Publications by authors named "Evgenios Neofytou"

28 Publications

  • Page 1 of 1

Method for selective ablation of undifferentiated human pluripotent stem cell populations for cell-based therapies.

JCI Insight 2021 Apr 8;6(7). Epub 2021 Apr 8.

Stanford Cardiovascular Institute.

Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy-based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.142000DOI Listing
April 2021

Endogenous Retrovirus-Derived lncRNA BANCR Promotes Cardiomyocyte Migration in Humans and Non-human Primates.

Dev Cell 2020 09 6;54(6):694-709.e9. Epub 2020 Aug 6.

Cardiovascular Institute, Stanford University, Stanford, CA 94305, USA; Departments of Medicine and Radiology, Stanford University, Stanford, CA 94305, USA. Electronic address:

Transposable elements (TEs) comprise nearly half of the human genome and are often transcribed or exhibit cis-regulatory properties with unknown function in specific processes such as heart development. In the case of endogenous retroviruses (ERVs), a TE subclass, experimental interrogation is constrained as many are primate-specific or human-specific. Here, we use primate pluripotent stem-cell-derived cardiomyocytes that mimic fetal cardiomyocytes in vitro to discover hundreds of ERV transcripts from the primate-specific MER41 family, some of which are regulated by the cardiogenic transcription factor TBX5. The most significant of these are located within BANCR, a long non-coding RNA (lncRNA) exclusively expressed in primate fetal cardiomyocytes. Functional studies reveal that BANCR promotes cardiomyocyte migration in vitro and ventricular enlargement in vivo. We conclude that recently evolved TE loci such as BANCR may represent potent de novo developmental regulatory elements that can be interrogated with species-matching pluripotent stem cell models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2020.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529962PMC
September 2020

Molecular Imaging of Infective Endocarditis With 6''-[F]Fluoromaltotriose Positron Emission Tomography-Computed Tomography.

Circulation 2020 May 26;141(21):1729-1731. Epub 2020 May 26.

Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Bio-X Program (M.W., G.G., E.C., M.N., T.H., M.T.G., J.C.W., S.S.G.), Stanford University School of Medicine, CA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.119.043924DOI Listing
May 2020

Pharmacological Silencing of MicroRNA-152 Prevents Pressure Overload-Induced Heart Failure.

Circ Heart Fail 2020 03 12;13(3):e006298. Epub 2020 Mar 12.

Department of Cardiothoracic Surgery (M.P., I.P.-G., I.K.), Stanford University School of Medicine, CA.

Background: MicroRNAs are small, noncoding RNAs that play a key role in gene expression. Accumulating evidence suggests that aberrant microRNA expression contributes to the heart failure (HF) phenotype; however, the underlying molecular mechanisms are not well understood. A better understanding of the mechanisms of action of microRNAs could potentially lead to targeted therapies that could halt the progression or even reverse HF.

Methods And Results: We found that microRNA-152 (miR-152) expression was upregulated in the failing human heart and experimental animal models of HF. Transgenic mice with cardiomyocyte-specific miR-152 overexpression developed systolic dysfunction (mean difference, -38.74% [95% CI, -45.73% to -31.74%]; <0.001) and dilated cardiomyopathy. At the cellular level, miR-152 overexpression perturbed mitochondrial ultrastructure and dysregulated key genes involved in cardiomyocyte metabolism and inflammation. Mechanistically, we identified Glrx5 (glutaredoxin 5), a critical regulator of mitochondrial iron homeostasis and iron-sulfur cluster synthesis, as a direct miR-152 target. Finally, a proof-of-concept of the therapeutic efficacy of targeting miR-152 in vivo was obtained by utilizing a locked nucleic acid-based inhibitor of miR-152 (LNA 152) in a murine model of HF subjected to transverse aortic constriction. We demonstrated that animals treated with LNA-152 (n=10) showed preservation of systolic function when compared with locked nucleic acid-control treated animals (n=9; mean difference, 18.25% [95% CI, 25.10% to 11.39%]; <0.001).

Conclusions: The upregulation of miR-152 expression in the failing myocardium contributes to HF pathophysiology. Preclinical evidence suggests that miR-152 inhibition preserves cardiac function in a model of pressure overload-induced HF. These findings offer new insights into the pathophysiology of HF and point to miR-152-Glrx5 axis as a potential novel therapeutic target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCHEARTFAILURE.119.006298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439562PMC
March 2020

Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as a Model to Study Trypanosoma cruzi Infection.

Stem Cell Reports 2019 06 16;12(6):1232-1241. Epub 2019 May 16.

