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2.
J Biopharm Stat ; 32(3): 496-510, 2022 05 04.
Article in English | MEDLINE | ID: mdl-35695137

ABSTRACT

The digital clinical trial is fast emerging as a pragmatic trial that can improve a trial's design including recruitment and retention, data collection and analytics. To that end, digital platforms such as electronic health records or wearable technologies that enable passive data collection can be leveraged, alleviating burden from the participant and study coordinator. However, there are challenges. For example, many of these data sources not originally intended for research may be noisier than traditionally obtained measures. Further, the secure flow of passively collected data and their integration for analysis is non-trivial. The Apple Heart Study was a prospective, single-arm, site-less digital trial designed to evaluate the ability of an app to detect atrial fibrillation. The study was designed with pragmatic features, such as an app for enrollment, a wearable device (the Apple Watch) for data collection, and electronic surveys for participant-reported outcomes that enabled a high volume of patient enrollment and accompanying data. These elements led to challenges including identifying the number of unique participants, maintaining participant-level linkage of multiple complex data streams, and participant adherence and engagement. Novel solutions were derived that inform future designs with an emphasis on data management. We build upon the excellent framework of the Clinical Trials Transformation Initiative to provide a comprehensive set of guidelines for data management of the digital clinical trial that include an increased role of collaborative data scientists in the design and conduct of the modern digital trial.


Subject(s)
Data Management , Wearable Electronic Devices , Humans , Prospective Studies , Surveys and Questionnaires
3.
Stat (Int Stat Inst) ; 11(1): e470, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36589778

ABSTRACT

An app-based clinical trial enrolment process can contribute to duplicated records, carrying data management implications. Our objective was to identify duplicated records in real time in the Apple Heart Study (AHS). We leveraged personal identifiable information (PII) to develop a dissimilarity score (DS) using the Damerau-Levenshtein distance. For computational efficiency, we focused on four types of records at the highest risk of duplication. We used the receiver operating curve (ROC) and resampling methods to derive and validate a decision rule to classify duplicated records. We identified 16,398 (4%) duplicated participants, resulting in 419,297 unique participants out of a total of 438,435 possible. Our decision rule yielded a high positive predictive value (96%) with negligible impact on the trial's original findings. Our findings provide principled solutions for future digital trials. When establishing deduplication procedures for digital trials, we recommend collecting device identifiers in addition to participant identifiers; collecting and ensuring secure access to PII; conducting a pilot study to identify reasons for duplicated records; establishing an initial deduplication algorithm that can be refined; creating a data quality plan that informs refinement; and embedding the initial deduplication algorithm in the enrolment platform to ensure unique enrolment and linkage to previous records.

5.
N Engl J Med ; 381(20): 1909-1917, 2019 11 14.
Article in English | MEDLINE | ID: mdl-31722151

ABSTRACT

BACKGROUND: Optical sensors on wearable devices can detect irregular pulses. The ability of a smartwatch application (app) to identify atrial fibrillation during typical use is unknown. METHODS: Participants without atrial fibrillation (as reported by the participants themselves) used a smartphone (Apple iPhone) app to consent to monitoring. If a smartwatch-based irregular pulse notification algorithm identified possible atrial fibrillation, a telemedicine visit was initiated and an electrocardiography (ECG) patch was mailed to the participant, to be worn for up to 7 days. Surveys were administered 90 days after notification of the irregular pulse and at the end of the study. The main objectives were to estimate the proportion of notified participants with atrial fibrillation shown on an ECG patch and the positive predictive value of irregular pulse intervals with a targeted confidence interval width of 0.10. RESULTS: We recruited 419,297 participants over 8 months. Over a median of 117 days of monitoring, 2161 participants (0.52%) received notifications of irregular pulse. Among the 450 participants who returned ECG patches containing data that could be analyzed - which had been applied, on average, 13 days after notification - atrial fibrillation was present in 34% (97.5% confidence interval [CI], 29 to 39) overall and in 35% (97.5% CI, 27 to 43) of participants 65 years of age or older. Among participants who were notified of an irregular pulse, the positive predictive value was 0.84 (95% CI, 0.76 to 0.92) for observing atrial fibrillation on the ECG simultaneously with a subsequent irregular pulse notification and 0.71 (97.5% CI, 0.69 to 0.74) for observing atrial fibrillation on the ECG simultaneously with a subsequent irregular tachogram. Of 1376 notified participants who returned a 90-day survey, 57% contacted health care providers outside the study. There were no reports of serious app-related adverse events. CONCLUSIONS: The probability of receiving an irregular pulse notification was low. Among participants who received notification of an irregular pulse, 34% had atrial fibrillation on subsequent ECG patch readings and 84% of notifications were concordant with atrial fibrillation. This siteless (no on-site visits were required for the participants), pragmatic study design provides a foundation for large-scale pragmatic studies in which outcomes or adherence can be reliably assessed with user-owned devices. (Funded by Apple; Apple Heart Study ClinicalTrials.gov number, NCT03335800.).


