Tao-Sheng Li

Relative Roles of Direct Regeneration Versus Paracrine Effects of Human Cardiosphere-Derived Cells Transplanted Into Infarcted Mice

Rationale: Multiple biological mechanisms contribute to the efficacy of cardiac cell therapy. Most prominent among these are direct heart muscle and blood vessel regeneration from transplanted cells, as opposed to paracrine enhancement of tissue preservation and/or recruitment of endogenous repair. Objective: Human cardiac progenitor cells, cultured as cardiospheres (CSps) or as CSp-derived cells (CDCs), have been shown to be capable of direct cardiac regeneration in vivo. Here we characterized paracrine effects in CDC transplantation and investigated their relative importance versus direct differentiation of surviving transplanted cells. Methods and Results: In vitro, many growth factors were found in media conditioned by human adult CSps and CDCs; CDC-conditioned media exerted antiapoptotic effects on neonatal rat ventricular myocytes, and proangiogenic effects on human umbilical vein endothelial cells. In vivo, human CDCs secreted vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor 1 when transplanted into the same SCID mouse model of acute myocardial infarction where they were previously shown to improve function and to produce tissue regeneration. Injection of CDCs in the peri-infarct zone increased the expression of Akt, decreased apoptotic rate and caspase 3 level, and increased capillary density, indicating overall higher tissue resilience. Based on the number of human-specific cells relative to overall increases in capillary density and myocardial viability, direct differentiation quantitatively accounted for 20% to 50% of the observed effects. Conclusions: Together with their spontaneous commitment to cardiac and angiogenic differentiation, transplanted CDCs serve as “role models,” recruiting endogenous regeneration and improving tissue resistance to ischemic stress. The contribution of the role model effect rivals or exceeds that of direct regeneration.

Direct comparison of different stem cell types and subpopulations reveals superior paracrine potency and myocardial repair efficacy with cardiosphere-derived cells

OBJECTIVES The goal of this study was to conduct a direct head-to-head comparison of different stem cell types in vitro for various assays of potency and in vivo for functional myocardial repair in the same mouse model of myocardial infarction. BACKGROUND Adult stem cells of diverse origins (e.g., bone marrow, fat, heart) and antigenic identity have been studied for repair of the damaged heart, but the relative utility of the various cell types remains unclear. METHODS Human cardiosphere-derived cells (CDCs), bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and bone marrow mononuclear cells were compared. RESULTS CDCs revealed a distinctive phenotype with uniform expression of CD105, partial expression of c-kit and CD90, and negligible expression of hematopoietic markers. In vitro, CDCs showed the greatest myogenic differentiation potency, highest angiogenic potential, and relatively high production of various angiogenic and antiapoptotic-secreted factors. In vivo, injection of CDCs into the infarcted mouse hearts resulted in superior improvement of cardiac function, the highest cell engraftment and myogenic differentiation rates, and the least-abnormal heart morphology 3 weeks after treatment. CDC-treated hearts also exhibited the lowest number of apoptotic cells. The c-kit(+) subpopulation purified from CDCs produced lower levels of paracrine factors and inferior functional benefit when compared with unsorted CDCs. To validate the comparison of cells from various human donors, selected results were confirmed in cells of different types derived from individual rats. CONCLUSIONS CDCs exhibited a balanced profile of paracrine factor production and, among various comparator cell types/subpopulations, provided the greatest functional benefit in experimental myocardial infarction.

Validation of the Cardiosphere Method to Culture Cardiac Progenitor Cells from Myocardial Tissue

Background At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs. Methodology/Principal Findings CPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP+ C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit+), endothelial cells (CD31+, CD34+), and mesenchymal cells (CD90+). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential. Conclusions/Significance This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used.

Cardiospheres Recapitulate a Niche-Like Microenvironment Rich in Stemness and Cell-Matrix Interactions, Rationalizing Their Enhanced Functional Potency for Myocardial Repair†‡§

Cardiac stem cells (CSCs) are promising candidates for use in myocardial regenerative therapy. We test the hypothesis that growing cardiac‐derived cells as three‐dimensional cardiospheres may recapitulate a stem cell niche‐like microenvironment, favoring cell survival and enhancing functional benefit after transplantation into the injured heart. CSCs and supporting cells from human endomyocardial biopsies were grown as cardiospheres and compared with cells cultured under traditional monolayer condition or dissociated from cardiospheres. Cardiospheres self‐assembled into stem cell niche‐like structures in vitro in suspension culture, while exhibiting greater proportions of c‐kit+ cells and upregulated expression of SOX2 and Nanog. Pathway‐focused polymerase chain reaction (PCR) array, quantitative real‐time PCR, and immunostaining revealed enhanced expression of stem cell‐relevant factors and adhesion/extracellular‐matrix molecules (ECM) in cardiospheres including IGF‐1, histone deacetylase 2 (HDAC2), Tert, integrin‐α2, laminin‐β1, and matrix metalloproteinases (MMPs). Implantation of cardiospheres in severe combined immunodeficiency (SCID) mouse hearts with acute infarction disproportionately improved cell engraftment and myocardial function, relative to monolayer‐cultured cells. Dissociation of cardiospheres into single cells decreased the expression of ECM and adhesion molecules and undermined resistance to oxidative stress, negating the improved cell engraftment and functional benefit in vivo. Growth of cardiac‐derived cells as cardiospheres mimics stem cell niche properties with enhanced “stemness” and expression of ECM and adhesion molecules. These changes underlie an increase in cell survival and more potent augmentation of global function following implantation into the infarcted heart. STEM CELLS 2010;28:2088–2098

