Robert C. Robbins

Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium

Under conditions of tissue injury, myocardial replication and regeneration have been reported. A growing number of investigators have implicated adult bone marrow (BM) in this process, suggesting that marrow serves as a reservoir for cardiac precursor cells. It remains unclear which BM cell(s) can contribute to myocardium, and whether they do so by transdifferentiation or cell fusion. Here, we studied the ability of c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1lo Lin- Sca-1+ long-term reconstituting haematopoietic stem cells to regenerate myocardium in an infarct model. Cells were isolated from transgenic mice expressing green fluorescent protein (GFP) and injected directly into ischaemic myocardium of wild-type mice. Abundant GFP+ cells were detected in the myocardium after 10 days, but by 30 days, few cells were detectable. These GFP+ cells did not express cardiac tissue-specific markers, but rather, most of them expressed the haematopoietic marker CD45 and myeloid marker Gr-1. We also studied the role of circulating cells in the repair of ischaemic myocardium using GFP+–GFP- parabiotic mice. Again, we found no evidence of myocardial regeneration from blood-borne partner-derived cells. Our data suggest that even in the microenvironment of the injured heart, c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1lo Lin- Sca-1+ long-term reconstituting haematopoietic stem cells adopt only traditional haematopoietic fates.

Human tissue-engineered blood vessels for adult arterial revascularization.

There is a crucial need for alternatives to native vein or artery for vascular surgery. The clinical efficacy of synthetic, allogeneic or xenogeneic vessels has been limited by thrombosis, rejection, chronic inflammation and poor mechanical properties. Using adult human fibroblasts extracted from skin biopsies harvested from individuals with advanced cardiovascular disease, we constructed tissue-engineered blood vessels (TEBVs) that serve as arterial bypass grafts in long-term animal models. These TEBVs have mechanical properties similar to human blood vessels, without relying upon synthetic or exogenous scaffolding. The TEBVs are antithrombogenic and mechanically stable for 8 months in vivo. Histological analysis showed complete tissue integration and formation of vasa vasorum. The endothelium was confluent and positive for von Willebrand factor. A smooth muscle–specific α-actin–positive cell population developed within the TEBV, suggesting regeneration of a vascular media. Electron microscopy showed an endothelial basement membrane, elastogenesis and a complex collagen network. These results indicate that a completely biological and clinically relevant TEBV can be assembled exclusively from an individuals own cells.

Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells

Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.

Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy

Human induced pluripotent stem cells generated from patients with familial dilated cardiomyopathy model cardiovascular disease in these patients. iPSCs Make the Heart Beat Faster Mutations in genes expressed in the heart can cause dilated cardiomyopathy (DCM), a form of heart disease in which a weakened and enlarged heart is unable to pump sufficient blood for the body’s needs. DCM can lead to progressive heart failure that eventually requires heart transplantation. This disease has been challenging to study because cardiomyocytes from the hearts of DCM patients are difficult to obtain and do not survive long. Mouse models of DCM are established and have provided important clues about the disease mechanisms for DCM. However, the mouse heart is very different in physiology compared to the human heart; for example, the mouse heart rate is 10 times faster than that of human. In a new study, Sun et al. generated induced pluripotent stem cells (iPSCs) from skin cells of patients in a family with inherited DCM. This family carries a deleterious mutation in TNNT2, a gene that is expressed specifically in the heart and regulates cardiomyocyte contraction. Using iPSCs, the authors generated a large number of individual-specific cardiomyocytes carrying the specific TNNT2 mutation and analyzed their functional properties. Compared to cardiomyocytes derived from iPSCs of healthy controls in the same family, cardiomyocytes derived from iPSCs of DCM patients exhibited an increased heterogeneous myofilament organization, susceptibility to stress, compromised ability to regulate calcium flux, and decreased contraction force. These results suggest that the mutation in TNNT2 causes abnormalities in the cardiomyocytes and contributes to the development of DCM disease. Using these DCM iPSC–derived cardiomyocytes, the researchers also showed that several current treatments that clinically benefit DCM disease improved DCM cardiomyocyte function in culture. The current study shows that human iPSC-derived cardiomyocytes could provide an important platform to investigate the specific disease mechanisms of DCM as well as other inherited cardiovascular disorders and for screening new drugs for cardiovascular disease. Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca2+), decreased contractility, and abnormal distribution of sarcomeric α-actinin. When stimulated with a β-adrenergic agonist, DCM iPSC–derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric α-actinin distribution. Treatment with β-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca2+ adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.

