A Strong Regenerative Ability of Cardiac Stem Cells Derived From Neonatal Hearts

Abstract

Background

Human stem cells (CSCs) promote myocardial regeneration in adult ischemic myocardium. The regenerative capacity of CSCs in the very young patients with non-ischemic congenital heart defects has not been explored. We hypothesized that isolated neonatal-derived CSCs may have a higher regenerative ability than adult-derived CSCs and might address the structural deficiencies of congenital heart disease.

Methods and Results

Human specimens were obtained during routine cardiac surgical procedures from right atrial appendage tissue discarded from two age groups: neonates and adults patients. We developed a reproducible isolation method that generated cardiosphere derived cells (CDCs), regardless of starting tissue weight or age. Neonatal-derived CDCs demonstrated increased number of cardiac progenitor cells expressing c-kit⁺, flk-1 and Islet-1 by flow cytometry and immunofluorescence. When transplanted into infarcted myocardium, neonatal-derived CDCs had a significantly higher ability to preserve myocardial function, prevent adverse remodeling and enhance blood vessel preservation and/or formation when compared to adult CDCs. Lastly, neonatal-derived CDCs were more cardiomyogenic than adult CDCs when co-cultured with neonatal cardiomyocytes and displayed enhanced angiogenic function compared to adult CDCs.

Conclusions

Neonatal-derived CDCs have a strong regenerative ability when compared to adult-derived CDCs that may depend on angiogenic cytokines and an increase prevalence of stem cells. This has important implications in the potential use of CDCs in future clinical trials.

INTRODUCTION

The identification and application of resident cardiac stem cells for regeneration of damaged myocardium challenges the previous dogma of a terminally differentiated, non-repairable heart. Recent human Phase I clinical trials in adult myocardial ischemic patients have demonstrated the beneficial regenerative abilities of two different resident cardiac stem cells: c-kit⁺ cardiac stem cells and cardiosphere derived cells (CDCs).^{1, 2} C-kit⁺ cells, which express the surface receptor tyrosine kinase, are self-renewing, clonogenic, and multipotent with the ability to differentiate into cardiomyocytes, smooth muscles, and endothelial cells.^{3, 4} In the cardiac stem cells in patients with ischemic cardiomyopathy (SCIPIO) trial, c-kit⁺ cell treatment in adult heart failure patients improved left ventricle function and reduced infarct size 12 months post-treatment. On the other hand, CDCs contain a heterogeneous pool of differentiated and undifferentiated cells which include c-kit⁺ cells.⁵ CDCs similarly differentiate into all three cardiac lineage cells as seen in animal cardiomyopathy models.^6–8 In the cardiosphere-derived autologous stem cells to reverse ventricular dysfunction (CADUCEUS ) trial, treatment with CDCs in adult ischemic patients decreased infarct size and increased myocardial mass. However, there was little effect on left ventricular ejection fraction. Even though these results are clinically promising, the number of patients in both treatment arms was small. Thus, larger, more appropriately powered studies are needed to verify the efficacy of these studies.

While adults with cardiac failure often have ischemic cardiomyopathy, pediatric cardiac heart failure is more varied and includes a spectrum of cardiomyopathies, congenital cardiac diseases, and arrthymias.⁹ The etiologies of heart failure in children may extend beyond myocardial ischemia to include volume or pressure overload to the systemic ventricle that may include both ventricles.⁹ Similar to adults, the prevalence of heart failure in children is on the rise during the last decade and the prognosis for patients who are admitted to the hospital with heart failure remains poor.¹⁰ Neonates with heart failure are the most medically and surgically challenging and have the highest congenital mortality rates.¹¹ Current medical therapies or devices do not address the fundamental loss or deconditioning of cardiac tissue in pediatric heart failure patients. To address this issue, resident cardiac stem cell therapy may be a strategy that regenerates the damaged myocardium by generating viable myocardium or by improving the myocardial environment through secreted growth factors. Cardiac cellular therapy may be tested either as a stand-alone cellular injection into the systemic ventricle or as an adjunct to the surgical reconstruction repair that may be planned for these patients, such as the Norwood or superior cavopulmonary connection for hypoplastic left heart syndrome (HLHS) patients.

