Cardiosphere-derived Cells Transplantation Research Articles

Isoproterenol pre-treatment improve the therapeutic efficacy of cardiosphere-derived cells transplantation for myocardial infarction.

This study aimed to investigate the effect of isoproterenol pre-treatment on the therapeutic efficacy of cardiosphere-derived cells (CDCs) transplantation for myocardial infarction (MI). Thirty 8-week-old male Sprague-Dawley (SD) rat model of MI was generated by ligation of the left anterior descending artery. The MI rats were treated with PBS (MI group, n=8), CDCs (MI + CDC group, n=8) and isoproterenol pre-treated CDCs (MI + ISO-CDC group, n=8), respectively. In the MI + ISO-CDC group, CDCs were pre-treated by 10-6 M isoproterenol and the cultured for additional 72 h, then injected to the myocardial infraction area like other groups. At 3 weeks after the operation, echocardiographic, hemodynamic, histological assessments and Western blot were performed to compare the CDCs differentiation degree and therapeutic effect. Isoproterenol treatment (10-6 M) simultaneously inhibited proliferation and induced apoptosis of CDCs, up-regulated proteins of vimentin, cTnT, α-sarcomeric actin and connexin 43, and down-regulated c-Kit proteins (all P<0.05). The echocardiographic and hemodynamic analysis demonstrated that the MI rats in the two CDCs transplantation groups had significantly better recovery of cardiac function than the MI group (all P<0.05). MI + ISO-CDC group had better recovery of cardiac function than the MI + CDC group, although the differences did not reach significant. Immunofluorescence staining showed that the MI + ISO-CDC group had more EdU-positive (proliferating) cells and cardiomyocytes in the infarct area than the MI + CDC group. MI + ISO-CDC group had significantly higher protein levels of c-Kit, CD31, cTnT, α-sarcomeric actin and α-SMA in the infarct area than the MI + CDC group. These results suggested that in CDCs transplantation, isoproterenol pre-treated CDCs can provide a better protective effect against MI than the untreated CDCs.

Epicardium-Derived Tbx18+ CDCs Transplantation Improve Heart Function in Infarcted Mice.

Cardiosphere-derived cells (CDCs) constitute a cardiac stem cell pool, a promising therapeutics in treating myocardial infarction (MI). However, the cell source of CDCs remains unclear. In this study, we isolated CDCs directly from adult mouse heart epicardium named primary epicardium-derived CDCs (pECDCs), which showed a different expression profile compared with primary epicardial cells (pEpiCs). Interestingly, pECDCs highly expressed T-box transcription factor 18 (Tbx18) and showed multipotent differentiation ability in vitro. Human telomerase reverse transcriptase (hTERT) transduction could inhibit aging-induced pECDCs apoptosis and differentiation, thus keeping a better proliferation capacity. Furthermore, immortalized epicardium CDCs (iECDCs) transplantation extensively promote cardiogenesis in the infracted mouse heart. This study demonstrated epicardium-derived CDCs that may derive from Tbx18+ EpiCs, which possess the therapeutic potential to be applied to cardiac repair and regeneration and suggest a new kind of CDCs with identified origination that may be followed in the developing and injured heart.