Consensus Conference Report Maximizing Use of Organs Recovered From the Cadaver Donor: Cardiac Recommendations: March 28–29, 2001, Crystal City, Va

The shortage of available donor hearts continues to limit cardiac transplantation. For this reason, strict criteria have limited the number of patients placed on the US waiting list to ≈6000 to 8000 per year. Because the number of available donor hearts has not increased beyond ≈2500 per year, the transplant waiting list mortality rate remains substantial. Suboptimal and variable utilization of donor hearts has compounded the problem in the United States. In 1999, the average donor yield from 55 US regions was 39%, ranging from 19% to 62%. This report provides the detailed cardiac recommendations from the conference on “Maximizing Use of Organs Recovered From the Cadaver Donor” held March 28 to 29, 2001, in Crystal City, Va. The specific objective of the report is to provide recommendations to improve the evaluation and successful utilization of potential cardiac donors. The report describes the accuracy of current techniques such as echocardiography in the assessment of donor heart function before recove...The shortage of available donor hearts continues to limit cardiac transplantation. For this reason, strict criteria have limited the number of patients placed on the US waiting list to 6000 to 8000 per year. Because the number of available donor hearts has not increased beyond 2500 per year, the transplant waiting list mortality rate remains substantial. Suboptimal and variable utilization of donor hearts has compounded the problem in the United States. In 1999, the average donor yield from 55 US regions was 39%, ranging from 19% to 62%. This report provides the detailed cardiac recommendations from the conference on “Maximizing Use of Organs Recovered From the Cadaver Donor” held March 28 to 29, 2001, in Crystal City, Va. The specific objective of the report is to provide recommendations to improve the evaluation and successful utilization of potential cardiac donors. The report describes the accuracy of current techniques such as echocardiography in the assessment of donor heart function before recovery and the impact of these data on donor yield. The rationale for and specific details of a donor-management pathway that uses pulmonary artery catheterization and hormonal resuscitation are provided. Administrative recommendations such as enhanced communication strategies among transplant centers and organ-procurement organizations, financial incentives for organ recovery, and expansion of donor database fields for research are also described. (Circulation. 2002;106:836-841.)

Novel Index for Invasively Assessing the Coronary Microcirculation

Background—A relatively simple, invasive method for quantitatively assessing the status of the coronary microcirculation independent of the epicardial artery is lacking. Methods and Results—By using a coronary pressure wire and modified software, it is possible to calculate the mean transit time of room-temperature saline injected down a coronary artery. The inverse of the hyperemic mean transit time has been shown to correlate with absolute flow. We hypothesize that distal coronary pressure divided by the inverse of the hyperemic mean transit time provides an index of microcirculatory resistance (IMR) that will correlate with true microcirculatory resistance (TMR), defined as the distal left anterior descending (LAD) pressure divided by hyperemic flow, measured with an external ultrasonic flow probe. A total of 61 measurements were made in 9 Yorkshire swine at baseline and after disruption of the coronary microcirculation, both with and without an epicardial LAD stenosis. The mean IMR (16.9±6.5 U to 25.9±14.4 U, P =0.002) and TMR (0.51±0.14 to 0.79±0.32 mm Hg · mL−1 · min−1, P =0.0001), as well as the % change in IMR (147±66%) and TMR (159±105%, P =NS versus IMR % change), increased significantly and to a similar degree after disruption of the microcirculation. These changes were independent of the status of the epicardial artery. There was a significant correlation between mean IMR and TMR values, as well as between the % change in IMR and % change in TMR. Conclusion—Measuring IMR may provide a simple, quantitative, invasive assessment of the coronary microcirculation.

Drug Screening Using a Library of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes Reveals Disease-Specific Patterns of Cardiotoxicity

Background— Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds. Methods and Results— Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome, familial hypertrophic cardiomyopathy, and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell–derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go–related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations. Conclusions— We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go–related gene test or healthy control hiPSC-CM/hESC-CM screening assays.