Previously, we have shown that c-kit⁺ cardiac stem cells are most abundant in the neonatal stage and subsequently decrease in prevalence with advancing age in congenital heart patients.⁷ We further demonstrated that human CDCs from congenital heart patients have cardiac regenerative abilities. Not addressed however, was whether the regenerative abilities of CDCs derived from neonates differed from CDCs derived from adults. Given the recent results of CADUCEUS trial, the mechanism(s) of the regenerative capacity of hCDCs is unclear and further studies are required to identify the characteristics of hCDCs that may lead to an improvement of left ventricular function. For instance, it is unclear whether age of the heart may play a role in the function of the hCDCs. Thus, we hypothesized that neonatal-derived CDCs would demonstrate an augmented regenerative capacity when compared to adult-derived CDCs because of a more abundant cardiac stem cell pool in neonatal hearts. In this study, CDCs were isolated from human neonates and adults and were examined for stem cell and cardiac lineage markers. Additionally, the regenerative capacity of the CDCs was tested in an immunodeficient rodent model of myocardial infarction. The overall goal of this work was to provide essential and practical data required for using neonatal-derived CDCs in future clinical trials.

MATERIALS and METHODS

Acquisition of Human Tissue Samples and Cell Culture

This study was approved by the Institutional Review Committee at Children’s Memorial Hospital. After parental consent was given, specimens (70±80 mg) from the right atrial (RA) appendage were obtained from neonate (n=43) and adult patients (n=13) during routine cardiac surgeries. The neonatal patients varied in diagnosis (Supplementary Table I). All tissue samples were processed for immunostaining and harvesting of human cardiac progenitor cells (CPCs) via cardiosphere development. Human CDCs were generated through the use of the protocol described by Smith et al. with modifications (See expanded methods in supplement).⁷

Flow Cytometry Analysis of CDCs

CDCs at P1 were evaluated by flow cytometry using a Becton-Dickinson FACS caliber (San Jose, CA) with 10,000 events collected. Cells were incubated with fluorochrome-conjugated primary antibodies against c-kit⁺, flk1, NKX2-5, Ki67⁺, Sca1⁺, Cardiac Troponin T (cTnT), CD105 and antibodies against hematopoietic lineage surface marker CD34 and CD45. Isotype controls were run for each immuno-subtype.

Infarct Model

Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending (LAD) coronary artery in immunodeficient male rats (250–300 g). The heart was exposed via a left thoracotomy, and the proximal LAD was ligated. Afterwards, 1 million neonate or adult CDCs or cardiac fibroblasts suspended in 250–400 μL of vehicle were, injected at multiple sites. Rats with induced infarction and without cell injection or rats injected with cardiac fibroblasts served as controls for the study.

Echocardiography

Transthoracic echocardiograms were performed on rats using a VisualSonics Vevo 770 ultrasound unit (VisualSonics, Toronto, Canada). Baseline echocardiograms were acquired at 1day before myocardial infarct surgery. Echocardiographic examinations were also performed at 7 days post-MI with additional echocardiograms acquired at 4 weeks post-MI. Two-dimensional and M-mode echocardiography were used to assess fractional area change (FAC). Images were obtained from the parasternal long axis and the parasternal short axis at the mid-papillary level.

Myocardial Histology

Hearts were excised under anesthesia, perfused with 4% paraformaldehyde, cryo-protected and then embedded. Sections were cut to 7 μm using a commercial cyrostat and used for isolectin B4 (Invitrogen; Carlsbad, CA), cardiac troponin T (Santa Cruz Biotechnology; Santa Cruz, CA) and anti-human nuclei (Millipore; Billerica, MA) staining. Sections were counterstained with nuclear DAPI (4′,6-diamidino-2-phenylindole) stain (Sigma; St. Louis, MO). To calculate infarct size, at least four Masson’s Trichrome stained sections at various levels along the long axis were analyzed for collagen deposition. The midline technique for infarct size determination was used as described previously¹².

Real Time RT-PCR

RNA was isolated from cardiac fibroblast and CDCs using a commercial RNeasy kit (Qiagen; Valencia, CA). Real Time PCR was run using a total of 5 ng. template cDNA for each sample. Each sample was run in duplicate using ABI FAST SYBR green supermix (Applied Biosystems; Foster City, CA) for multiple genes including: Angiogenin (ANG), Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor-1 (FGF-1), Fibroblast Growth Factor-2 (FGF-1), Platelet Derived Growth Factor-B (PDGF-B), Insulin-like Growth Factor (IGF-1) and ribosomal protein 13A (RPL13A). Relative RNA abundance was calculated using the following equation: 2^−ΔΔcT.

