Repeated Administrations of Cardiac Progenitor Cells Are Markedly More Effective Than a Single Administration: A New Paradigm in Cell Therapy

Isolation, culture, and transfection of c-kit^POS CPCs

CPCs were isolated from the entire heart (atria and ventricles) of adult male Fischer 344 rats as described⁴ and phenotyped by FACS analysis of fixed cells. The cells were injected into rat hearts at passages 6 to 8. The rationale for using male donor CPCs was that the fate of transplanted CPCs can be tracked by detecting Y-chromosome^pos cells, although previous studies have suggested that bone marrow-derived mesenchymal stromal cells differ significantly between male and female sources, with female cells usually performing more robustly.³⁷

Surgical procedures, treatment protocol, and preliminary studies

The rat model of old MI has been described previously.⁶ Adult female Fischer 344 rats (age, 4 months; weight 175 ± 2 g) underwent a 2-h occlusion of the left anterior descending coronary artery followed by reperfusion. Thirty days after surgery, the animals were randomly allocated to three treatment groups: vehicle (control), single dose, or multiple doses (Fig. 1). Randomization was performed using the MS Excel random group generator.

All rats received an echo-guided intraventricular injection, which was performed using the Vevo 2100 Imaging System (VisualSonics) equipped with a 20-MHz transducer, a Vevo Image Station with Injection Mount and micro-manipulation controls, and a Chemyx NanoJet Stereotaxic syringe pump (Chemyx Inc. TX). Before the procedure, rats were anesthetized with 3% isoflurane. The anterior chest was shaved and the animals were placed on the imaging table in the right lateral decubitus position with the left lateral side facing the injection mount. Light anesthesia was maintained with 1% isoflurane. With the imaging transducer aligned perpendicularly to the injection mount, the left ventricle was initially imaged in the parasternal plane and a good 2D long-axis view was procured by adjusting the angle and position of the imaging table. The transducer was then turned 90 degrees clockwise and the left ventricle was scanned in the 2D short-axis and color Doppler views from apex to base to determine the optimal site for needle insertion, a site that did not include the infarct scar or a coronary artery. Under the guidance of a real-time B-mode view, a 30 G injection needle (1” length) was then carefully inserted from the left lateral side using the micro-manipulation controls and advanced into the center of the LV cavity.

Rats received an infusion of CPCs (12 × 10⁶ cells in 5 ml of DPBS (Dulbecco's phosphate-buffered saline, Gibco) or vehicle (5 ml of DPBS) into the LV cavity with the injection pump at a rate of 1.25 ml/min for 4 min. The single-dose and multiple-dose groups received CPCs whereas the vehicle group received DPBS. Thirty-five days later, this procedure was repeated; rats received a second infusion of CPCs (multiple-dose group) or an infusion of DPBS (single-dose and vehicle groups). Thirty-five days later, rats received a third infusion of CPCs (multiple-dose group) or another infusion of DPBS (single-dose and vehicle groups). mCherry-labeled CPCs were used for the 1^st treatment, non-labeled CPCs for the 2^nd treatment, and GFP-labeled CPCs for the 3^rd treatment. To monitor formation of new cells, in all groups 5-bromo-2’-deoxyuridine (BrdU, Sigma) was given for 35 days after the 1^st treatment and 5-iodo-2’-deoxyuridine (IdU, Sigma) for 35 days after the 3^rd treatment (both were given in the drinking water at a final concentration of 0.1%). At 35 days after the 3^rd treatment, rats were subjected to hemodynamic studies and euthanized for histologic studies or PCR analysis (Fig. 1).

Before initiating the protocol, pilot studies were conducted to identify a dose of CPCs that would results in LV myocardial retention similar to that observed after intracoronary infusion of 1 million CPCs, the dose used in our previous studies in rats.^{3, 4, 6} Rats underwent a 90-min coronary occlusion and reperfusion. Four hours after reperfusion, they received 1.0 ×10⁶ CPCs (in 1 ml of DPBS) via intracoronary infusion as in our previous studies^3–6, 3.0, 9.0, or 12.0 ×10⁶ CPCs (in 5 ml of DPBS over 4 min) using echo-guided injection into the LV cavity, or 3.0×10⁶ CPCs i.v. Rats were euthanized 24 h after CPC delivery and the heart was harvested to measure the number of CPCs in the tissue by real-time PCR.