Stanford Cardiovascular Institute, 265 Campus Drive, Rm G1120B, Stanford, CA 94305, USA; Department of Medicine, Division of Cardiovascular Medicine, Stanford University, School of Medicine, Stanford, CA 94305, USA; Department of Radiology, Stanford University, School of Medicine, Stanford, CA 94305, USA. Electronic address:

Chagas disease (ChD) is one of the most neglected tropical diseases, with cardiomyopathy being the main cause of death in Trypanosoma cruzi-infected patients. As the parasite actively replicates in cardiomyocytes (CMs), the heart remains a key target organ in the pathogenesis of ChD. Here we modeled ChD using human induced pluripotent stem cell-derived CMs (iPSC-CMs) to understand the complex interplay between the parasite and host cells. We showed that iPSC-CMs can get infected with the T. cruzi Y strain and that all parasite cycle stages can be identified in our model system. Importantly, characterization of T. cruzi-infected iPSC-CMs showed significant changes in their gene expression profile, cell contractility, and distribution of key cardiac markers. Moreover, these infected iPSC-CMs exhibited a pro-inflammatory profile as indicated by significantly elevated cytokine levels and cell-trafficking regulators. We believe our iPSC-CM model is a valuable platform to explore new treatment strategies for ChD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2019.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565757PMC
June 2019

Myocardial viability of the peri-infarct region measured by T1 mapping post manganese-enhanced MRI correlates with LV dysfunction.

Int J Cardiol 2019 Apr 31;281:8-14. Epub 2019 Jan 31.

Department of Medicine (Cardiovascular Medicine) and Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, United States of America. Electronic address:

Background: Manganese-enhanced MRI (MEMRI) detects viable cardiomyocytes based on the intracellular manganese uptake via L-type calcium-channels. This study aimed to quantify myocardial viability based on manganese uptake by viable myocardium in the infarct core (IC), peri-infarct region (PIR) and remote myocardium (RM) using T1 mapping before and after MEMRI and assess their association with cardiac function and arrhythmogenesis.

Methods: Fifteen female swine had a 60-minute balloon ischemia-reperfusion injury in the LAD. MRI (Signa 3T, GE Healthcare) and electrophysiological study (EPS) were performed 4 weeks later. MEMRI and delayed gadolinium-enhanced MRI (DEMRI) were acquired on LV short axis. The DEMRI positive total infarct area was subdivided into the regions of MEMRI-negative non-viable IC and MEMRI-positive viable PIR. T1 mapping was performed to evaluate native T1, post-MEMRI T1, and delta R1 (R1-R1, where R1 equals 1/T1) of each territory. Their correlation with LV function and EPS data was assessed.

Results: PIR was characterized by intermediate native T1 (1530.5 ± 75.2 ms) compared to IC (1634.7 ± 88.4 ms, p = 0.001) and RM (1406.4 ± 37.9 ms, p < 0.0001). Lower post-MEMRI T1 of PIR (1136.3 ± 99.6 ms) than IC (1262.6 ± 126.8 ms, p = 0.005) and higher delta R1 (0.23 ± 0.08 s) of PIR than IC (0.18 ± 0.09 s, p = 0.04) indicated higher myocardial manganese uptake of PIR compared to IC. Post-MEMRI T1 (r = -0.57, p = 0.02) and delta R1 (r = 0.51, p = 0.04) of PIR correlated significantly with LVEF.

Conclusions: PIR is characterized by higher manganese uptake compared to the infarct core. In the subacute phase post-IR, PIR viability measured by post-MEMRI T1 correlates with cardiac function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijcard.2019.01.101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434939PMC
April 2019

Photoacoustic Imaging of Embryonic Stem Cell-Derived Cardiomyocytes in Living Hearts with Ultrasensitive Semiconducting Polymer Nanoparticles.

Adv Funct Mater 2018 Jan 8;28(1). Epub 2017 Nov 8.

Stanford Cardiovascular Institute, Stanford, CA, 94305, United States.

The last decade has seen impressive progress in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) that makes them ideal tools to repair injured hearts. To achieve an optimal outcome, advanced molecular imaging methods are essential to accurately track these transplanted cells in the heart. Herein, we demonstrate for the first time that a class of photoacoustic nanoparticles (PANPs) incorporating semiconducting polymers (SPs) as contrast agents can be used in the photoacoustic imaging (PAI) of transplanted hESC-CMs in living mouse hearts. This is achieved by virtue of two benefits of PANPs. First, strong PA signals and specific spectral features of SPs allow PAI to sensitively detect and distinguish a small number of PANP-labeled cells (2,000) from background tissues . Second, the PANPs show a high efficiency for hESC-CM labeling without adverse effects on cell structure, function, and gene expression. Assisted by ultrasound imaging, the delivery and engraftment of hESC-CMs in living mouse hearts can be assessed by PANP-based PAI with high spatial resolution (~100 μm). In summary, this study explores and validates a novel application of SPs as a PA contrast agent to track labeled cells with high sensitivity and accuracy , highlighting the advantages of integrating PAI and PANPs to advance cardiac regenerative therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adfm.201704939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247950PMC
January 2018

Radiolabeled Duramycin: Promising Translational Imaging of Myocardial Apoptosis.