Assessment of CDC Differentiation Potential

A co-culture system using neonatal cardiomyocytes and either neonate or adult CDCs was used to determine cardiomyogenic potential. Briefly, neonatal rats were sacrificed and cardiomyocytes isolated using a commercial kit (Worthington Biomedical Corp, Lakewood, NJ). Cells were seeded at a density of 50,000 cells per well of an eight well chamber slide and after 24 hours, 5,000 neonate or adult CDCs were added for an additional 14 days. Differentiation of CDCs toward cardiac lineages was assessed using fluorescence microscopy by staining cells for cTnT, human mitochondria (Millipore; Billerica, MA) and a nuclear DAPI (4′,6-diamidino-2-phenylindole) stain (Sigma; St. Louis, MO).

Statistical Analysis and Interpretation

A one-way ANOVA with appropriate post-hoc testing was used for the interpretation of in vivo and PCR data sets. A p value less than 0.05 indicated statistical significance. A Student’s t-test was used for histological data sets.

RESULTS

Characterization of CDCs derived from Neonate and Adult Cardiac Biopsies

CSCs were isolated and expanded from right atrial (RA) tissue using a modified version of the cardiosphere method. RA heart samples were taken from neonates (n=43) and adults (n=13) and ranged in weight size from 25 to 200 mg. Immunostaining of intact cardiospheres identified cells that expressed c-kit⁺, NKX2-5, GATA-4, and TnI (Figure 1A–H). The c-kit⁺ cells were located in the inner and outer core of the cardiopshere. The cardiosphere expression pattern for GATA-4, Islet-1 (ISL1) and TnI was similarly varied throughout the cardiosphere. We saw no difference in the expression of these markers in cardiospheres derived from neonates or adults.

Figure 1.

Generation and characterization of cardiospheres derived from neonatal patients and adult patients. Intact cardiospheres were sectioned and stained for c-kit⁺ (A), NKX2-5 (B), serial staining with c-kit+ and NKX2-5 (C), GATA4 (D), cTNI (E), and serial staining with GATA4 and cTNI (F). (Nuclei are shown in blue, Magnification 10x). Generally, there was no difference in expression levels and localization between neonatal and adult cardiospheres.

From these cardiospheres, CDCs could be expanded beyond 30 days and yielded greater than 2×10⁶ cells. Even neonatal right atrial samples weighing between 20 to 40 mgs generated similar cell numbers using the current CDC modifications. CDCs were examined for their expression of stemness markers and cardiac lineage commitment markers to determine whether the expanded CDCs remained in an uncommitted state or differentiated state. Neonatal-derived CDCs displayed statistical significantly higher levels of c-kit⁺ and flk-1 compared to adults (c-kit⁺, n=4 in each group: 22.6 ± 6.1 vs. 4.9 ± 3%, p=0.04; flk-1, n=3 in each group: 26.2 ± 5.1 vs. 7.7 ± 3.4%, p = 0.04, Fig 2A). Since the ISL1 antibody was not conducive for flow cytometric analysis, ISL1 expression was determined by quantitative immunofluorescence which showed a statistically significant increase of ISL1 cells when compared to adults (36.6 ± 1.5 vs. 3.2 ± 1.9%, p = 0.0001, n=3, Fig 2B). Differences did not reach statistical significance in other lineage and commitment markers: Sca-1, CD105, CD31, Ki67 and NKX2-5 (supplementary Table 2). These results indicated that the neonatal-derived CDCs had a higher prevalence of cardiac progenitor cells in comparison to adult CDCs.

Figure 2.

Phenotypic characterization of CDCs isolated from neonatal and adult cardiac biopsies. A) CDCs derived from neonatal cardiac biopsies display significantly upregulation expression of c-kit and flk-1 by flow cytometry compared to adult CDCs (c-kit⁺, n=4: 22.6 ± 6.1 vs. 4.9 ± 3%; flk-1, n=3: 26.2 ± 5.1 vs. 7.7 ± 3.4%, p = 0.04 for both markers). Other phenotypic markers including: SCA-1, CD105, CD31, Ki67 and cTNT showed no change. B and C) ISL1 expression was determined by immunoflouresence between neonatal and adult CDCs. Neonatal CDCs showed augmented nuclear localized ISL1 expression compared to adult CDCs (36.6 ± 1.5 vs. 3.2 ± 1.9%, p = 0.0001, n=3).