Echocardiographic studies

All echocardiographic analyses were performed by investigators who were blinded to treatment allocation. Serial echocardiograms were obtained at five time-points: baseline (three days before coronary artery occlusion), 30 days after MI (before the 1^st treatment), 35 days after the 1^st treatment (before the 2^nd treatment), 35 days after the 2^nd treatment (before the 3^rd treatment), and 35 days after the 3^rd treatment (before euthanasia)(Fig. 1). The echocardiographic studies were performed as described^3–6 using a Vevo 2100 Imaging System equipped with a 20-MHz transducer.

Hemodynamic studies

All hemodynamic analyses were performed by investigators who were blinded to treatment allocation. The hemodynamic studies were conducted 35 days after the 3^rd treatment, just before euthanasia (Fig. 1). The protocol has been described.^4–6

Histologic studies

The protocol for histologic analyses has been described.^{4–7, 38}

Immunohistochemistry was performed in formalin-fixed, paraffin-embedded, 4-µm-thick heart sections. To assess the fate of the transplanted male CPCs, single-labeled Y chromosome was detected by fluorescence in situ hybridization (FISH) according to the modified manufacturer’s protocol (ID Labs, London, ON).^{4, 39}

Measurement of transplanted cells by real-time PCR

To determine the absolute number of transplanted CPCs, a quantitative real-time PCR-based method was used as previously described^{17, 18} in a subset of hearts from the single-dose group (n=8) and the multiple-dose group (n=11), which were frozen just after excision.

Statistical analysis

All data are expressed as means ± SEM. Echocardiographic data were analyzed with two-way repeated-measures ANOVA followed by Student’s t-tests with Bonferroni correction for intra- and inter-group comparisons, as appropriate. All parametric data including morphometric, histologic, immunohistochemical, and hemodynamic data were analyzed by one-way ANOVA followed by Student’s t-tests with Bonferroni correction for inter-group comparisons.^40–42 Mortality was analyzed by the chi-square test. All analyses were conducted with SigmaStat 3.5. P values <0.05 were considered significant.

Characterization of CPCs

Supplementary Figures IIA and IIB are representative confocal images of mCherry- and GFP-labeled c-kit^POS CPCs. Similar to our previous studies^{4, 5}, FACS analysis showed that 82.9 ± 2.2% (n=7) of the cells were c-kit positive at the passage when they were injected (passages 6–8); in addition, 80.8 ± 5.4% (n=5) were mCherry positive and 97.6 ± 0.4% (n=2) GFP positive (Supplementary Figs. IIC and D). CPCs were negative for CD45 (0.5 ± 0.1%, n=5) and CD31 (0.4 ± 0.0%, n=5), and expressed mesenchymal markers (CD 90, 19.7 ± 9.8%, n=5; CD105, 68.1 ± 6.5%, n=5; CD73, 92.2 ± 4.6%, n=5; CD29, 99.5 ± 0.1%, n=5) (Supplementary Fig. IID). The mean population doubling time was 28.9 ± 2.8 h (n=5).

Preliminary studies

We have previously found that 1×10⁶ CPCs, delivered intracoronarily, produce an improvement in LV function in this rat model.⁶ Preliminary studies were conducted in 19 rats to select a dose of CPCs that would result in a comparable degree of myocardial retention when delivered into the LV cavity. As shown in Table 1, intraventricular infusion of 3×10⁶, 9×10⁶, and 12×10⁶ CPCs resulted, 24 h later, in a dose-dependent retention of cells. Since the number of CPCs retained 24 h after the 12×10⁶ dose (112,983±56,300 cells/heart, n=6) was similar to that retained 24 h after intracoronary infusion of 1×10⁶ CPCs (118,924±24,458, n=3), and since, as mentioned above, the latter treatment is effective in enhancing LV function in this rat model⁶, we selected 12×10⁶ CPCs as the dose to be used in the present study. The equivalency of the intraventricular CPC dose used herein and the intracoronary CPC dose used previously is further supported by the fact that the benefits of 12×10⁶ CPCs in the single-dose group (vide infra) were comparable to those observed previously in this same model after intracoronary infusion of 1×10⁶ CPCs.⁶ In contrast to the intraventricular infusion, no CPCs could be found in the heart after intravenous infusion of 3×10⁶ CPCs (Table 1).