JACC Cardiovasc Imaging 2018 12 14;11(12):1834-1836. Epub 2018 Feb 14.

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California; Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcmg.2017.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383561PMC
December 2018

Comparison of Non-human Primate versus Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Treatment of Myocardial Infarction.

Stem Cell Reports 2018 02 1;10(2):422-435. Epub 2018 Feb 1.

Stanford Cardiovascular Institute, Stanford, CA 94305-5454, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA 94305, USA; Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Non-human primates (NHPs) can serve as a human-like model to study cell therapy using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, whether the efficacy of NHP and human iPSC-CMs is mechanistically similar remains unknown. To examine this, RNU rats received intramyocardial injection of 1 × 10 NHP or human iPSC-CMs or the same number of respective fibroblasts or PBS control (n = 9-14/group) at 4 days after 60-min coronary artery occlusion-reperfusion. Cardiac function and left ventricular remodeling were similarly improved in both iPSC-CM-treated groups. To mimic the ischemic environment in the infarcted heart, both cultured NHP and human iPSC-CMs underwent 24-hr hypoxia in vitro. Both cells and media were collected, and similarities in transcriptomic as well as metabolomic profiles were noted between both groups. In conclusion, both NHP and human iPSC-CMs confer similar cardioprotection in a rodent myocardial infarction model through relatively similar mechanisms via promotion of cell survival, angiogenesis, and inhibition of hypertrophy and fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2018.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830958PMC
February 2018

Thalidomide treatment prevents chronic graft rejection after aortic transplantation in rats - an experimental study.

Transpl Int 2017 Nov 14;30(11):1181-1189. Epub 2017 Aug 14.

Department of Pediatrics (Cardiology) and the Cardiovascular Institute, Stanford University, Stanford, CA, USA.

Cardiac allograft vasculopathy (CAV) affects approximately 30% of cardiac transplant patients at 5 years post-transplantation. To date, there are few CAV treatment or prevention options, none of which are highly effective. The aim of the study was to investigate the effect of thalidomide on the development of CAV. The effect of thalidomide treatment on chronic rejection was assessed in rat orthotopic aortic transplants in allogeneic F344 or syngeneic Lew rats (n = 6 per group). Animals were left untreated or received thalidomide for 30 days post-transplant, and evidence of graft CAV was determined by histology (trichrome and immunohistochemistry) and intragraft cytokine measurements. Animals that received thalidomide treatment post-transplant showed markedly reduced luminal obliteration, with concomitant rescue of smooth muscle cells (SMCs) in the aortic media of grafts. Thalidomide counteracted neointimal hyperplasia by preventing dedifferentiation of vascular SMCs. Measurement of intragraft cytokine levels after thalidomide treatment revealed downregulation of matrix metalloproteinase 8 and monocyte chemotactic protein 1, cytokines involved in tissue remodelling and inflammation, respectively. Importantly, no negative side effects of thalidomide were observed. Thalidomide treatment prevents CAV development in a rodent model and is therefore potentially useful in clinical applications to prevent post-transplant heart rejection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tri.13004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643223PMC
November 2017

Brief Report: External Beam Radiation Therapy for the Treatment of Human Pluripotent Stem Cell-Derived Teratomas.

Stem Cells 2017 08 10;35(8):1994-2000. Epub 2017 Jul 10.

Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 10 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. Stem Cells 2017;35:1994-2000.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.2653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568072PMC
August 2017

Specific Imaging of Bacterial Infection Using 6″-F-Fluoromaltotriose: A Second-Generation PET Tracer Targeting the Maltodextrin Transporter in Bacteria.

J Nucl Med 2017 10 10;58(10):1679-1684. Epub 2017 May 10.