CDCs Derived from Neonates Display Augmented Regenerative Potential

To determine whether the increased number of cells expressing c-kit⁺, flk-1, and ISL1 in neonatal-derived CDCs correlated with improved regenerative potential, two sets of experiments were performed. First, CDCs co-cultured with rat neonatal cardiomyocytes were used to quantify cardiomyogenic potential. After two weeks of co-culturing, there was a threefold increase in cTnT expressing cardiomyocytes derived from neonatal-derived CDCs compared to adults (Figure 3C;neonate 3.4 ± 0.7 vs adult 0.9 ± 0.5 cells per total, p = 0.04). Positive cells were generally found surrounding dense neonatal cardiomyocyte clusters (Figure 3A–B). These results indicated the robust cardiomyogenic potential of the neonatal-derived CDCs in vitro that may contribute to their eventual functionality in vivo.

Figure 3.

The efficacy of CDC differentiation into cardiomyocytes in vitro. Adult (A) and neonatal (B) CDCs were co-cultured with rat neonatal cardiomyocytes for 14 days to induce cardiomyogenesis. Sections were stained with a human specific mitochondria (green) and cTNT (red) to depict CDC differentiation into mature cardiomocytes (nuclei are blue; Magnification 40x). Neonatal CDCs demonstrated an increased capacity for differentiation compared to adult CDCs (Neo 3.4 ± 0.7 vs Adult 0.9 ± 0.5 cells per total, p = 0.04, n=3) as evidenced by an increased number of structurally competent cardiac cells expressing human mitochondria.

The second experiment involved an observer blinded study to determine the regenerative potential of transplanted CDCs within acutely infarcted myocardium of immunodeficient rats. Soon after the left anterior descending (LAD) artery was ligated, CDCs, human fibroblast cells or Iscove’s Modified Dulbeccos’s Medium (IMDM; control) were injected into the peri-infarct or infarct regions of the left ventricle. The baseline left ventricular ejection fraction calculated by fractional area change (FAC), were similar among all rats receiving CDCs, cardiac fibroblasts, or IMDM (Figure 4). Echocardiography performed at 7 days revealed a higher FAC in hearts receiving neonatal-derived CDCs than in those receiving adult-derived CDCs, cardiac fibroblasts or controls (neonate, n =4: 56.4 ± 1.4% vs adult, n=4: 39.5 ± 3.6%, p<0.001; cardiac fibroblast, n=4: 32.9 ± 4.6%, p<0.01 and control, n=5: 30.6 ± 3.1%, p < 0.01). Differences amongst adult-derived CDCs, cardiac fibroblasts or control treated hearts did not reach statistical significance, but there was a trend for more functional recovery in the adult-derived CDCs. The improved left ventricular function was maintained from day 7 to 28 and represented the sustained effect of the neonatal-derived CDCs (neonate, n =4: 53.9 ± 5.1% vs. adult, n=4: 35.8 ± 3.4%, p<0.05; cardiac fibroblast, n=5: 29.9 ± 4%, p<0.05 and control, n=5: 30 ± 1.9%, p < 0.05).

Figure 4.

Neonatal CDCs preserved cardiac function after myocardial infarction in immunodeficient rats were treated with either control, cardiac fibroblast, neonatal CDCs or adult CDCs. Echocardiographic anaylsis was performed pre-surgically and post-surgically at day 7 and day 28. A) Left ventricle blood pool fractional area change in diastole (FAC, ejection fraction) was significantly improved in infarcted hearts treated with neonatal CDCs compared to control, cardiac fibroblast and adult CDC groups at both day 7 and day 28. Baseline echocardiograms showed no pre-surgical variation amongst the four groups (Day 7: Neonate, n =4: 56.4 ± 1.4% vs Adult, n=4: 39.5 ± 3.6%; Cardiac Fibroblast, n=4: 32.9 ± 4.6% and Control, n=5: 30.6 ± 3.1%; *p < 0.001, **p < 0.01; Day 28: Neonate, n =4: 53.9 ± 5.1% vs Adult, n=4: 35.8 ± 3.4%; Cardiac Fibroblast, n=5: 29.9 ± 4% and Control, n=5: 30 ± 1.9%; *p < 0.05). B) Infarct size was improved following treatment with neonate CDCs compared to control four weeks after infarction (Control, n=4: 44.4 ± 1.9, Cardiac Fibroblasts, n=3: 40.9 ± 3.4, Neonatal, n=4: 23.7 ± 4.9, Adult, n=4: 34 ± 2.7, **p < 0.01 control vs. neonate). There was no significant prevention of infarct expansion using adult CDCs as a therapeutic platform.