Exclusions and gross measurements

As shown in Supplementary Table I, of the 85 rats that were subjected to coronary artery occlusion/reperfusion, 13 died within seven days after MI. At 30 days after MI, the remaining 72 rats were assigned to one of the three treatment groups (vehicle, single dose, or multiple doses); nine of these animals died after CPC infusion because of intrathoracic bleeding from the LV injection site. Thus, a total 63 rats completed the protocol; of these, two were excluded because of prespecified criteria (one because LV EF decreased <15 units after MI and before treatment, as shown by echocardiography [Supplementary Fig. IA] and one because of small scar size [<10% of risk region], as shown by trichrome stain [Supplementary Fig. IB]). Therefore, a total of 61 rats (16 in the vehicle group, 20 in the single-dose group, and 25 in the multiple-dose group) were included in the final analysis. Of these, 50 rats (16 in the vehicle, 16 in the single-dose, and 18 in the multiple-dose group) underwent echocardiographic and hemodynamic studies, 42 (16, 12, and 14, respectively) were used for pathologic and immunohistochemical analyses, and 8 (4 in the single-dose and 4 in the multiple-dose group) were used for qPCR analysis of CPC retention. An additional 11 rats (4 in the single-dose group and 7 in the multiple-dose group) were used for qPCR analysis without echocardiographic or hemodynamic analyses; two of these rats (in the multiple-dose group) were excluded because the CPC counts were >2 SD above the average, likely as a result of accidental injection of cells into the LV wall. Therefore, data from 17 rats (8 in the single-dose and 9 in the multiple-dose group) were used for the qPCR analysis of CPC retention (Supplementary Table I).

There were no significant differences in body weight among the three groups throughout the experimental protocol (Supplementary Table II). Similarly, there were no significant differences among the groups with respect to LV weight or LV volume measured at postmortem examination (Supplementary Table II).

Echocardiographic measurements

Echocardiographic measurements are summarized in Supplementary Table III; representative echocardiographic recordings are illustrated in Supplementary Fig. III. Before the 1^st treatment (30 days after MI), LV end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were markedly increased from baseline (Fig. 2A), whereas diastolic thickness and systolic thickening fraction (ThF) in the infarcted wall (Fig. 3A) and LV EF (Figs. 4A and B) were markedly decreased. There were no significant differences among the vehicle, single-dose, and multiple-dose groups in any of these variables, indicating that before the 1^st treatment, the severity of post-MI LV remodeling and dysfunction was comparable in all groups.

After the 1^st treatment, however, the three groups exhibited a different course. As expected, the vehicle-treated (control) group continued to show progressive deterioration of both regional and global LV function (Figs. 2–4; Supplementary Fig. III). Thus, in the 105-day interval between the 1^st treatment and euthanasia, LVESV increased progressively compared with pretreatment values (at 30 days after MI) (Fig. 2B), whereas diastolic thickness of the infarcted wall (IWTd) (Fig. 3B), systolic ThF in the infarcted wall (Fig. 3B), and LV EF (Fig. 4D) all decreased progressively.

The single-dose group exhibited a significant improvement in indices of regional and global LV function after the 1^st infusion (which consisted of CPCs); however, no further significant improvement was observed after the 2^nd and 3^rd infusions (which consisted of vehicle) (Figs. 2–4). For example, compared with the vehicle group, administration of CPCs in the single-dose group resulted, 35 days later, in a decrease in LVESV (Figs. 2A and B) and in the percentage of the LV circumference that was akinetic (akinetic length) (Figs. 3C and D) and in an increase in the diastolic thickness in the infarcted wall (Figs. 3A and B), in the ThF in the infarcted LV wall (Figs. 3A and B), as well as in LV EF (Fig. 4); after the 2^nd and 3^rd infusions (vehicle), none of these variables changed significantly (Figs. 2–4).