Department of Radiology, Stanford University School of Medicine, Stanford, California

6″-F-fluoromaltotriose is a PET tracer that can potentially be used to image and localize most bacterial infections, much like F-FDG has been used to image and localize most cancers. However, unlike F-FDG, 6″-F-fluoromaltotriose is not taken up by inflammatory lesions and appears to be specific to bacterial infections by targeting the maltodextrin transporter that is expressed in gram-positive and gram-negative strains of bacteria. 6″-F-fluoromaltotriose was synthesized with high radiochemical purity and evaluated in several clinically relevant bacterial strains in cultures and in living mice. 6″-F-fluoromaltotriose was taken up in both gram-positive and gram-negative bacterial strains. 6″-F-fluoromaltotriose was also able to detect in a clinically relevant mouse model of wound infection. The utility of 6″-F-fluoromaltotriose to help monitor antibiotic therapies was also evaluated in rats. 6″-F-fluoromaltotriose is a promising new tracer that has significant diagnostic utility, with the potential to change the clinical management of patients with infectious diseases of bacterial origin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2967/jnumed.117.191452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5632736PMC
October 2017

Potential Strategies to Address the Major Clinical Barriers Facing Stem Cell Regenerative Therapy for Cardiovascular Disease: A Review.

JAMA Cardiol 2016 11;1(8):953-962

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California2Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California4Department of Radiology, Stanford University School of Medicine, Stanford, California.

Importance: Although progress continues to be made in the field of stem cell regenerative medicine for the treatment of cardiovascular disease, significant barriers to clinical implementation still exist.

Objectives: To summarize the current barriers to the clinical implementation of stem cell therapy in patients with cardiovascular disease and to discuss potential strategies to overcome them.

Evidence Review: Information for this review was obtained through a search of PubMed and the Cochrane database for English-language studies published between January 1, 2000, and July 25, 2016. Ten randomized clinical trials and 8 systematic reviews were included.

Findings: One of the major clinical barriers facing the routine implementation of stem cell therapy in patients with cardiovascular disease is the limited and inconsistent benefit observed thus far. Reasons for this finding are unclear but may be owing to poor cell retention and survival, as suggested by numerous preclinical studies and a small number of human studies incorporating imaging to determine cell fate. Additional studies in humans using imaging to determine cell fate are needed to understand how these factors contribute to the limited efficacy of stem cell therapy. Treatment strategies to address poor cell retention and survival are under investigation and include the following: coadministration of immunosuppressive and prosurvival agents, delivery of cardioprotective factors packaged in exosomes rather than the cells themselves, and use of tissue-engineering strategies to provide structural support for cells. If larger grafts are achieved using these strategies, it will be imperative to carefully monitor for the potential risks of tumorigenicity, immunogenicity, and arrhythmogenicity.

Conclusions And Relevance: Despite important achievements to date, stem cell therapy is not yet ready for routine clinical implementation. Significant research is still needed to address the clinical barriers outlined herein before the next wave of large clinical trials is under way.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1001/jamacardio.2016.2750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378463PMC
November 2016

Multipotency and cardiomyogenic potential of human adipose-derived stem cells from epicardium, pericardium, and omentum.

Stem Cell Res Ther 2016 Jun 13;7(1):84. Epub 2016 Jun 13.

Department of Cardiothoracic Surgery, Stanford University School of Medicine, Falk Cardiovascular Research Center, 300 Pasteur Dr, Stanford, CA, 94305, USA.

Background: Acute myocardial infarction (MI) leads to an irreversible loss of proper cardiac function. Application of stem cell therapy is an attractive option for MI treatment. Adipose tissue has proven to serve as a rich source of stem cells (ADSCs). Taking into account the different morphogenesis, anatomy, and physiology of adipose tissue, we hypothesized that ADSCs from different adipose tissue depots may exert a diverse multipotency and cardiogenic potential.

Methods: The omental, pericardial, and epicardial adipose tissue samples were obtained from organ donors and patients undergoing heart transplantation at our institution. Human foreskin fibroblasts were used as the control group. Isolated ADSCs were analyzed for adipogenic and osteogenic differentiation capacity and proliferation potential. The immunophenotype and constitutive gene expression of alkaline phosphatase (ALP), GATA4, Nanog, and OCT4 were analyzed. DNA methylation inhibitor 5-azacytidine was exposed to the cells to stimulate the cardiogenesis. Finally, reprogramming towards cardiomyocytes was initiated with exogenous overexpression of seven transcription factors (ESRRG, GATA4, MEF2C, MESP1, MYOCD, TBX5, ZFPM2) previously applied successfully for fibroblast transdifferentiation toward cardiomyocytes. Expression of cardiac troponin T (cTNT) and alpha-actinin (Actn2) was analyzed 3 weeks after initiation of the cardiac differentiation.

Results: The multipotent properties of isolated plastic adherent cells were confirmed with expression of CD29, CD44, CD90, and CD105, as well as successful differentiation toward adipocytes and osteocytes; with the highest osteogenic and adipogenic potential for the epicardial and omental ADSCs, respectively. Epicardial ADSCs demonstrated a lower doubling time as compared with the pericardium and omentum-derived cells. Furthermore, epicardial ADSCs revealed higher constitutive expression of ALP and GATA4. Increased Actn2 and cTNT expression was observed after the transduction of seven reprogramming factors, with the highest expression in the epicardial ADSCs, as compared with the other ADSC subtypes and fibroblasts.