We further examined the impact of CDCs on the infarcted myocardium by examining myocardial sections via histology analysis after 28 days of transplantation. By tracking the CDCs with a human nuclear marker, we observed modest engraftment (Figure 5C; neonate, n=0.2: 3.6 ± 1.5 vs adult, n=4: 1.5 ± 0.4 CDCs per total per field, p = 0.04) of CDCs in the peri-infarct and infarct regions (Figure 5A–B). Immunophenotypic characterization revealed that the engrafted neonatal–derived CDCs stained positive for the cTnT and demonstrated structural maturation. Although cardiac differentiation occurred infrequently, these results suggested that part of the regenerative potential of neonatal-derived CDCs may be contributed to the differentiation of these cells to working cardiomyocytes (Figure 5D). We did not find any adult-derived CDCs that differentiated into mature cardiomyocytes staining positive for cTnT.

Figure 5.

The efficacy of CDC differentiation to cardiomyocytes in vivo. A) Neonate or B) adult CDCs engraftment at 4 weeks after transplantation in a immuodeficient rat model of MI. CDCs were tracked using human anti-nuclei (green) and co-stained with cTnT (red) to detect differentiation into cardiomyocytes. C) Generally there was a trend for increased engraftment/survival of neonatal CDCs compared to adult CDCs, however. D) Only CDCs derived from neonates demonstrated a tendency to differentiate into a typical cardiomyocytes phenotype as demonstrated by broad cTNT expression and formation of striations. We did not find any adult CDCs with this phenotype (Magnification 20x)

To further assess the relative roles of direct differentiation, we measured bioluminescence in mice that were injected with luciferase-labeled CDCs. This method was able to actively track the luciferase-labeled CDCs during various time intervals post injection to provide a qualitative measure of transplant cell survival. After one week, the luciferase signal had dropped by approximately 30% of that on day one and by three weeks, little to no detectable signal was determined (Supplementary Figure S1). Similar results have been previously reported using this same luciferase methodology but using different patient population derived CDCs. ^{13, 14} Even though this method is nonquantitative and prone to limitations by tissue attenuation and reporter gene silencing, this data suggests that CDCs are most likely noncontributory after 3–4 weeks post transplantation.

Additionally, we quantified regeneration by staining sections with Masson’s Trichrome to determine the extent of infarct expansion after LAD ligation. A typical Masson’s Trichrome staining pattern in hearts transplanted with control, cardiac fibroblast or CDCs is shown in Figure 4. Positive red-stained regions (viable tissue) within the predominately blue-stained (fibrous tissue) infarct zone are evident in all hearts transplanted. A trend for reduced infarct expansion was noticed with the neonatal-derived CDCs in comparison to the adult-derived CDCs and cardiac fibroblasts. Additionally, neonatal-derived CDCs transplanted hearts had a smaller infarct size within the infarct zone than hearts treated with control media (control, n=4: 44.4 ± 1.9 cardiac fibroblasts, n=3: 40.9 ± 3.4, neonatal, n=4: 23.7 ± 4.9, adult, n=4: 34 ± 2.7, p < 0.01 control vs. neonate). Taken together, these data indicated that neonatal-derived CDCs resulted in enhanced functional recovery and more favorable remodeling in the infarcted myocardium than adult-derived CDCs.