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HomeCirculation ResearchVol. 123, No. 10In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published25 Oct 2018https://doi.org/10.1161/RES.0000000000000238Circulation Research. 2018;123:1103is related toLack of Cardiac Improvement After Cardiosphere-Derived Cell Transplantation in Aging Mouse Heartsis related toTranscriptome Analysis Reveals Nonfoamy Rather Than Foamy Plaque Macrophages Are Proinflammatory in Atherosclerotic Murine ModelsMicrobial Transplantation With Human Gut Commensals Containing CutC Is Sufficient to Transmit Enhanced Platelet Reactivity and Thrombosis PotentialTranscriptome of Atherosclerotic Macrophages (p 1127)Download figureDownload PowerPointNonfoamy plaque macrophages are more proinflammatory than their foamy counterparts, report Kim et al.Atherosclerosis is characterized by the deposition of fat and the accumulation of immune cells such as macrophages in the vessel wall. These macrophages phagocytose the lipid particles and ultimately turn into fat-filled foam cells. Given this central role of macrophages in atherosclerosis, Kim and colleagues wanted to investigate their characteristics more thoroughly. To that end, they developed a new flow-cytometry technique that could distinguish between nonfoamy plaque macrophages (those that have accumulated little to no lipids) and fully lipid-laden foamy cells. They separated these two populations of macrophages by differences in their granularity and fluorescent labeling by the lipid probe BODIPY493/503, and compared their transcriptomes. They found that nonfoamy macrophages tended to express high levels of proinflammatory genes, while foamy macrophages had high levels of genes involved in lipid processing and as well as the resolution of inflammation. The team also confirmed the results obtained with mouse cells in human plaque macrophages, showing that the nonfoamy cell population was markedly proinflammatory.CDC Transplantation in Aging Mouse Hearts (p e21)Download figureDownload PowerPointCardiosphere-derived cells do not improve heart function in aged mice, say Zhao et al.Several clinical and preclinical studies are currently evaluating the potential of different cell types for promoting the repair and regeneration of injured and aged heart tissue. Of the candidate cells under consideration, cardiosphere-derived cells (CDCs), which are balls of cells that form during ex vivo culture of myocardial tissue, have been shown capable of differentiating into all three major cell types of the heart: cardiac myocytes, endothelium, and smooth muscle cells. Although recent work has shown that in aged rats CDCs can improve myocardial performance, Zhao and colleagues now report that this result is not reproducible in aged mice, where intramyocardial injections of CDCs provided no perceivable benefits over control treatments. To test the effects of CDCs, Zhao et al injected these cells in the left ventricles of nineteen 21-month-old mice. A separate group of 15 mice received control injections of phosphate-buffered saline. A month after these treatments, there was no difference between these two groups in terms of their left ventricle function, exercise capacity, or other markers of myocardial health. In view of the inconsistent results obtained with CDCs, the authors advise that further preclinical evaluation with these cells may be required to determine their efficacy.Microbial Transplantation Confers Thrombosis Risk (p 1164)Download figureDownload PowerPointGut microbes containing the gene cutC confer thrombosis risk, report Skye et al.Transfer of gut microbes from one organism to another can, in some cases, lead to the transfer of phenotypes, such as disease susceptibility. It is thought that the metabolites produced by the microbes somehow affect host physiology. Indeed, one such bacterial metabolite—trimethylamine N-oxide (TMAO)—has been linked to an increase in platelet reactivity, thrombosis risk, and cardiovascular disease risk in animal models and humans. The microbial gene responsible for the production of TMA (which gets converted to TMAO in the host liver) has been identified, but whether the presence of this gene—cutC—in human microbial specimens is sufficient to affect thrombosis has not been demonstrated. Skye and colleagues now show that mice that received an oral dose of a defined set of human gut microbes—all of which lacked cutC—did not exhibit increased platelet activation and thrombosis, while the addition of just one species containing cutC to that microbial set heightened platelet aggregation in the recipient mice. Together, these results provide further evidence that microbial TMA is linked to thrombosis risk and suggest that targeting cutC or TMA may be a novel avenue for preventing or treating cardiovascular disease. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesLack of Cardiac Improvement After Cardiosphere-Derived Cell Transplantation in Aging Mouse HeartsZhen-Ao Zhao, et al. Circulation Research. 2018;123:e21-e31Transcriptome Analysis Reveals Nonfoamy Rather Than Foamy Plaque Macrophages Are Proinflammatory in Atherosclerotic Murine ModelsKyeongdae Kim, et al. Circulation Research. 2018;123:1127-1142Microbial Transplantation With Human Gut Commensals Containing CutC Is Sufficient to Transmit Enhanced Platelet Reactivity and Thrombosis PotentialSarah M. Skye, et al. Circulation Research. 2018;123:1164-1176 October 26, 2018Vol 123, Issue 10 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000238 Originally publishedOctober 25, 2018 PDF download Advertisement

Lack of Cardiac Improvement After Cardiosphere-Derived Cell Transplantation in Aging Mouse Hearts.