In contrast, the multiple-dose group exhibited a progressive improvement in regional and global LV function after each CPC infusion (Figs. 2–4). Each additional treatment produced additional benefit, and the magnitude of the improvement observed after the 2^nd and 3^rd infusions was roughly comparable to that observed after the 1^st infusion (Figs. 2–4). Thus, compared with pretreatment values (at 30 days after MI), the diastolic thickness of the infarcted wall increased by 0.10 ± 0.02 mm, 0.20 ± 0.02 mm, and 0.32 ± 0.03 mm after the 1^st, 2^nd, and 3^rd infusion of CPCs, respectively (P <0.01 for 2^nd vs. 1^st and 3^rd vs. 2^nd) (Fig. 3B); systolic ThF in the infarcted wall increased by 8.5 ± 2.4%, 16.5 ± 2.8%, and 22.3 ± 3.9% (P <0.05 for 2^nd vs. 1^st and 3^rd vs. 2^nd)(Fig. 3B); LVESV decreased by 10.2 ± 4.0 µL, 18.2 ± 3.5 µL, and 24.2 ± 5.5 µL (P <0.05 for 2^nd vs. 1^st) (Fig. 2B); LV EF increased by 3.4 ± 0.5%, 7.4 ± 0.9%, and 13.0 ± 0.8% (P <0.01 for 2^nd vs. 1^st and 3^rd vs. 2^nd) (Fig. 4D); and the percentage of the LV circumference that was akinetic (akinetic length) decreased by 5.6 ± 2.6%, 10.5 ± 2.4%, and 13.6 ± 2.0% (Fig. 3D).

As a result of this stepwise improvement, the cumulative beneficial effects of cell therapy after three CPC infusions were much greater than the effects seen after one CPC infusion. For example, the total (cumulative) increase in LV EF between pretreatment and end of the study in the multiple-dose group (13.0 ± 0.8%) was approximately triple that in the single-dose group (4.7 ± 0.8%) (P<0.01) (Fig. 4D). The total (cumulative) increase in infarcted wall ThF was ~2.5 greater in the former (22%) than in the latter (9%) (P<0.05; Fig. 3B). Similarly, the total (cumulative) decrease in akinetic length was ~2.5 times greater in the multiple-dose than in the single-dose group (−13.6% vs. −5.6%, P<0.05) (Fig. 3D). The greater therapeutic efficacy of multiple CPC infusions is further demonstrated by the fact that, at the end of the protocol, there were statistically significant differences in infarcted wall thickness, infarcted wall ThF, LV EF, LVESV, and akinetic length not only between the multiple-dose group and the vehicle group, but also between the multiple-dose group and the single-dose group (P<0.01 for all comparisons) (Figs. 2–4). An additional difference is that in the multiple-dose group, the systolic ThF in the posterior (noninfarcted) LV wall was significantly greater than in the vehicle group after the 2^nd and 3^rd treatment (P<0.01); in contrast, in the single-dose group systolic ThF in the posterior wall was significantly (P<0.01) greater than in the vehicle group only after the 3^rd treatment (Fig. 3A).

For LV EF, repeated-measures two-way ANOVA demonstrated a significant interaction between time and group (F=35.57, P<0.001), indicating that the relationship between LV function and time depended on treatment group. Similar conclusions were achieved for LVESV, LV stroke volume, diastolic thickness of the infarcted wall, systolic ThF in the infarcted wall, and akinetic length (Figs. 2 and 3). Trend analysis indicated that i) in the vehicle group, LV EF declined significantly over time (P<0.001); ii) in the single-dose group, a single linear trend could not capture the EF profile over time because EF increased after the 1^st treatment (P<0.001) but plateaued after the 2^nd and 3^rd treatment; and iii) in the multiple-dose group, LV EF increased significantly over time (P<0.001) and the LV EF profile could be captured by a linear trend. Similar conclusions were achieved when trend analysis was used for the other variables illustrated in Figs. 2 and 3.

In summary, the patterns observed in the three treatment groups were quite different: indices of regional and global LV function improved after each treatment (by a similar amount) in the multiple-dose group, improved only after the 1^st treatment in the single-dose group, and either worsened or were unchanged after each treatment in the vehicle group.

Echocardiography did not demonstrate any significant effect of CPCs on LV dilatation. As shown in Fig. 2A, LVEDV remained relatively stable in vehicle-treated rats during the three treatments and was not affected by either single or multiple administrations of CPCs (Fig. 2A). The observation that CPCs did not reverse LV dilatation is consistent with our previous studies in this rat model of old MI.⁶

Hemodynamic measurements

The hemodynamic studies, which were performed just before euthanasia, also demonstrated superior LV function in the multiple-dose group compared with the single-dose group (Figs. 5A and B; Supplementary Table IV). Both of these groups exhibited a significant improvement in most hemodynamic parameters compared with the vehicle group; however, the efficacy of multiple treatments was superior to that of a single treatment, as demonstrated by the fact that LV EF (a load-dependent index of LV systolic function) and preload recruitable stroke work and end-systolic elastance (two load-independent indices) were significantly greater in the multiple-dose vs. the single-dose group, whereas ESV was significantly smaller (Fig. 5B). Thus, two independent methods of functional assessment (echocardiography and hemodynamic studies with a conductance catheter) consistently demonstrated that multiple administrations of CPCs produced a greater improvement in regional and global LV function compared with a single administration.