Novel Role for the Potent Endogenous Inotrope Apelin in Human Cardiac Dysfunction

Background—Apelin is among the most potent stimulators of cardiac contractility known. However, no physiological or pathological role for apelin–angiotensin receptor-like 1 (APJ) signaling has ever been described. Methods and Results—We performed transcriptional profiling using a spotted cDNA microarray with 12 814 unique clones on paired samples of left ventricle obtained before and after placement of a left ventricular assist device in 11 patients. The significance analysis of microarrays and a novel rank consistency score designed to exploit the paired structure of the data confirmed that natriuretic peptides were among the most significantly downregulated genes after offloading. The most significantly upregulated gene was the G-protein–coupled receptor APJ, the specific receptor for apelin. We demonstrate here using immunoassay and immunohistochemical techniques that apelin is localized primarily in the endothelium of the coronary arteries and is found at a higher concentration in cardiac tissue after mechanical offloading. These findings imply an important paracrine signaling pathway in the heart. We additionally extend the clinical significance of this work by reporting for the first time circulating human apelin levels and demonstrating increases in the plasma level of apelin in patients with left ventricular dysfunction. Conclusions—The apelin-APJ signaling pathway emerges as an important novel mediator of cardiovascular control.

MicroRNA-210 as a Novel Therapy for Treatment of Ischemic Heart Disease

Background— MicroRNAs are involved in various critical functions, including the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. We hypothesize that microRNA-210 can rescue cardiac function after myocardial infarction by upregulation of angiogenesis and inhibition of cellular apoptosis in the heart. Methods and Results— Using microRNA microarrays, we first showed that microRNA-210 was highly expressed in live mouse HL-1 cardiomyocytes compared with apoptotic cells after 48 hours of hypoxia exposure. We confirmed by polymerase chain reaction that microRNA-210 was robustly induced in these cells. Gain-of-function and loss-of-function approaches were used to investigate microRNA-210 therapeutic potential in vitro. After transduction, microRNA-210 can upregulate several angiogenic factors, inhibit caspase activity, and prevent cell apoptosis compared with control. Afterward, adult FVB mice underwent intramyocardial injections with minicircle vector carrying microRNA-210 precursor, minicircle carrying microRNA-scramble, or sham surgery. At 8 weeks, echocardiography showed a significant improvement of left ventricular fractional shortening in the minicircle vector carrying microRNA-210 precursor group compared with the minicircle carrying microRNA-scramble control. Histological analysis confirmed decreased cellular apoptosis and increased neovascularization. Finally, 2 potential targets of microRNA-210, Efna3 and Ptp1b, involved in angiogenesis and apoptosis were confirmed through additional experimental validation. Conclusion— MicroRNA-210 can improve angiogenesis, inhibit apoptosis, and improve cardiac function in a murine model of myocardial infarction. It represents a potential novel therapeutic approach for treatment of ischemic heart disease.

Embryonic Stem Cell Immunogenicity Increases Upon Differentiation After Transplantation Into Ischemic Myocardium

Background—We investigated whether differentiation of embryonic stem cells (ESCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection. Methods and Results—In one series, 129/SvJ-derived mouse ESCs (ES-D3 line) were transplanted by direct myocardial injection (1×106 cells) into murine hearts of both allogeneic (BALB/c, n=20) and syngeneic (129/SvJ, n=12) recipients after left anterior artery ligation. Hearts were procured at 1, 2, 4, and 8 weeks after ESC transplantation and analyzed by immunohistochemistry to assess immune cell infiltration (CD3, CD4, CD8, B220, CD11c, Mac-1, and Gr-1) and ESC differentiation (hematoxylin and eosin). In a second series (allogeneic n=5, sham n=3), ESC transplantation was performed similarly; however after 2 weeks, left anterior descending artery-ligated and ESC-injected hearts were heterotopically transplanted into naive BALB/c recipients. After an additional 2 weeks, donor hearts were procured and analyzed by immunohistochemistry. In the first series, the size of all ESC grafts remained stable and there was no evidence of ESC differentiation 2 weeks after transplantation; however, after 4 weeks, both allogeneic and syngeneic ESC grafts showed the presence of teratoma. By 8 weeks, surviving ESCs could be detected in the syngeneic but not in the allogeneic group. Mild inflammatory cellular infiltrates were found in allogeneic recipients at 1 and 2 weeks after transplantation, progressing into vigorous infiltration at 4 and 8 weeks. The second series demonstrated similar vigorous infiltration of immune cells as early as 2 weeks after heterotopic transplantation. Conclusion—In vivo differentiated ESCs elicit an accelerated immune response as compared with undifferentiated ESCs. These data imply that clinical transplantation of allogeneic ESCs or ESC derivatives for treatment of cardiac failure might require immunosuppressive therapy.