Aging is one of the most significant risk factors for cardiovascular diseases, and the incidence of myocardial ischemia increases dramatically with age. Some studies have reported that cardiosphere-derived cells (CDCs) could benefit the injured heart. Nevertheless, the convincing evidence on CDC-induced improvement of aging heart is still limited. In this study, we tested whether the CDCs isolated from neonatal mice could benefit cardiac function in aging mice. We evaluated cardiac function of PBS- (n=15) and CDC-injected (n=19) aging mice. Echocardiography indicated that left ventricular (LV) ejection fraction (57.46%±3.57% versus 57.86%±2.44%) and LV fraction shortening (30.67%±2.41% versus 30.51%±1.78%) showed similar values in PBS- and CDC-injected mice. The diastolic wall thickness of LV was significantly increased after CDC injection, resulting in reduced diastolic LV volume. The pulse-wave Doppler and tissue Doppler imaging indicated that aging mice receiving PBS or CDC injection presented similar values of the peak early transmitral flow velocity, the peak late transmitral flow velocity, the ratio of the peak early transmitral flow velocity to the peak late transmitral flow velocity, and the ratio of the peak early transmitral flow velocity to the peak early diastolic mitral annular velocity, respectively. Pressure-volume loop experiment indicated that the LV end-diastolic pressure-volume relationship and end-systolic pressure-volume relationship were comparable in both PBS- and CDC-injected mice. Postmortem analysis of aging mouse hearts showed similar fibrotic degree in the 2 groups. In addition, the aging markers showed comparable expression levels in both PBS- and CDC-injected mice. The systemic aging performance measures, including exercise capacity, hair regrowth capacity, and inflammation, showed no significant improvement in CDC-injected mice. Finally, the telomere length was comparable between PBS- and CDC-injected mice. Together, these results indicate that CDCs do not improve heart function and systemic performances in aging mice.

Metformin promotes the survival of transplanted cardiosphere-derived cells thereby enhancing their therapeutic effect against myocardial infarction

BackgroundTransplantation of cardiosphere-derived cells (CDCs) has been shown to exert a therapeutic effect in patients with myocardial infarction (MI). However, poor survival of transplanted CDCs limits their beneficial effect. Metformin (MET) activates AMP-activated protein kinase (AMPK) which is associated with cell survival. The aim of this study is to determine whether MET improves CDC survival in the transplantation microenvironment and enhances the therapeutic effect of CDC transplantation against MI.MethodsCDCs were isolated and expanded from transgenic β-actin-GFP mice. CDCs were pretreated with MET and intramyocardially injected into wild-type C57 mouse heart with MI injury. The survival of CDCs was quantified, and the infarct size and cardiac function of treated hearts were evaluated.ResultsCDC transplantation modestly reduced infarct size and improved cardiac function in the post-MI heart, which was further improved by MET treatment. MET pretreatment significantly increased the survival of CDCs transplanted into the myocardium. MET also reduced CDC apoptosis induced by oxidative stress in vitro. The anti-apoptotic effect of MET was blocked by the AMPK inhibitor compound C. MET increased AMPK phosphorylation and upregulated endothelial nitric oxide synthase (eNOS) in CDCs under oxidative stress, which might be associated with the anti-apoptotic effect of MET.ConclusionsMET improves the survival of transplanted CDCs in the myocardium, thereby enhancing their therapeutic effect against MI injury. The pro-survival function of MET on CDCs might be associated with an AMPK-eNOS-dependent mechanism.

Allogeneic cardiosphere-derived cells for myocardial regeneration: current progress and recent results.

Early-phase clinical testing of autologous cardiosphere-derived cells (CDCs) has yielded intriguing results, consistent with therapeutic myocardial regeneration. However, autologous therapy is associated with significant technical, timing, economic and logistic constraints, prompting researchers to explore the potential of allogeneic CDC therapy. CDCs exhibit a favorable immunologic antigenic profile and are hypoimmunogenic in vitro. Preclinical studies in immunologically mismatched animals demonstrate that allogeneic CDC transplantation without immunosuppression is safe and produces sustained functional and structural benefits through stimulation of endogenous regenerative pathways. Currently, allogeneic human CDCs are being tested clinically in the ALLSTAR and DYNAMIC trials. Potential establishment of clinical safety and efficacy of allogeneic CDCs combined with generation of highly standardized, 'off-the-shelf' allogeneic cellular products would facilitate broad clinical adoption of cell therapy.