Morphometric and histological analysis

As illustrated in Fig. 6A, the vehicle group exhibited LV dilatation and an extremely thin infarcted wall with a confluent scar. In the single-dose and multiple-dose groups, the infarcted wall was thicker and the amount of viable tissue within the risk region was greater compared with the vehicle group (Fig. 6A).

In each heart, a detailed quantitative analysis was performed on two sections (one from each of two mid-ventricular slices); the results are summarized in Fig. 6B and Supplementary Table V. Although the size of the risk region was similar among the three groups, both the single-dose and the multiple-dose groups exhibited a greater amount of viable myocardium within the risk region (60.6 ± 2.2% and 67.7 ± 2.9% of the risk region, respectively) than the vehicle group (51.4 ± 2.3%, P<0.05 vs. the single-dose group and P<0.01 vs. the multiple-dose group). Unlike the single-dose group, however, the multiple-dose group also exhibited a significant increase in the thickness of the infarcted wall and in the percentage of LV weight that was accounted for by viable tissue (P<0.05 and <0.01, respectively, vs. the vehicle group) (Fig. 6B). There was a trend for the percentage of scarred tissue to be smaller and the infarcted wall thickness and percentage of viable myocardium to be greater in the multiple-dose compared with the single-dose group, but the differences were not statistically significant (Fig. 6B). Taken together, the morphometric data confirm the echocardiographic finding of greater infarcted wall thickness in the multiple-dose than in the single-dose group (Fig. 3A) and reveal a difference between single and multiple treatments with respect to the amount of viable myocardium: both treatments increased viable tissue and decreased scar within the risk region, but when these variables were expressed as percentages of the entire left ventricle, only multiple treatments were associated with an increase in the total amount of viable tissue and a decrease in the total scar burden.

Myocardial fibrosis plays a key role in the pathology of LV remodeling after MI.⁴³ In the noninfarcted region, collagen content was significantly less in the multiple-dose compared with the single-dose group (P<0.05) (Figs. 6C and D). This reduced collagen deposition in the myocardium may have contributed, at least in part, to the functional benefits of CPC therapy.

Myocyte cross-sectional area and myocyte and vessel density

Myocyte cross-sectional area and myocyte density were assessed by staining myocytes with an anti-alpha-sarcomeric actin (α-SA) antibody and myocyte membranes with FITC-conjugated WGA to facilitate counting individual myocytes (Fig. 7 and Supplementary Fig. IV). In the noninfarcted region, there were no significant differences among groups (Figs. 8B and C). However, in the risk region myocyte cross-sectional area was significantly smaller and myocyte density was significantly greater in the two treated groups compared with the vehicle group (Figs. 7B and C); in the multiple-dose group, these changes were more pronounced, such that both myocyte cross-sectional area and density were significantly different from the single-dose group (P<0.05) (Figs. 7B and C). Consistent with these data, analysis of the distribution of cardiomyocyte cross-sectional area demonstrated no difference in the noninfarcted region but a shift to the left in the risk region in the single-dose group and, even more so, in the multiple-dose group (Supplementary Fig. IV). Although other explanations are possible, increased myocyte density is consistent with increased formation of new myocytes.

Vascular density was assessed by staining tissue sections with FITC-conjugated Isolectin B4 (Supplementary Fig. V). In all groups, vascular density was significantly less in the risk region than in the noninfarcted region, but no differences were noted among the three groups.