Subject(s)
Atrial Fibrillation/diagnosis , Electrocardiography/instrumentation , Mobile Applications , Telemedicine/instrumentation , Wearable Electronic Devices , Adult , Aged , Algorithms , Confidentiality , Female , Humans , Male , Middle Aged , Predictive Value of Tests , Prospective Studies
6.
Am Heart J ; 207: 66-75, 2019 01.
Article in English | MEDLINE | ID: mdl-30392584

ABSTRACT

BACKGROUND: Smartwatch and fitness band wearable consumer electronics can passively measure pulse rate from the wrist using photoplethysmography (PPG). Identification of pulse irregularity or variability from these data has the potential to identify atrial fibrillation or atrial flutter (AF, collectively). The rapidly expanding consumer base of these devices allows for detection of undiagnosed AF at scale. METHODS: The Apple Heart Study is a prospective, single arm pragmatic study that has enrolled 419,093 participants (NCT03335800). The primary objective is to measure the proportion of participants with an irregular pulse detected by the Apple Watch (Apple Inc, Cupertino, CA) with AF on subsequent ambulatory ECG patch monitoring. The secondary objectives are to: 1) characterize the concordance of pulse irregularity notification episodes from the Apple Watch with simultaneously recorded ambulatory ECGs; 2) estimate the rate of initial contact with a health care provider within 3 months after notification of pulse irregularity. The study is conducted virtually, with screening, consent and data collection performed electronically from within an accompanying smartphone app. Study visits are performed by telehealth study physicians via video chat through the app, and ambulatory ECG patches are mailed to the participants. CONCLUSIONS: The results of this trial will provide initial evidence for the ability of a smartwatch algorithm to identify pulse irregularity and variability which may reflect previously unknown AF. The Apple Heart Study will help provide a foundation for how wearable technology can inform the clinical approach to AF identification and screening.


Subject(s)
Algorithms , Atrial Fibrillation/diagnosis , Atrial Flutter/diagnosis , Electrocardiography, Ambulatory/instrumentation , Mobile Applications , Smartphone , Wearable Electronic Devices , Atrial Fibrillation/epidemiology , Atrial Flutter/epidemiology , Humans , Patient Acceptance of Health Care/statistics & numerical data , Patient Reported Outcome Measures , Prospective Studies , Telemedicine , Time Factors
7.
Proc Natl Acad Sci U S A ; 110(31): 12643-8, 2013 Jul 30.
Article in English | MEDLINE | ID: mdl-23858471

ABSTRACT

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.


Subject(s)
Bone and Bones/metabolism , Cartilage/metabolism , Hematopoiesis/physiology , Hematopoietic Stem Cells/metabolism , Signal Transduction/physiology , Stem Cell Niche/physiology , Animals , Antigens, Differentiation/biosynthesis , Antigens, Differentiation/genetics , Bone and Bones/cytology , Cartilage/cytology , Cytokines/genetics , Cytokines/metabolism , Gene Expression Regulation/physiology , Hematopoietic Stem Cells/cytology , Mice , Mice, Transgenic , Stromal Cells/cytology , Stromal Cells/metabolism
8.
Circulation ; 126(11 Suppl 1): S20-8, 2012 Sep 11.
Article in English | MEDLINE | ID: mdl-22965984

ABSTRACT

BACKGROUND: Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. METHODS AND RESULTS: We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. CONCLUSIONS: The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.