Conclusions: Human epicardial ADSCs revealed a higher cardiomyogenic potential as compared with the pericardial and omental ADSC subtypes as well as the fibroblast counterparts. Epicardial ADSCs may thus serve as the valuable subject for further studies on more effective methods of adult stem cell differentiation toward cardiomyocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13287-016-0343-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907285PMC
June 2016

The Selective JAK1/3-Inhibitor R507 Mitigates Obliterative Airway Disease Both With Systemic Administration and Aerosol Inhalation.

Transplantation 2016 05;100(5):1022-31

1 TSI Laboratory, University Heart Center Hamburg, Hamburg, Germany. 2 Cardiovascular Research Center Hamburg (CVRC) and DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Luebeck, Hamburg, Germany. 3 Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany. 4 Department of Surgery, TSI Laboratory, University of California San Francsico (UCSF), San Francsico, CA. 5 Cardiovascular Medicine and Stanford Cardiovascular Institute, Stanford University, Stanford, CA. 6 Department of Anaesthesiology, University Hospital Hamburg, Hamburg, Germany. 7 Rigel Pharmaceuticals, South San Francisco, CA. 8 Aerogen Ltd, Galway, Ireland. 9 Department of Clinical Chemistry, University Medical Center Hamburg, Hamburg, Germany. 10 Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden. 11Department of Cardiothoracic Surgery, Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA.

Background: The efficacy of selective Janus kinase 1/3 inhibitor R507 to prevent obliterative airway disease was analyzed in preclinical airway transplantation models.

Methods: Orthotopic trachea transplantations were performed between Lewis donors and Brown Norway rat recipients. Oral everolimus (4 mg/kg once per day) or oral respective inhaled R507 (60 mg/kg twice per day, each) was used for immunosuppression. Grafts were retrieved after 6 or 60 days. Toxicity and anti-inflammatory effects of R507 were analyzed on human airway epithelial cells.

Results: In 6-day animals, oral and inhaled R507 more potently diminished mononuclear graft infiltration than everolimus and preserved ciliated pseudostratified columnar respiratory epithelium. Everolimus and R507 similarly suppressed systemic cellular and humoral immune activation. In untreated rats, marked obliterative airway disease developed over 60 days. Oral and inhaled R507 was significantly more effective in reducing airway obliteration and preserved the morphology of the airway epithelium. Luciferase-positive donors revealed that a substantial amount of smooth muscle cells within the obliterative tissue was of donor origin. Only everolimus but not R507, adversely altered kidney function and lipid profiles. The R507 aerosol did not show airway toxicity in vitro but effectively suppressed activation of inflammatory signaling pathways induced by IL-1β.

Conclusions: The Janus kinase 1/3 inhibitor R507 is a very well-tolerated immunosuppressant that similarly diminished obliterative airway disease with systemic or inhaled administration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/TP.0000000000001110DOI Listing
May 2016

Comparison of Magnetic Resonance Imaging and Serum Biomarkers for Detection of Human Pluripotent Stem Cell-Derived Teratomas.

Stem Cell Reports 2016 Feb 14;6(2):176-87. Epub 2016 Jan 14.

Stanford Cardiovascular Institute, Stanford University School of Medicine, Lorry Lokey Stem Cell Research Building, 265 Campus Drive, Stanford, CA 94305, USA; Division of Cardiology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA; Molecular Imaging Program, Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

The use of cells derived from pluripotent stem cells (PSCs) for regenerative therapies confers a considerable risk for neoplastic growth and teratoma formation. Preclinical and clinical assessment of such therapies will require suitable monitoring strategies to understand and mitigate these risks. Here we generated human-induced pluripotent stem cells (iPSCs), selected clones that continued to express reprogramming factors after differentiation into cardiomyocytes, and transplanted these cardiomyocytes into immunocompromised rat hearts post-myocardial infarction. We compared magnetic resonance imaging (MRI), cardiac ultrasound, and serum biomarkers for their ability to delineate teratoma formation and growth. MRI enabled the detection of teratomas with a volume >8 mm(3). A combination of three plasma biomarkers (CEA, AFP, and HCG) was able to detect teratomas with a volume >17 mm(3) and with a sensitivity of more than 87%. Based on our findings, a combination of serum biomarkers with MRI screening may offer the highest sensitivity for teratoma detection and tracking.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2015.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4750097PMC
February 2016

Human Engineered Heart Muscles Engraft and Survive Long Term in a Rodent Myocardial Infarction Model.