Survival of transplanted CPCs

Survival of transplanted CPCs was assessed by two independent methods. The CPCs administered during the 1^st infusion were labeled with mCherry and those infused during the 3^rd infusion with GFP (Fig. 1). As shown in Table 2, at the end of the study (35 days after the 3^rd CPC infusion and 105 days after the 1^st infusion), very few GFP^POS cells and even fewer mCherry^POS cells were found in the left ventricle using immunohistochemistry (n=14). In addition, the number of CPCs remaining in the heart at the end of the protocol was measured using quantitative real-time PCR in two subsets of rats (Supplementary Table VI). Transplanted CPCs were detected only in five of the eight rats analyzed in the single-dose group and nine of the eleven rats analyzed in the multiple-dose group. In the hearts with detectable CPCs, the number of these cells was extremely low: 3,330±1,451 in the entire heart in the single-dose group (n=5) and 5,078±1,409 in the multiple-dose group (n=7) (P=NS). Assuming that no cells were present in hearts with no detectable CPCs, the total content of CPCs averaged 2,081 ± 1,060 cells in the single-dose group (n=8) and 3,950±1,310 in the multiple-dose group (n=9) (P=NS). Thus, the number of transplanted cells that were engrafted in the heart was very low; it tended to increase after three CPC infusions vs. a single infusion, but the difference was not statistically significant, possibly because of the large variability.

Analysis of cell proliferation

In previous studies, we have observed a pronounced and prolonged proliferative response to a single CPC administration.^{4, 6} To determine the effect of multiple CPC doses on cell proliferation, rats were given BrdU for 35 days after the 1^st treatment (days 1–35 after start of therapy) and IdU for 35 days after the 3^rd treatment (days 71–105 after start of therapy) (Fig. 1). In all three groups, the number of BrdU^POS and IdU^POS cells was significantly greater in the risk region than in the noninfarcted region (Figs. 8C–F), indicating greater cell proliferation and/or greater infiltration of proliferating cells in the former - a finding consistent with our previous observations.^{4, 6}

In the noninfarcted region, there were no significant differences among the three treatment groups with respect to either BrdU^POS or IdU^POS cells (Fig. 8C–F). In contrast, significant differences were noted in the risk region. In vehicle-treated hearts, 13.8 ± 1.0% of nuclei in the risk region were BrdU^POS (Fig. 8C) and 22.2 ± 1.9% IdU^POS (Fig. 8D) at the end of the study, indicating that a substantial number of new cells were formed between days 1–105 (BrdU^POS cells) and between days 71–105 after the beginning of treatment (IdU^POS cells). These values are consistent with our previous results.^{4, 6}

A single dose of CPCs did not result in a significant increase in BrdU^POS cells (Fig. 8C); this, however, does not rule out a proliferative response to CPCs because BrdU was given only for the first 35 days after CPC infusion and cells may have proliferated early after CPC infusion and then died by the time rats were euthanized 105 days later. A single dose of CPCs did produce a significant increase in the number of IdU^POS cells (Fig. 8D), indicating that at 71–105 days after the administration of CPCs, there was increased cell proliferation. The ability of a single CPC infusion to promote cell proliferation several months later is consistent with our previous findings in this rat model.^{4, 6} The number of BrdU^POS/IdU^POS (double positive) cells was not significantly increased 105 days after a single CPC administration (Fig. 8E), but again, an early proliferative response may have been missed if significant numbers of BrdU^POS cells died before euthanasia. This possibility is supported by the fact that, in the vehicle-treated group, the number of IdU^POS cells (22.2 ± 1.9 % in risk region and 14.6 ± 0.8 % in noninfarcted region) was greater than the number of BrdU^POS cells (13.8 ± 1.0 % in risk region and 10.8 ± 1.0 % in noninfarcted region) (Figs. 8C and D).

In rats that received multiple doses of CPCs, the number of BrdU^POS cells was markedly increased vs. both the single-dose group and the vehicle group (Fig. 8C), suggesting that the 2^nd and 3^rd infusions of CPCs induced a robust proliferative response (on days 36–105 after the beginning of CPC treatment). The number of IdU^POS cells was significantly increased vs. the vehicle group but not vs. the single-dose group (Fig. 8D), suggesting that while three CPC doses produced a robust proliferation on days 71–105 after the beginning of treatment, this proliferation was not greater than that induced by a single treatment given earlier. Unlike the single-dose group, the multiple-dose group exhibited a significant increase in the number of BrdU^POS/IdU^POS (double positive) cells (Fig. 8E), which constituted ~1/2 of all IdU^POS cells (Fig. 8D), suggesting that with repeated CPC infusions, ~1/2 of the new cells formed on days 71–105 after the beginning of treatment were derived from cells formed on days 1–70. When all newly-formed (BrdU^POS or IdU^POS) cells were combined, their number was significantly increased after both a single and multiple CPC infusions (Fig. 8F); the increase tended to be greater after multiple CPC infusions, but the difference vs. the single-dose group was not significant.

