, pages 9444 - 9459
S. SHARMA ET AL.: "A Deep Proteome Analysis Identifies the Complete Secretome as the Functional Unit of Human Cardiac Progenitor Cells", CIRCULATION RESEARCH, vol. 120, 2017, pages 816 - 834
S. SHARMA ET AL.: "Cardiosphere-derived cells from pediatric end-stage heart failure patients have enhanced functional activity due to the heat shock response regulating the secretome", STEM CELLS, vol. 33, 2015, pages 1213 - 1229, XP055499959, DOI: 10.1002/stem.1937
S. WEI ET AL.: "Hsa-miR-623 suppresses tumor progression in human lung adenocarcinoma", CELL DEATH & DISEASE, vol. 7, 2016, pages e2388
U. AGARWAL ET AL.: "Age-Dependent Effect of Pediatric Cardiac Progenitor Cells After Juvenile Heart Failure", STEM CELLS TRANSLATIONAL MEDICINE, vol. 5, 2016, pages 883 - 892
U. AGARWAL ET AL.: "Experimental, Systems, and Computational Approaches to Understanding the MicroRNA-Mediated Reparative Potential of Cardiac Progenitor Cell-Derived Exosomes From Pediatric Patients", CIRCULATION RESEARCH, vol. 120, 2017, pages 701 - 712
V. N. S. GARIKIPATIF. SHOJA-TAHERIM. E. DAVISR. KISHORE: "Extracellular Vesicles and the Application of System Biology and Computational Modeling in Cardiac Repair", CIRCULATION RESEARCH, vol. 123, 2018, pages 188 - 204
W. D. GRAY ET AL.: "Identification of therapeutic covariant microRNA clusters in hypoxia-treated cardiac progenitor cell exosomes using systems biology", CIRC RES, vol. 116, 2015, pages 255 - 263
X. L. WANG ET AL.: "MiR-378b Promotes Differentiation of Keratinocytes through NKX3.1", PLOS ONE, vol. 10, 2015, pages e0136049
XU JKIMBALL TRLORENZ JNBROWN DABAUSKIN ARKLEVITSKY RHEWETT TEBREIT SNMOLKENTIN JD: "GDF15/MIC-1 functions as a protective and antihypertrophic factor released from the myocardium in association with SMAD protein activation", CIRCULATION RESEARCH, vol. 98, no. 3, 2006, pages 342 - 50, XP002565988, DOI: 10.1161/01.RES.0000202804.84885.d0
Y. LI ET AL.: "Epigenetic deregulation of miR-29a and miR-1256 by isoflavone contributes to the inhibition of prostate cancer cell growth and invasion", EPIGENETICS, vol. 7, 2012, pages 940 - 949, XP055463687, DOI: 10.4161/epi.21236
Y. ODUK ET AL.: "VEGF nanoparticles repair the heart after myocardial infarction", AMERICAN JOURNAL OF PHYSIOLOGY. HEART AND CIRCULATORY PHYSIOLOGY, vol. 314, 2018, pages H278 - h284
Y. X. WANG ET AL.: "MiR-384 inhibits human colorectal cancer metastasis by targeting KRAS and CDC42", ONCOTARGET, vol. 7, 2016, pages 84826 - 84838

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210009935A1 (en) * 2019-07-12 2021-01-14 Hitachi, Ltd. Cell culture monitoring device and cell culture system
WO2022204045A1 (fr) * 2021-03-22 2022-09-29 Spiritus Therapeutics, Inc. Utilisations diagnostiques et thérapeutiques d'exosomes puissants purifiés contenant un cargo de signature basé sur une maladie et basé sur une thérapie

Similar Documents

Publication Publication Date Title
Saha et al. Circulating exosomes derived from transplanted progenitor cells aid the functional recovery of ischemic myocardium
Park et al. Dual stem cell therapy synergistically improves cardiac function and vascular regeneration following myocardial infarction
Bronckaers et al. Angiogenic properties of human dental pulp stem cells
Kane et al. Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and in vivo
JP2022071069A (ja) 単離腎細胞を含有するオルガノイド及びその使用
US11045502B2 (en) Method of isolating cells for therapy and prophylaxis
US10526581B2 (en) Modulation of cardiac stem-progenitor cell differentiation, assays and uses thereof
Lee et al. A novel cell-free strategy for promoting mouse liver regeneration: utilization of a conditioned medium from adipose-derived stem cells
Li et al. Endothelial nitric oxide synthase promotes bone marrow stromal cell migration to the ischemic myocardium via upregulation of stromal cell-derived factor-1α
Suzuki et al. Therapeutic angiogenesis by transplantation of induced pluripotent stem cell-derived Flk-1 positive cells
JP2023026662A (ja) 血管形成の調節
US9987310B2 (en) Cardiac progenitor cells and methods of use therefor
Odent et al. Combinatorial approach for improving the outcome of angiogenic therapy in ischemic tissues
Ieronimakis et al. Direct isolation, culture and transplant of mouse skeletal muscle derived endothelial cells with angiogenic potential
Balistrieri Endothelial progenitor cells
WO2020097440A1 (fr) Procédés de prédiction de récupération fonctionnelle de tissu à l'aide d'exosomes circulants dérivés de cellules transplantées
Gao et al. Human-derived decellularized extracellular matrix scaffold incorporating autologous bone marrow stem cells from patients with congenital heart disease for cardiac tissue engineering
Hendawy et al. Heterogeneity of adipose stromal vascular fraction cells from the different harvesting sites in rats
Baio et al. A hyper-crosslinked carbohydrate polymer scaffold facilitates lineage commitment and maintains a reserve pool of proliferating cardiovascular progenitors
Hilage et al. Characterization and angiogenic potential of CD146+ endometrial stem cells
Iqbal Characterization of Potential Mechanisms that Render the Combination of First Trimester Umbilical Cord-Derived Perivascular Cells (FTM HUCPVC) and Endothelial Progenitor Cells (EPC) a Promising Cellular Therapeutic Strategy Following Ischemic Cardiac Injury
Hongbao et al. Stem Cell Markers Research Literatures
Meregalli STUDY OF MULTIPOTENT RENAL PKHHIGH STEM-LIKE CELLS, ISOLATED FROM HUMAN NEPHROSPHERES: REGENERATIVE ABILITIES AND TRANSCRIPTOMIC PROFILE
Mund et al. Defining endothelial progenitor cells
TW201741451A (zh) 一種用於生物工程化組織的組合物與方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19818303

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19818303

Country of ref document: EP

Kind code of ref document: A1