Circ Res 2015 Sep 19;117(8):720-30. Epub 2015 Aug 19.

From the Division of Cardiology, Department of Medicine, Stanford Cardiovascular Institute (J.R., A.E., E.T., U.R., O.J.A., O.S., N.G.K., E.N., M.W., P.S.T., J.D.G., J.C.W.) and Department of Pathology (A.J.C.), Stanford University School of Medicine, CA; Department for Research and Development, Veterans Administration Palo Alto Health Care System, CA (P.S.T.); Institute of Pharmacology, Heart Research Center, University Medical Center, Georg-August-University and German Center for Cardiovascular Research, Göttingen, Germany (M.T., T.M., W.H.Z.); and Center for Biomedicine and Genetics (V.C.C., L.A.C.) and Center for Applied Technology Development, Beckman Research Institute (A.J.C.), City of Hope, Duarte, CA.

Rationale: Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure.

Objective: Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model.

Methods And Results: We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring.

Conclusions: EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.115.306985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679370PMC
September 2015

Celecoxib Nanoparticles for Therapeutic Angiogenesis.

ACS Nano 2015 Sep 10;9(9):9416-26. Epub 2015 Aug 10.

Department of Chemistry, Stanford University , Stanford, California 94305-5080, United States.

Controllable induction of blood vessel formation (angiogenesis) presents an important therapeutic goal in ischemic diseases and is also beneficial in various normal physiological processes. In this study, we have shown that nanoparticles of celecoxib, a lipophilic nonsteroidal anti-inflammatory drug, effectively evoke therapeutic angiogenesis in animal models, in both normal and ischemic organs. Celecoxib is widely considered to inhibit angiogenesis, although a recent study suggests that it can instead promote blood vessel growth in cancer cell lines. The hydrophobic nature of this drug necessitates its administration in nanoparticulate form in order to elicit a perceivable pharmacological response. We developed a facile method for nanoparticle formation by solvent extraction from microemulsions in supercritical carbon dioxide. This method exploits a spontaneous formation of nanometric domains within the microemulsion system and their rapid conversion to nanoparticles by supercritical fluid. The resultant nanoparticles were administered subcutaneously to mice in a biocompatible hydrogel, and caused a 4-fold increase in blood vessel count in normally perfused skin compared with drug-free particles. They were at least as effective in inducing angiogenesis as nanoparticles of deferoxamine, a well-established neovascularization promoter. Next, we evaluated their effect on ischemic tissues in murine model of myocardial infarction. We found that celecoxib nanoparticles were able to induce a significant vascularization of ischemic myocardium and hamper the progression of heart failure, which points toward a new approach for treating ischemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsnano.5b04137DOI Listing
September 2015

Hurdles to clinical translation of human induced pluripotent stem cells.

J Clin Invest 2015 Jul 1;125(7):2551-7. Epub 2015 Jul 1.

Human pluripotent stem cells are known to have the capacity to renew indefinitely, being intrinsically able to differentiate into many different cell types. These characteristics have generated tremendous enthusiasm about the potential applications of these cells in regenerative medicine. However, major challenges remain with the development and testing of novel experimental stem cell therapeutics in the field. In this Review, we focus on the nature of the preclinical challenges and discuss potential solutions that could help overcome them. Furthermore, we discuss the use of allogeneic versus autologous stem cell products, including a review of their respective advantages and disadvantages, major clinical requirements, quality standards, time lines, and costs of clinical grade development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI80575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4563685PMC
July 2015

Mesenchymal stromal cell therapy: different sources exhibit different immunobiological properties.

Transplantation 2015 Jun;99(6):1113-8

1 TSI Laboratory, University Heart Center Hamburg, Hamburg, Germany. 2 Department of Cardiothoracic Surgery, Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA. 3 Cardiovascular Research Center Hamburg (CVRC) and DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Luebeck, Germany. 4 Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/TP.0000000000000734DOI Listing
June 2015

Immunobiology of fibrin-based engineered heart tissue.

Stem Cells Transl Med 2015 Jun 6;4(6):625-31. Epub 2015 May 6.