Taken together, these data indicate that a single CPC infusion promoted a sustained proliferative response in the risk region (or a greater infiltration of inflammatory cells in the risk region) that was still evident 2–3 months (71–105 days) later, and that multiple CPC infusions tended to further augment this response.

Analysis of new myocyte formation

Formation of new mature myocytes was assessed by measuring the number of BrdU^POS or IdU^POS cells that expressed cTnI and exhibited a morphology typical of adult myocytes (Fig. 8). (Thus, the small cells that express alpha-sarcomeric actin but lack the morphology of mature cardiomyocytes [cells that we observed after CPC transplantation in our previous studies^{4, 6}] were not included in this analysis.)

In Fig. 8, the number of new mature myocytes (BrdU^POS/cTnI^POS or IdU^POS/cTnI^POS cells) is expressed both as a percent of total nuclei (Figs. 8G–J) and as a percent of total myocytes (Figs. 8K–N). In all groups, these cells tended to be more abundant in the noninfarcted than in the risk region but, in absolute terms, they were rare. For example, in the vehicle-treated group, the number of BrdU^POS or IdU^POS mature myocytes was <0.5% of the nuclei and <0.5% of myocytes both in the risk region and in the noninfarcted region (Fig. 8G–N). The density of these cells was not significantly increased by a single CPC infusion (Fig. 8G–N). Three CPC infusions were associated with a significant increase in BrdU^POS and IdU^POS mature myocytes in the risk region (P<0.05 vs. vehicle-treated rats) (Figs. 8G–N), but even in this group, the percentage of these cells was very low (<0.5% of nuclei and <2% of myocytes in the risk region and <1 % of nuclei and <1% of myocytes in the noninfarcted region).

Taken together, these data indicate that the number of newly-formed mature myocytes was very small in the absence of CPC infusion and did not change after a single infusion; although this number increased significantly after three CPC infusions, it was still extremely low in absolute terms, and thus not sufficient to account for the improvement in function.

Analysis of Y-chromosome^POS cells

To assess the fate of transplanted male CPCs, the number of Y-chromosome^pos cells was measured by FISH. The advantage of using the Y-chromosome to track transplanted cells in long-term studies is that, unlike EGFP or beta-galactosidase, its presence is not affected by changes in gene expression.^44–46 Y-chromosome^pos cells were detected both in the single-dose and in the multiple-dose groups, but they were significantly more abundant in the multiple-dose group, both in the risk and in the noninfarcted region (Fig. 9C). The vast majority (>90%) of Y-chromosome^pos cells, however, did not show a myocyte morphology: in the risk region, 90.7 ± 2.0 and 93.5 ± 1.4% of Y-chromosome^pos cells were nonmyocytes in the single- and multiple-dose groups, respectively; in the noninfarcted region, 71.4 ± 7.2% and 78.4 ± 6.0% were nonmyocytes. Consequently, the number of Y-chromosome^pos myocytes was minuscule both in the single-dose and in the multiple-dose groups: 0.25 ± 0.05% and 0.37 ± 0.07% of total nuclei, respectively, in the risk region, and 0.25 ± 0.11 % and 0.54 ± 0.17%, respectively, in the noninfarcted region (Fig. 9D). Similar values were obtained when Y-chromosome^pos myocytes were expressed as a percentage of total myocytes: they accounted for <1% of myocytes in the risk region and <0.5% in the noninfarcted region (Fig. 9E).

Taken together, these data indicate that, regardless of the number of CPC infusions, very few transplanted cells (or their progeny) engraft in the heart and, of these, only a very small fraction differentiate into mature cardiomyocytes. Multiple CPC infusions produce an increase in CPC engraftment that is statistically significant but, in absolute terms, is quite small.

Analysis of BrdU and IdU positivity revealed that only a fraction of the Y-chromosome^pos myocytes had incorporated BrdU or IdU (Figs. 9F–K). For example, in the noninfarcted region of the multiple-dose group, Y-chromosome^pos myocytes accounted for 0.54 ± 0.17% of all nuclei (Fig. 9D), whereas Y-chromosome^pos/BrdU^POS myocytes accounted for 0.04±0.04% of all nuclei and Y-chromosome^pos/IdU^POS myocytes for 0.09±0.06% of all nuclei (Fig. 9H). This finding suggests that not all CPCs underwent cell cycling before differentiating into mature myocytes.