Therapeutic efficacy of cardiosphere-derived cells in a transgenic mouse model of non-ischaemic dilated cardiomyopathy.

Cardiosphere-derived cells (CDCs) produce regenerative effects in the post-infarct setting. However, it is unclear whether CDCs are beneficial in non-ischaemic dilated cardiomyopathy (DCM). We tested the effects of CDC transplantation in mice with cardiac-specific Gαq overexpression, which predictably develop progressive cardiac dilation and failure, with accelerated mortality. Wild-type mouse CDCs (10(5) cells) or vehicle only were injected intramyocardially in 6-, 8-, and 11-week-old Gαq mice. Cardiac function deteriorated in vehicle-treated mice over 3 months of follow-up, accompanied by oxidative stress, inflammation and adverse ventricular remodelling. In contrast, CDCs preserved cardiac function and volumes, improved survival, and promoted cardiomyogenesis while blunting Gαq-induced oxidative stress and inflammation in the heart. The mechanism of benefit is indirect, as long-term engraftment of transplanted cells is vanishingly low. Cardiosphere-derived cells reverse fundamental abnormalities in cell signalling, prevent adverse remodelling, and improve survival in a mouse model of DCM. The ability to impact favourably on disease progression in non-ischaemic heart failure heralds new potential therapeutic applications of CDCs.

Exosomes as Critical Agents of Cardiac Regeneration Triggered by Cell Therapy

SummaryThe CADUCEUS trial of cardiosphere-derived cells (CDCs) has shown that it may be possible to regenerate injured heart muscle previously thought to be permanently scarred. The mechanisms of benefit are known to be indirect, but the mediators have yet to be identified. Here we pinpoint exosomes secreted by human CDCs as critical agents of regeneration and cardioprotection. CDC exosomes inhibit apoptosis and promote proliferation of cardiomyocytes, while enhancing angiogenesis. Injection of exosomes into injured mouse hearts recapitulates the regenerative and functional effects produced by CDC transplantation, whereas inhibition of exosome production by CDCs blocks those benefits. CDC exosomes contain a distinctive complement of microRNAs, with particular enrichment of miR-146a. Selective administration of a miR-146a mimic reproduces some (but not all) of the benefits of CDC exosomes. The findings identify exosomes as key mediators of CDC-induced regeneration, while highlighting the potential utility of exosomes as cell-free therapeutic candidates.

Abstract 18790: Therapeutic Efficacy of Cardiosphere-Derived Cells in a Transgenic Mouse Model of Nonischemic Dilated Cardiomyopathy

Introduction: Cardiosphere-derived cells (CDCs) produce regenerative effects and improve function in the post-infarct setting. However, it is unclear whether CDCs are beneficial in non-ischemic dilated cardiomyopathy (DCM). We tested the effects of CDC transplantation in mice with cardiac-specific Gαq overexpression, which leads to intense myocardial oxidative/nitrosative stress and inflammation. These mice predictably develop progressive cardiac dilation and failure, with accelerated mortality. Methods and Results: A total of 71 mice were investigated in this study. Wild-type syngeneic mouse CDCs (10 5 cells total) or vehicle only were injected intramyocardially at five left ventricular sites in 6-, 8- and 11-week old Gαq mice. Cardiac function deteriorated in vehicle-treated mice over 3 months of follow-up, accompanied by oxidative stress, inflammation and adverse ventricular remodeling. Left ventricular (LV) ejection fraction and LV end-diastolic and end-systolic volumes were progressively worsened over 3 months observational period in vehicle-treated mice compared to CDC-treated group (p&lt;0.05). This was associated with upregulation of reactive oxygen species- and nitric oxide-producing enzymes [NAD(P)H oxidase and inducible nitric oxide synthase], accumulation of proinflammatory oxidant product, 3-Nitrotyrosine, intense infiltration of inflammatory cells [CD68 + macrophages and CD3 + T cells], increased apoptosis and activation of pathologic remodeling pathway, PKC-PKD-CREB. Concomitantly, marked collagen I and III deposition and fibrosis were observed in the vehicle-treated Gαq mouse heart. In contrast, CDCs preserved cardiac function and volumes (p&lt;0.05), and promoted cardiomyocyte cycling (p&lt;0.01) while blunting Gαq-induced oxidative stress, inflammation and fibrosis in the heart. Survival was enhanced by CDCs (p&lt;0.005) in Gαq mice that had been subjected to doxoribicin stress. Conclusions: CDCs reverse fundamental abnormalities in cell signaling, prevent adverse remodeling and improve survival in a mouse model of DCM. These findings motivate testing of CDCs as a potential therapeutic to prevent disease progression in patients with early-stage DCM.