Subject(s)
Fetal Stem Cells/transplantation , Genetic Therapy/methods , Hindlimb/blood supply , Ischemia/surgery , Mutagenesis, Insertional/methods , Myocardial Infarction/surgery , Animals , Cell Differentiation , Cell Division , Chromosomes, Human, Pair 19/genetics , Endothelial Cells/cytology , Female , Fetal Heart/cytology , Fetal Stem Cells/cytology , Fetal Stem Cells/metabolism , Gene Expression Regulation , Genes, Reporter , Humans , Integrases , Intracellular Signaling Peptides and Proteins , Ischemia/physiopathology , Luciferases, Firefly/genetics , Luminescent Proteins/genetics , Magnetic Resonance Imaging , Mice , Mice, SCID , Proteins/genetics , Random Allocation , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Thymidine Kinase/genetics , Transgenes , Vesicular Transport Proteins , Viral Proteins/genetics , Virus Integration , Red Fluorescent Protein
9.
J Am Coll Cardiol ; 60(14): 1278-87, 2012 Oct 02.
Article in English | MEDLINE | ID: mdl-22841153

ABSTRACT

OBJECTIVES: The goal of this study was to demonstrate the enhancement of human cardiac progenitor cell (hCPC) reparative and regenerative potential by genetic modification for the treatment of myocardial infarction. BACKGROUND: Regenerative potential of stem cells to repair acute infarction is limited. Improved hCPC survival, proliferation, and differentiation into functional myocardium will increase efficacy and advance translational implementation of cardiac regeneration. METHODS: hCPCs isolated from the myocardium of heart failure patients undergoing left ventricular assist device implantation were engineered to express green fluorescent protein (hCPCe) or Pim-1-GFP (hCPCeP). Functional tests of hCPC regenerative potential were performed with immunocompromised mice by using intramyocardial adoptive transfer injection after infarction. Myocardial structure and function were monitored by echocardiographic and hemodynamic assessment for 20 weeks after delivery. hCPCe and hCPCeP expressing luciferase were observed by using bioluminescence imaging to noninvasively track persistence. RESULTS: hCPCeP exhibited augmentation of reparative potential relative to hCPCe control cells, as shown by significantly increased proliferation coupled with amelioration of infarction injury and increased hemodynamic performance at 20 weeks post-transplantation. Concurrent with enhanced cardiac structure and function, hCPCeP demonstrated increased cellular engraftment and differentiation with improved vasculature and reduced infarct size. Enhanced persistence of hCPCeP versus hCPCe was revealed by bioluminescence imaging at up to 8 weeks post-delivery. CONCLUSIONS: Genetic engineering of hCPCs with Pim-1 enhanced repair of damaged myocardium. Ex vivo gene delivery to modify stem cells has emerged as a viable option addressing current limitations in the field. This study demonstrates that efficacy of hCPCs from the failing myocardium can be safely and significantly enhanced through expression of Pim-1 kinase, setting the stage for use of engineered cells in pre-clinical settings.


Subject(s)
Genetic Engineering , Myocardial Infarction/therapy , Myocytes, Cardiac/transplantation , Proto-Oncogene Proteins c-pim-1/metabolism , Animals , Cell Proliferation , Echocardiography , Gene Expression , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hemodynamics , Humans , Luminescent Measurements , Mice , Myocytes, Cardiac/enzymology , Neovascularization, Pathologic , Proto-Oncogene Proteins c-pim-1/genetics , Stem Cell Transplantation , Stem Cells/enzymology
10.
Circ Cardiovasc Imaging ; 5(4): 481-90, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22565608

ABSTRACT

BACKGROUND: Human cardiac progenitor cells have demonstrated great potential for myocardial repair in small and large animals, but robust methods for longitudinal assessment of their engraftment in humans is not yet readily available. In this study, we sought to optimize and evaluate the use of positron emission tomography (PET) reporter gene imaging for monitoring human cardiac progenitor cell (hCPC) transplantation in a mouse model of myocardial infarction. METHODS AND RESULTS: hCPCs were isolated and expanded from human myocardial samples and stably transduced with herpes simplex virus thymidine kinase (TK) PET reporter gene. Thymidine kinase-expressing hCPCs were characterized in vitro and transplanted into murine myocardial infarction models (n=57). Cardiac echocardiographic, magnetic resonance imaging and pressure-volume loop analyses revealed improvement in left ventricular contractile function 2 weeks after transplant (hCPC versus phosphate-buffered saline, P<0.03). Noninvasive PET imaging was used to track hCPC fate over a 4-week time period, demonstrating a substantial decline in surviving cells. Importantly, early cell engraftment as assessed by PET was found to predict subsequent functional improvement, implying a "dose-effect" relationship. We isolated the transplanted cells from recipient myocardium by laser capture microdissection for in vivo transcriptome analysis. Our results provide direct evidence that hCPCs augment cardiac function after their transplantation into ischemic myocardium through paracrine secretion of growth factors. CONCLUSIONS: PET reporter gene imaging can provide important diagnostic and prognostic information regarding the ultimate success of hCPC treatment for myocardial infarction.