Magnetic Targeting Enhances Engraftment and Functional Benefit of Iron-Labeled Cardiosphere-Derived Cells in Myocardial Infarction

Rationale: The success of cardiac stem cell therapies is limited by low cell retention, due at least in part to washout via coronary veins. Objective: We sought to counter the efflux of transplanted cells by rendering them magnetically responsive and imposing an external magnetic field on the heart during and immediately after injection. Methods and Results: Cardiosphere-derived cells (CDCs) were labeled with superparamagnetic microspheres (SPMs). In vitro studies revealed that cell viability and function were minimally affected by SPM labeling. SPM-labeled rat CDCs were injected intramyocardially, with and without a superimposed magnet. With magnetic targeting, cells were visibly attracted toward the magnet and accumulated around the ischemic zone. In contrast, the majority of nontargeted cells washed out immediately after injection. Fluorescence imaging revealed more retention of transplanted cells in the heart, and less migration into other organs, in the magnetically targeted group. Quantitative PCR confirmed that magnetic targeting enhanced cell retention (at 24 hours) and engraftment (at 3 weeks) in the recipient hearts by ≈3-fold compared to nontargeted cells. Morphometric analysis revealed maximal attenuation of left ventricular remodeling, and echocardiography showed the greatest functional improvement, in the magnetic targeting group. Histologically, more engrafted cells were evident with magnetic targeting, but there was no incremental inflammation. Conclusions: Magnetic targeting enhances cell retention, engraftment and functional benefit. This novel method to improve cell therapy outcomes offers the potential for rapid translation into clinical applications.

Intramyocardial Injection of Autologous Cardiospheres or Cardiosphere-Derived Cells Preserves Function and Minimizes Adverse Ventricular Remodeling in Pigs With Heart Failure Post-Myocardial Infarction

OBJECTIVES The purpose of this study was to test the safety and efficacy of direct injection of cardiosphere-derived cells (CDCs) and their 3-dimensional precursors, cardiospheres, for cellular cardiomyoplasty in a mini-pig model of heart failure after myocardial infarction. BACKGROUND Intracoronary administration of CDCs has been demonstrated to reduce infarct size and improve hemodynamic indexes in the mini-pig model, but intramyocardial injection of CDCs or cardiospheres has not been assessed in large animals. METHODS Autologous cardiospheres or CDCs grown from endomyocardial biopsies were injected through thoracotomy 4 weeks after anteroseptal myocardial infarction. Engraftment optimization with luciferase-labeled CDCs guided the choice of cell dose (0.5 million cells/site) and target tissue (20 peri-infarct sites). Pigs were randomly allocated to placebo (n = 11), cardiospheres (n = 8), or CDCs (n = 10). Functional data were acquired before injection and again 8 weeks later, after which organs were harvested for histopathology. RESULTS Beyond the immediate perioperative period, all animals survived to protocol completion. Ejection fraction was equivalent at baseline, but at 8 weeks was higher than placebo in both of the cell-treated groups (placebo vs. CDC, p = 0.01; placebo vs. cardiospheres, p = 0.01). Echocardiographic and hemodynamic indexes of efficacy improved disproportionately with cardiospheres; likewise, adverse remodeling was more attenuated with cardiospheres than with CDCs. Provocative electrophysiologic testing showed no differences among groups, and no tumors were found. CONCLUSIONS Dosage-optimized direct injection of cardiospheres or CDCs is safe and effective in preserving ventricular function in porcine ischemic cardiomyopathy. Although CDCs and cardiospheres have equivalent effects on left ventricular ejection fraction, cardiospheres are superior in improving hemodynamics and regional function, and in attenuating ventricular remodeling.