University Heart Center Hamburg, Transplant and Stem Cell Immunobiology Laboratory, Hamburg, Germany; Department of Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany; DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, and Cardiovascular Research Center and Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Stanford Cardiovascular Institute and Department of Cardiothoracic Surgery, Stanford University School of Medicine, Falk Cardiovascular Research Center, Stanford, California, USA

Unlabelled: Different tissue-engineering approaches have been developed to induce and promote cardiac regeneration; however, the impact of the immune system and its responses to the various scaffold components of the engineered grafts remains unclear. Fibrin-based engineered heart tissue (EHT) was generated from neonatal Lewis (Lew) rat heart cells and transplanted onto the left ventricular surface of three different rat strains: syngeneic Lew, allogeneic Brown Norway, and immunodeficient Rowett Nude rats. Interferon spot frequency assay results showed similar degrees of systemic immune activation in the syngeneic and allogeneic groups, whereas no systemic immune response was detectable in the immunodeficient group (p < .001 vs. syngeneic and allogeneic). Histological analysis revealed much higher local infiltration of CD3- and CD68-positive cells in syngeneic and allogeneic rats than in immunodeficient animals. Enzyme-linked immunospot and immunofluorescence experiments revealed matrix-directed TH1-based rejection in syngeneic recipients without collateral impairment of heart cell survival. Bioluminescence imaging was used for in vivo longitudinal monitoring of transplanted luciferase-positive EHT constructs. Survival was documented in syngeneic and immunodeficient recipients for a period of up to 110 days after transplant, whereas in the allogeneic setting, graft survival was limited to only 14 ± 1 days. EHT strategies using autologous cells are promising approaches for cardiac repair applications. Although fibrin-based scaffold components elicited an immune response in our studies, syngeneic cells carried in the EHT were relatively unaffected.

Significance: An initial insight into immunological consequences after transplantation of engineered heart tissue was gained through this study. Most important, this study was able to demonstrate cell survival despite rejection of matrix components. Generation of syngeneic human engineered heart tissue, possibly using human induced pluripotent stem cell technology with subsequent directed rejection of matrix components, may be a potential future approach to replace diseased myocardium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5966/sctm.2013-0202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449088PMC
June 2015

Transdermal deferoxamine prevents pressure-induced diabetic ulcers.

Proc Natl Acad Sci U S A 2015 Jan 22;112(1):94-9. Epub 2014 Dec 22.

Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305;

There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1413445112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291638PMC
January 2015

Wireless power transfer to deep-tissue microimplants.

Proc Natl Acad Sci U S A 2014 Jun 19;111(22):7974-9. Epub 2014 May 19.

Departments of Electrical Engineering and

The ability to implant electronic systems in the human body has led to many medical advances. Progress in semiconductor technology paved the way for devices at the scale of a millimeter or less ("microimplants"), but the miniaturization of the power source remains challenging. Although wireless powering has been demonstrated, energy transfer beyond superficial depths in tissue has so far been limited by large coils (at least a centimeter in diameter) unsuitable for a microimplant. Here, we show that this limitation can be overcome by a method, termed midfield powering, to create a high-energy density region deep in tissue inside of which the power-harvesting structure can be made extremely small. Unlike conventional near-field (inductively coupled) coils, for which coupling is limited by exponential field decay, a patterned metal plate is used to induce spatially confined and adaptive energy transport through propagating modes in tissue. We use this method to power a microimplant (2 mm, 70 mg) capable of closed-chest wireless control of the heart that is orders of magnitude smaller than conventional pacemakers. With exposure levels below human safety thresholds, milliwatt levels of power can be transferred to a deep-tissue (>5 cm) microimplant for both complex electronic function and physiological stimulation. The approach developed here should enable new generations of implantable systems that can be integrated into the body at minimal cost and risk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1403002111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050616PMC
June 2014

Protein-polymer hybrid nanoparticles for drug delivery.

Small 2012 Dec 9;8(23):3573-8. Epub 2012 Aug 9.

Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.

Amphiphilic bovine serum albumin-poly(methyl methacrylate) conjugate forms nanoparticles with the uniform size of ~100 nm by self-assembling. Loaded with the hydrophobic anti-tumor drug camptothecin, the nanoparticle efficiently delivers drugs into cancer cells, and thus inhibits ~79% of tumor growth in animals compared with free drug.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/smll.201200889DOI Listing
December 2012

Drug release from electric-field-responsive nanoparticles.

ACS Nano 2012 Jan 30;6(1):227-33. Epub 2011 Nov 30.

Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.

We describe a new temperature and electric field dual-stimulus responsive nanoparticle system for programmed drug delivery. Nanoparticles of a conducting polymer (polypyrrole) are loaded with therapeutic pharmaceuticals and are subcutaneously localized in vivo with the assistance of a temperature-sensitive hydrogel (PLGA-PEG-PLGA). We have shown that drug release from the conductive nanoparticles is controlled by the application of a weak, external DC electric field. This approach represents a novel interactive drug delivery system that can show an externally tailored release profile with an excellent spatial, temporal, and dosage control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/nn203430mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489921PMC
January 2012

Adipose tissue-derived stem cells display a proangiogenic phenotype on 3D scaffolds.

J Biomed Mater Res A 2011 Sep 31;98(3):383-93. Epub 2011 May 31.