Differential Secretion of Angiogenic Factors may Account for Improved Regenerative Potential

To determine if neonatal-derived CDCs secreted different angiogenic factors in comparison to adult-derived CDCs and cardiac fibroblasts, cells were cultured under hypoxic conditions to simulate ischemia and RT-PCR was performed to assess the secretion profiles. VEGF (cardiac fibroblasts: 0.04 ± 0.004, neonatal: 0.6 ± 0.14, adult: 0.2 ± 0.06, p = 0.04) and ANG (cardiac fibroblasts: 0.11 ± 0.01, neonatal: 0.61 ± 0.04, adult: 0.22 ± 0.03, p = 0.04) show statistical differences that suggests augmented secretion of pro-angiogenic factors in neonatal-derived CDCs (Fig 6A–B, n=3 for all) IGF-1 and FGF-1and showed no change (Figure 6C–F). FGF-2 in neonatal-derived CDCs was only enhanced compared to cardiac fibroblast was but differences did not reach statistical significance compared to adult-derived CDCs (cardiac fibroblasts: 0.13 ± 0.02, neonatal: 1.42 ± 0.56, adult: 0.5 ± 0.16, *p < 0.05). To confirm these results in vivo, immunofluorescence for blood vessel preservation and/or formation in infarcted hearts treated with neonatal-derived CDCs or adult-derived CDCs was performed using isolectin B4. Hearts treated with neonatal-derived CDCs displayed higher levels of blood vessel preservation and/or formation (neonatal: 8.5 ± 0.04 vs. adult: 4.8 ± 0.5, p = 0.04, n = 4; Figure 6G ). Blood vessels were generally found in the peri-infarct wall, though fewer blood vessels were also found spanning the infarct within the lateral wall (Figure Figure 6H–I).

Figure 6.

Enhanced angiogenic potential of CDCs derived from neonates. To determine whether CDCs derived for neonatal paitients demonstrate an enhanced angiogenic secretion profile, expression levels of several trophic factors were analyzed using RT-PCR. CDCs derived from neonatal cardiac biopsies display upregulated expression of (A) ANG and (B)VEGF by RT-PCR compared to adult CDCs and cardiac fibroblasts (VEGF-CFb: 0.04 ± 0.004, Neonatal: 0.6 ± 0.14, Adult: 0.2 ± 0.06, *p < 0.05 and **p < 0.01) and ANG-CFb: 0.11 ± 0.01, Neonatal: 0.61 ± 0.04, Adult: 0.22 ± 0.03, *p < 0.05 and ** p < 0.01). mRNA expression levels for other factors including: (C) IFG-1, (D) PDGF-B and (E) FGF-1 show no change. (F) FGF-2 in neonatal CDCs was only enhanced compared to cardiac fibroblast was but differences between neonate and adult CDCs did not reach statistical significance (CFb: 0.13 ± 0.02 Neonatal: 1.42 ± 0.56, Adult: 0.5 ± 0.16, *p < 0.05). To determine if the augmentation of pro-angiogenic factors translated to increase blood vessel preservation and/or formation, immuoflouresence was performed using Isolectin B4 (red; Magnification 20x). Infarcted hearts treated with (H) neonatal CDCs displayed a significantly higher level of blood vessel preservation and/or formation compared to (I) adult CDCs (Neonatal: 8.5 ± 0.04 vs. Adult: 4.8 ± 0.5, *p = 0.04, n = 4)

Discussion

Our results suggest that neonatal-derived CDCs can be reproducibly isolated and expanded from as little as 20 mgs of myocardial tissue, which can be harvested during routine pediatric cardiac surgery. Neonatal-derived CDCs has a higher prevalence of different cardiac stem cells in comparison to adult-derived CSDs. Additionally, the transplanted neonatal-derived CDCs can significantly improve cardiac function in comparison to adult-derived CDCs when tested in immunodeficient in a rodent model of myocardial infarction. The cardiac regeneration observed with neonatal-derived CDCs may be due to differentiation into functional cardiomyocytes or may be due to increased blood vessel preservation and/or formation resulting from secreted angiogenic factors. These novel findings of human CDCs support the strong regenerative ability of neonatal-derived CDCs, a potentially new therapy for congenital heart failure patients. Equally important is the observation that all CDCs may not be equivalent in their functional ability and may need to be optimized depending on patient age.