Cellular Bioenergetics Is an Important Determinant of the Molecular Imaging Signal Derived From Luciferase and the Sodium-Iodide Symporter

Molecular imaging is useful for longitudinal assessment of engraftment. However, it is not known which factors, other than cell number, can influence the molecular imaging signal obtained from reporter genes. The effects of cell dissociation/suspension on cellular bioenergetics and the signal obtained by firefly luciferase and human sodium-iodide symporter labeling of cardiosphere-derived cells were investigated. (18)Fluorodeoxyglucose uptake, ATP levels, (99m)Tc-pertechnetate uptake, and bioluminescence were measured in vitro in adherent and suspended cardiosphere-derived cells. In vivo dual-isotope single-photon emission computed tomography/computed tomography imaging or bioluminescence imaging (BLI) was performed 1 hour and 24 hours after cardiosphere-derived cell transplantation. Single-photon emission computed tomography quantification was performed using a phantom for signal calibration. Cell loss between 1 hour and 24 hours after transplantation was quantified by quantitative polymerase chain reaction and ex vivo luciferase assay. Cell dissociation followed by suspension for 1 hour resulted in decreased glucose uptake, cellular ATP, (99m)Tc uptake, and BLI signal by 82%, 43%, 42%, and 44%, respectively, compared with adherent cells, in vitro. In vivo (99m)Tc uptake was significantly lower at 1 hour compared with 24 hours after cell transplantation in the noninfarct (P<0.001; n=3) and infarct (P<0.001; n=4) models, despite significant cell loss during this period. The in vivo BLI signal was significantly higher at 1 hour than at 24 hours (P<0.01), with the BLI signal being higher when cardiosphere-derived cells were suspended in glucose-containing medium compared with saline (PBS). Adhesion is an important determinant of cellular bioenergetics, (99m)Tc-pertechnetate uptake, and BLI signal. BLI and sodium-iodide symporter imaging may be useful for in vivo optimization of bioenergetics in transplanted cells.

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

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. 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. 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.

Constitutive HIF-1α Expression Blunts the Beneficial Effects of Cardiosphere-Derived Cell Therapy in the Heart by Altering Paracrine Factor Balance

Hypoxia-inducible factor-1alpha (HIF-1α) expression promotes angiogenesis and can influence stem cell engraftment. We investigated the effect of stable over-expression of constitutively active HIF-1α on cardiosphere-derived cell (CDC) engraftment and left ventricular function. CDCs were transduced with a lentivirus expressing a constitutively active mutant of human HIF-1α (LVHIF-1α). Two million male rat CDCs were injected into the infarct following ligation of the mid-LAD in female syngeneic rats. Left ventricular ejection fraction (EF) and circumferential strain were measured by echocardiography at 1 and 4 weeks post-MI in the following groups: PBS group (n = 7), CELL group (n = 7), and CELL-HIF group (n = 7). HIF-1α, VEGF, endothelin-1 expression, and CDC engraftment were measured by quantitative PCR. At 30 days, EF was unchanged in the CELL-HIF group (p = NS), increased in the CELL group (p = 0.025), and decreased in the PBS group (p = 0.021), but engraftment was similar (2.4% ± 3.3% vs 1.7% ± 0.8%, p = NS). Mean circumferential strain of the infarcted region was unchanged in the CELL-HIF group, but improved in the CELL group (p = 0.02). Endothelin-1 and VEGF expression were higher in HIF-CDCs exposed to hypoxia, compared with non-transduced CDCs. HIF-1α expression in CDCs blunted the beneficial functional effects of CDC transplantation, suggesting that paracrine factor balance may play an important role in cardiac regeneration.