Subject(s)
Myocardial Infarction/diagnostic imaging , Myocardial Infarction/therapy , Myocytes, Cardiac/transplantation , Positron-Emission Tomography/methods , Stem Cell Transplantation , Analysis of Variance , Animals , Cell Survival , Disease Models, Animal , Echocardiography , Gene Transfer Techniques , Genes, Reporter/genetics , Genetic Therapy , Guanine/analogs & derivatives , Humans , Immunohistochemistry , Laser Capture Microdissection , Linear Models , Magnetic Resonance Imaging , Mice , Mice, SCID , Myocardial Contraction/physiology , Myocardial Infarction/physiopathology , Paracrine Communication/physiology , Phenotype , Recovery of Function , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , Viral Proteins/metabolism
11.
Circulation ; 124(11 Suppl): S27-34, 2011 Sep 13.
Article in English | MEDLINE | ID: mdl-21911815

ABSTRACT

BACKGROUND: Although stem cell therapy has provided a promising treatment for myocardial infarction, the low survival of the transplanted cells in the infarcted myocardium is possibly a primary reason for failure of long-term improvement. Therefore, the development of novel prosurvival strategies to boost stem cell survival will be of significant benefit to this field. METHODS AND RESULTS: Cardiac progenitor cells (CPCs) were isolated from transgenic mice, which constitutively express firefly luciferase and green fluorescent protein. The CPCs were transduced with individual lentivirus carrying the precursor of miR-21, miR-24, and miR-221, a cocktail of these 3 microRNA precursors, or green fluorescent protein as a control. After challenge in serum free medium, CPCs treated with the 3 microRNA cocktail showed significantly higher viability compared with untreated CPCs. After intramuscular and intramyocardial injections, in vivo bioluminescence imaging showed that microRNA cocktail-treated CPCs survived significantly longer after transplantation. After left anterior descending artery ligation, microRNA cocktail-treated CPCs boost the therapeutic efficacy in terms of functional recovery. Histological analysis confirmed increased myocardial wall thickness and CPC engraftment in the myocardium with the microRNA cocktail. Finally, we used bioinformatics analysis and experimental validation assays to show that Bim, a critical apoptotic activator, is an important target gene of the microRNA cocktail, which collectively can bind to the 3'UTR region of Bim and suppress its expression. CONCLUSIONS: We have demonstrated that a microRNA prosurvival cocktail (miR-21, miR-24, and miR-221) can improve the engraftment of transplanted cardiac progenitor cells and therapeutic efficacy for treatment of ischemic heart disease.


Subject(s)
Cell- and Tissue-Based Therapy/methods , Graft Survival/physiology , MicroRNAs/genetics , MicroRNAs/therapeutic use , Myocardial Infarction/therapy , Stem Cell Transplantation/methods , Stem Cells/cytology , Animals , Cell Differentiation/physiology , Cells, Cultured , Female , Injections, Intramuscular , Lentivirus , Mice , Mice, SCID , Mice, Transgenic , Models, Animal , Myocytes, Cardiac/cytology , Myocytes, Cardiac/physiology , Stem Cells/physiology , Transduction, Genetic , Treatment Outcome
12.
Circulation ; 124(11 Suppl): S46-54, 2011 Sep 13.
Article in English | MEDLINE | ID: mdl-21911818

ABSTRACT

BACKGROUND: Under normoxic conditions, hypoxia-inducible factor (HIF)-1α is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure. METHODS AND RESULTS: PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1α protein and angiogenesis genes, respectively. CONCLUSIONS: We demonstrated that HIF-1α upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.


Subject(s)
Embryonic Stem Cells/transplantation , Gene Knockdown Techniques , Genetic Therapy/methods , Mixed Function Oxygenases/genetics , Myocardial Infarction/therapy , Neovascularization, Physiologic/physiology , Procollagen-Proline Dioxygenase/genetics , Animals , Apoptosis/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Female , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Mice, Inbred Strains , Mixed Function Oxygenases/metabolism , Models, Animal , Myoblasts, Cardiac/metabolism , Myoblasts, Cardiac/pathology , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardium/metabolism , Myocardium/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Procollagen-Proline Dioxygenase/metabolism , Stem Cell Transplantation , Treatment Outcome
13.
Nat Biotechnol ; 29(9): 829-34, 2011 Aug 14.
Article in English | MEDLINE | ID: mdl-21841799

ABSTRACT

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.