The heterogeneity of CDCs may be particularly favorable for cardiovascular applications because they contain uncommitted stem cells normally residing within the heart.⁵ In our study, neonatal-derived CDCs displayed an increased prevalence of c-kit⁺, flk-1 and ISL1 stem cells when compared to adult-derived CDCs. C-kit⁺ and ISL1 expressing stem cells have been extensively characterized in the heart, but it is still unclear whether these cells represent distinct categories of undifferentiated cells with unique functional activities.^{3, 15} The increased prevalence of cardiac stem cells observed in our study may be explained by our previous work that demonstrated higher number of stem cells in neonates. ⁷ These types of stem cells have been shown to replenish the population of cardiomyocytes and cardiac vascular cells that die during a heart’s lifetime.¹⁶ Furthermore neonatal-derived CDCs have the unique ability to secrete variable growth factors such as VEGF and angiogenin. Both of these factors are highly pro-angiogenic and may be involved in restricting adverse remodeling and cardiomyopathic progression via blood vessel preservation and/or formation. The more pro-angiogenic secretion profile of neonatal-derived CDCs compared to adult-derived CDCs may be due to differences in CDC phenotypes and stem cell numbers. Understanding the molecular basis of this ability may have important implications on how to further improve the activity of adult-derived CDCs. Overall, these neonatal-derived CDCs are highly regenerative.

Implications for Pediatric Heart Failure

To date, there has been no direct comparison between the regenerative abilities of neonatal and adult derived-human stem cells. Recently, rodent cardiac c-kit⁺ cells derived from neonates have shown an increased cardiomyogenic potential than adult derived c-kit⁺ cells but the underlying molecular basis at this time is unknown.¹⁷ Additionally, two relevant clinical trials offer insight into cell based therapy in human adult ischemic patients. The SCIPIO trial demonstrated that the delivery of a pure population of c-kit⁺, lineage-negative cardiac stem cells to adult patients with myocardial infarction augment ejection fraction (12.3%) and decreased infarct size (30%) over 12 months. The CADUCEUS trial reported their Phase I clinical results in ischemic adult patients using CDCs and demonstrated decreased scar formation by 30 to 70% and increased left ventricle mass. Unfortunately, this finding did not result in any statistically significant change in ejection fraction with the treatment of adult-derived CDCs. The results of these two trials suggest that c-kit⁺ cells may play an important role in the augmentation of cardiac function, which may explain the differences we noticed with neonatal-derived CDCs where there was a higher prevalence of cardiac stem cells, with c-kit⁺ cells being one of them. Our results also suggest that all derived CDCs are not equivalent in their functional ability to improve LV ejection fraction. One explanation is that neonatal-derived hCDCs releases more VEGF and angiogenin which triggers more blood vessel formation and/or preservation when compared to adult-derived CDCs.^{18, 19} Lastly, neonatal-derived CDCs may stimulate regeneration at multiple levels which include preventing apoptosis¹⁸, secreting trophic factors²⁰, and stimulating the endogenous cardiac stem cell pool^{21, 22}. Despite the lack of more direct cardiomyocyte differentiation, CDCs may improve regeneration at multiple levels that need further exploration.

The goal of cellular therapy for pediatric heart failure patients is to regenerate lost or dysfunctional myocardium. In contrast to adults, pediatric heart failure etiologies include a variety of conditions that encompass ischemia, cardiomyopathies, and congenital lesions. ⁹ Since neonates are the most medically and surgically challenging and carry the highest surgical mortality rates, we have focused on generating clinical protocols that have focused mainly on hypoplastic left heart syndrome (HLHS) patients. The physiology of HLHS is dominated by the absence of an anatomic left ventricle such that the morphologic right ventricle provides the entire cardiac output, which ultimately may lead to right ventricular dysfunction.¹¹ The etiology of right ventricular dysfunction is multi-factorial, including pressure and volume overload, intrinsic right ventricle muscle dysfunction, and myocardial ischemia. The unique ability of neonatal-derived CDCs to function at multiple levels, including generating myocardium and releasing angiogenic factors, may make these cells ideal to treat HLHS patients. Additionally, CDCs can be frozen for future use allowing repeat injections when cardiac dysfunction may arise later in time.

Conclusion

The strong regenerative ability of neonatal-derived CDCs warrants further investigation as a treatment for pediatric heart failure patients. Given the ease and expandability of these cells from right atrial tissue, the use of neonatal-derived CDCs may provide an attractive cellular based therapy either as a stand-alone intervention or as an adjunct to surgical repair for congenital heart patients.