Safety and Efficacy of Allogeneic Cell Therapy in Infarcted Rats Transplanted with Mismatched Cardiosphere-Derived Cells

Background— Cardiosphere-derived cells (CDCs) are an attractive cell type for tissue regeneration, and autologous CDCs are being tested clinically. However, autologous therapy necessitates patient-specific tissue harvesting and cell processing, with delays to therapy and possible variations in cell potency. The use of allogeneic CDCs, if safe and effective, would obviate such limitations. We compared syngeneic and allogeneic CDC transplantation in rats from immunologically-mismatched inbred strains. Methods and Results— In vitro, CDCs expressed major histocompatibility complex class I but not class II antigens or B7 costimulatory molecules. In mixed-lymphocyte cocultures, allogeneic CDCs elicited negligible lymphocyte proliferation and inflammatory cytokine secretion. In vivo, syngeneic and allogeneic CDCs survived at similar levels in the infarcted rat heart 1 week after delivery, but few syngeneic (and even fewer allogeneic) CDCs remained at 3 weeks. Allogeneic CDCs induced a transient, mild, local immune reaction in the heart, without histologically evident rejection or systemic immunogenicity. Improvements in cardiac structure and function, sustained for 6 months, were comparable with syngeneic and allogeneic CDCs. Allogeneic CDCs stimulated endogenous regenerative mechanisms (cardiomyocyte cycling, recruitment of c-kit+ cells, angiogenesis) and increased myocardial vascular endothelial growth factor, insulin-like growth factor-1, and hepatocyte growth factor equally with syngeneic CDCs. Conclusions— Allogeneic CDC transplantation without immunosuppression is safe, promotes cardiac regeneration, and improves heart function in a rat myocardial infarction model, mainly through stimulation of endogenous repair mechanisms. The indirect mechanism of action rationalizes the persistence of benefit despite the evanescence of transplanted cell survival. This work motivates the testing of allogeneic human CDCs as a potential off-the-shelf product for cellular cardiomyoplasty.

Dedifferentiation and Proliferation of Mammalian Cardiomyocytes

Background It has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle. Methodology/Principal Findings Here we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1) cardiomyocyte purification from rat hearts, and 2) genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs), while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP) continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit+. Conclusions/Significance Contradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness, including the expression of c-kit and the capacity for multipotency.

Regeneration of Infarcted Myocardium by Intramyocardial Implantation of Ex Vivo Transforming Growth Factor-β–Preprogrammed Bone Marrow Stem Cells

Background—Recent studies have shown that bone marrow–derived stem cells differentiate into the phenotype of cardiomyocytes in vivo and in vitro. We tried to regenerate infarcted myocardium by implanting ex vivo transforming growth factor (TGF)-&bgr;–preprogrammed CD117 (c-kit)–positive (CD117+) stem cells intramyocardially. Methods and Results—CD117+ cells were isolated from the bone marrow mononuclear cells of GFP-transgenic or normal C57/BL6 mice. The myogenic differentiation of CD117+ cells was achieved by cultivation with TGF-&bgr;. Using an acute myocardial infarction model, we also tried to regenerate infarcted myocardium by implanting untreated (newly isolated) or preprogrammed (24 hours of cultivation with 5 ng/mL TGF-&bgr;1) CD117+ cells intramyocardially. TGF-&bgr; increased the cellular expression of myosin, troponins, connexin-43, GATA-4, and NKx-2.5, which suggested that it induced the myogenic differentiation of CD117+ cells. Compared with the effects of PBS injection only, the microvessel density in the infarcted myocardium was increased significantly 3 months after the implantation of either TGF-&bgr;–preprogrammed or untreated CD117+ cells. Moreover, many of the TGF-&bgr;–preprogrammed CD117+ cells were stained positively for myosin, whereas few of the untreated CD117+ cells were. Histological analysis revealed newly regenerated myocardium in the left ventricular anterior wall after the implantation of TGF-&bgr;–preprogrammed cells but not untreated cells. Furthermore, the left ventricular percent fraction shortening was significantly higher after the implantation of TGF-&bgr;–preprogrammed cells than after the implantation of untreated CD117+ cells. Conclusions—TGF-&bgr; conducted the myogenic differentiation of CD117+ stem cells by upregulating GATA-4 and NKx-2.5 expression. Therefore, the intramyocardial implantation of TGF-&bgr;–preprogrammed CD117+ cells effectively assisted the myocardial regeneration and induced therapeutic angiogenesis, contributing to functional cardiac regeneration.

Malfunction of nuclease ERCC1-XPF results in diverse clinical manifestations and causes Cockayne syndrome, xeroderma pigmentosum, and Fanconi anemia.

Cockayne syndrome (CS) is a genetic disorder characterized by developmental abnormalities and photodermatosis resulting from the lack of transcription-coupled nucleotide excision repair, which is responsible for the removal of photodamage from actively transcribed genes. To date, all identified causative mutations for CS have been in the two known CS-associated genes, ERCC8 (CSA) and ERCC6 (CSB). For the rare combined xeroderma pigmentosum (XP) and CS phenotype, all identified mutations are in three of the XP-associated genes, ERCC3 (XPB), ERCC2 (XPD), and ERCC5 (XPG). In a previous report, we identified several CS cases who did not have mutations in any of these genes. In this paper, we describe three CS individuals deficient in ERCC1 or ERCC4 (XPF). Remarkably, one of these individuals with XP complementation group F (XP-F) had clinical features of three different DNA-repair disorders--CS, XP, and Fanconi anemia (FA). Our results, together with those from Bogliolo et al., who describe XPF alterations resulting in FA alone, indicate a multifunctional role for XPF.