Subject(s)
Antibodies, Monoclonal/metabolism , Pluripotent Stem Cells/cytology , Polysaccharides/metabolism , Stage-Specific Embryonic Antigens/metabolism , Teratoma/pathology , Animals , Biomarkers , Cell Differentiation , Cells, Cultured , Computational Biology , Flow Cytometry , Gene Expression Regulation , Humans , Immunohistochemistry , Mice , Mice, Inbred BALB C , Microarray Analysis , Pluripotent Stem Cells/chemistry , Real-Time Polymerase Chain Reaction , Teratoma/metabolism
14.
J Biol Chem ; 286(37): 32697-704, 2011 Sep 16.
Article in English | MEDLINE | ID: mdl-21719696

ABSTRACT

Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.


Subject(s)
Adipose Tissue/metabolism , Gene Expression Regulation , Induced Pluripotent Stem Cells/metabolism , Stem Cell Transplantation , Adipose Tissue/cytology , Animals , Disease Models, Animal , Dogs , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Humans , Induced Pluripotent Stem Cells/cytology , Male , Mice , Mice, SCID , Myocardial Ischemia/therapy , Oligonucleotide Array Sequence Analysis , Stromal Cells/cytology , Stromal Cells/metabolism , Transplantation, Autologous , Transplantation, Heterologous
15.
Biomaterials ; 32(20): 4647-58, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21477858

ABSTRACT

Polyethylenimine (PEI) based polymers are efficient agents for cell transfection. However, their use has been hampered due to high cell death associated with transfection thereby resulting in low efficiency of gene delivery within the cells. To circumvent the problem of cellular toxicity, metal binding peptides were linked to PEI. Eight peptide-PEI derivatives were synthesized to improve cell survival and transfection efficiency. TAT linked PEI was used as a control polymer. Peptides linked with PEI amines formed nanogels as shown by electron microscopy and atomic force microscopic measurements. Polymers were characterized by spectroscopic methods and their ability to form complexes with plasmids was tested using electrophoretic studies. These modifications improved polymer biocompatibility as well as cell survival markedly, when compared to PEI alone. A subset of the modified peptide-polymers also showed significantly higher transfection efficiency in primary human cells with respect to the widely used transfection agent, lipofectamine. Study of the underlying mechanism of the observed phenomena revealed lower levels of 'reactive oxygen species' (ROS) in the presence of the peptide-polymers when compared to PEI alone. This was further corroborated with global gene expression analysis which showed upregulation of multiple genes and pathways involved in regulating intracellular oxidative stress.


Subject(s)
Gene Transfer Techniques , Peptides/chemistry , Peptides/metabolism , Polyethyleneimine , Polymers , Amino Acid Sequence , Animals , Genetic Therapy/methods , HEK293 Cells , Humans , Materials Testing , Molecular Sequence Data , Molecular Structure , Oxidative Stress , Peptides/genetics , Polyethyleneimine/chemistry , Polyethyleneimine/metabolism , Polymers/chemistry , Polymers/metabolism , Stromal Cells/cytology , Stromal Cells/physiology
17.
Adv Drug Deliv Rev ; 62(12): 1175-86, 2010 Sep 30.
Article in English | MEDLINE | ID: mdl-20816906

ABSTRACT

Stem cell therapy has the potential to regenerate injured tissue. For stem cells to achieve their full therapeutic potential, stem cells must differentiate into the target cell, reach the site of injury, survive, and engraft. To fully characterize these cells, evaluation of cell morphology, lineage specific markers, cell specific function, and gene expression must be performed. To monitor survival and engraftment, cell fate imaging is vital. Only then can organ specific function be evaluated to determine the effectiveness of therapy. In this review, we will discuss methods for evaluating the function of transplanted cells for restoring the heart, nervous system, and pancreas. We will also highlight the specific challenges facing these potential therapeutic areas.


Subject(s)
Cell Lineage , Hematopoietic Stem Cells/physiology , Stem Cell Transplantation , Stem Cells/physiology , Animals , Cell Differentiation , Heart Diseases/metabolism , Heart Diseases/therapy , Humans , Nervous System Diseases/metabolism , Nervous System Diseases/therapy , Pancreatic Diseases/metabolism , Pancreatic Diseases/therapy , Regeneration
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