Department of Cardiothoracic Surgery, Stanford University School of Medicine, California, USA.

Ischemic heart disease is the leading cause of death worldwide. Recent studies suggest that adipose tissue-derived stem cells (ASCs) can be used as a potential source for cardiovascular tissue engineering due to their ability to differentiate along the cardiovascular lineage and to adopt a proangiogenic phenotype. To understand better ASCs' biology, we used a novel 3D culture device. ASCs' and b.END-3 endothelial cell proliferation, migration, and vessel morphogenesis were significantly enhanced compared to 2D culturing techniques. ASCs were isolated from inguinal fat pads of 6-week-old GFP+/BLI+ mice. Early passage ASCs cells (P3-P4), PKH26-labeled murine b.END-3 cells or a co-culture of ASCs and b.END-3 cells were seeded at a density of 1 × 10(5) on three different surface configurations: (a) a 2D surface of tissue culture plastic, (b) Matrigel, and (c) a highly porous 3D scaffold fabricated from inert polystyrene. VEGF expression, cell proliferation, and tubulization, were assessed using optical microscopy, fluorescence microscopy, 3D confocal microscopy, and SEM imaging (n = 6). Increased VEGF levels were seen in conditioned media harvested from co-cultures of ASCs and b.END-3 on either Matrigel or a 3D matrix. Fluorescence, confocal, SEM, bioluminescence revealed improved cell, proliferation, and tubule formation for cells seeded on the 3D polystyrene matrix. Collectively, these data demonstrate that co-culturing ASCs with endothelial cells in a 3D matrix environment enable us to generate prevascularized tissue-engineered constructs. This can potentially help us to surpass the tissue thickness limitations faced by the tissue engineering community today.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbm.a.33113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161132PMC
September 2011

Engineered pullulan-collagen composite dermal hydrogels improve early cutaneous wound healing.

Tissue Eng Part A 2011 Mar 17;17(5-6):631-44. Epub 2010 Nov 17.

Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305, USA.

New strategies for skin regeneration are needed to address the significant medical burden caused by cutaneous wounds and disease. In this study, pullulan-collagen composite hydrogel matrices were fabricated using a salt-induced phase inversion technique, resulting in a structured yet soft scaffold for skin engineering. Salt crystallization induced interconnected pore formation, and modification of collagen concentration permitted regulation of scaffold pore size. Hydrogel architecture recapitulated the reticular distribution of human dermal matrix while maintaining flexible properties essential for skin applications. In vitro, collagen hydrogel scaffolds retained their open porous architecture and viably sustained human fibroblasts and murine mesenchymal stem cells and endothelial cells. In vivo, hydrogel-treated murine excisional wounds demonstrated improved wound closure, which was associated with increased recruitment of stromal cells and formation of vascularized granulation tissue. In conclusion, salt-induced phase inversion techniques can be used to create modifiable pullulan-collagen composite dermal scaffolds that augment early wound healing. These novel biomatrices can potentially serve as a structured delivery template for cells and biomolecules in regenerative skin applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.tea.2010.0298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398002PMC
March 2011

IFATS collection: Adipose stromal cells adopt a proangiogenic phenotype under the influence of hypoxia.

Stem Cells 2009 Jan;27(1):266-74

Department of Surgery, Stanford University School of Medicine, California 94305-5148, USA.

Evolving evidence suggests a possible role for adipose stromal cells (ASCs) in adult neovascularization, although the specific cues that stimulate their angiogenic behavior are poorly understood. We evaluated the effect of hypoxia, a central mediator of new blood vessel development within ischemic tissue, on proneovascular ASC functions. Murine ASCs were exposed to normoxia (21% oxygen) or hypoxia (5%, 1% oxygen) for varying lengths of time. Vascular endothelial growth factor (VEGF) secretion by ASCs increased as an inverse function of oxygen tension, with progressively higher VEGF expression at 21%, 5%, and 1% oxygen, respectively. Greater VEGF levels were also associated with longer periods in culture. ASCs were able to migrate towards stromal cell-derived factor (SDF)-1, a chemokine expressed by ischemic tissue, with hypoxia augmenting ASC expression of the SDF-1 receptor (CXCR4) and potentiating ASC migration. In vivo, ASCs demonstrated the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this was supported by in vitro studies showing increasing ASC proliferation with greater degrees of hypoxia. Hypoxia did not significantly alter the expression of endothelial surface markers by ASCs. However, these cells did assume an endothelial phenotype as evidenced by their ability to tubularize when seeded with differentiated endothelial cells on Matrigel. Taken together, these data suggest that ASCs upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1634/stemcells.2008-0276DOI Listing
January 2009