Human Cardiospheres Are a Source of Stem Cells with Cardiomyogenic Potential

Several laboratories have shown that collections of adult cardiac stem cells can be grown directly from myocardial tissue.(1–7) Expanded cells are multipotent and clonogenic.(3–5) Spherical aggregates of cells grown from heart biopsies, termed cardiospheres, self-assemble in suspension culture and are enriched in stemness.(1, 6, 7) Andersen and colleagues, in work on neonatal murine heart tissue, used novel culture methods as a basis to question the cardiogenic potential of cardiosphere-cultured stem cells.(8) We contend that their sweeping conclusions are not applicable to studies using established methods (1, 2, 6, 7). To confirm the cardiogenic nature of human cardiosphere-derived cells (CDCs) in vivo, we evaluated the ability of CDCs to engraft and form new cardiomyocytes after transplantation into infarcted SCID mice. Immunostaining for human nuclear antigen (1:50, Chemicon MAB1281) and lentivirally-mediated GFP or beta-galactosidase labelling were used to track cells after injection. While the majority of CDCs could be found throughout the infarct (57±3% of the total engrafted) and the immediate border zone (30±5%), stable engraftment also existed in the remote myocardium (13±3%). Figure 1A and 1B show GFP-labelled CDCs within the border and infarct zones. Counterstaining for cardiac troponin I (cTnI; Chemicon MAB3150) demonstrates that many CDCs have differentiated into cardiomyocytes. As shown in Figure 1, portions of the mouse heart were reconstituted by human CDCs (Figure 1A), while CDC-derived cardiomyocytes in the dense central infarct zone remained small with little cTnI expression in their cytoplasm (Figure 1B). Human nuclear antigen expression also demonstrates differentiated cardiomyocytes, derived from transplanted human CDCs, within the border zone (Figure 1C). These human-derived cells express Cx43, suggesting, but not proving, functional integration within the infarcted tissue (Figure 1D). Such functional integration has, however, been rigorously documented in vitro with myocyte-CDC co-culture.(7) Further, beta-galactosidase-labelled CDCs expressing markers of endothelial (vWF, Figure 1E) and smooth muscle (αSMA; Figure 1F) lineages highlight the in vivo multilineage potential of CDCs. Figure 1 Cardiogenic differentiation of transplanted cells cultured from humans biopsies using the cardiosphere method. Human CDCs were injected into SCID mice at the time of myocardial infarction. (A): Example images of the infarct border zone demonstrating engraftment ... These results challenge the conclusions of Andersen et al. by demonstrating in vivo cardiomyogenesis of transplanted cells cultured using established cardiosphere methods. Our lab and others have shown that CDCs also decrease infarct size and improve myocardial function(1, 7), with the different sub-populations within CDCs acting synergistically to improve myocardial function.(9) Although cardiac differentiation of transplanted CDCs does occur consistently, long-term persistence of transplanted CDCs is relatively low (10), with important contributions of paracrine effects (both tissue preservation and recruitment of endogenous regeneration) to the functional benefit of CDC transplantation.(11). In this light, Andersen and colleagues(8) may have overstated the relevance of their findings, considering that their work made use of several unprecedented experimental procedures with novel experimental findings that occurred exclusively when neonatal rat heart was used as a tissue source. These investigators stored the myocardial tissue prior to processing in a novel buffer, potentially damaging the very cells of interest; performed prolonged enzymatic digestion to collect cells from explant culture, possibly favoring the survival of contaminating cells; and finally, failed to characterize their cells in vivo.(12) If the authors seek to convincingly critique the cardiosphere approach, they would be well-advised to reproduce established methods. As such, their work is unrelated to adult cardiosphere culture and, as performed, provides no justification for the indictment of the clinical utility of cardiospheres or CDCs, which are already being tested in human subjects. (13)