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

Abstract

Rationale: The effects of c-kit(POS) cardiac progenitor cells (CPCs, and adult cell therapy in general) on left ventricular (LV) function have been regarded as modest or inconsistent.

Objective: To determine whether 3 CPC infusions have greater efficacy than 1 infusion.

Methods and results: Rats with a 30-day-old myocardial infarction received 1 or 3 CPC infusions into the LV cavity, 35 days apart. Compared with vehicle-treated rats, the single-dose group exhibited improved LV function after the first infusion (consisting of CPCs) but not after the second and third (vehicle). In contrast, in the multiple-dose group, regional and global LV function improved by a similar degree after each CPC infusion, resulting in greater cumulative effects. For example, the total increase in LV ejection fraction was approximately triple in the multiple-dose group versus the single-dose group (P<0.01). The multiple-dose group also exhibited more viable tissue and less scar, less collagen in the risk and noninfarcted regions, and greater myocyte density in the risk region.

Conclusions: This is the first demonstration that repeated CPC administrations are markedly more effective than a single administration. The concept that the full effects of CPCs require repeated doses has significant implications for both preclinical and clinical studies; it suggests that the benefits of cell therapy may be underestimated or even overlooked if they are measured after a single dose, and that repeated administrations are necessary to evaluate the effectiveness of a cell product properly. In addition, we describe a new method that enables studies of repeated cell administrations in rodents.

Keywords: cardiac progenitor cells; cell therapy; collagen; left ventricular function; myocardial infarction; reperfusion injury; ventricular remodeling.

Figure 1. Experimental protocol

Echo, echocardiogram; CPCs-mCherry, mCherry-labeled CPCs; CPCs-GFP, GFP-labeled CPCs; Pre-Rx, pretreatment (30 days after MI); 1^st, 2^nd, 3^rd Rx: 1^st, 2^nd, and 3^rd treatment.

Figure 2. Echocardiographic assessment of LV volume

A. LV end-diastolic volume (EDV), end-systolic volume (ESV), and stroke volume (SV) in the vehicle, single-dose, and multiple-dose groups at baseline (BSL), before the 1^st treatment (Pre-Rx) (i.e., 30 days after MI), 35 days after the 1^st treatment (1^st Rx), 35 days after 2^nd treatment (2^nd Rx), and 35 days after the 3^rd treatment (3^rd Rx); B. Cumulative changes in ESV (left) and SV (right) vs. pretreatment (Pre-Rx) values. Data are means ± SEM.

Figure 3. Echocardiographic assessment of regional LV function

A. End-diastolic thickness of the infarcted LV wall (IWTd), thickening fraction in the infarcted LV wall (IW ThF), end-diastolic thickness of the posterior (noninfarcted) LV wall (PWTd), and thickening fraction in the posterior (non-infarcted) wall (PW ThF) in the vehicle, single-dose, and multiple-dose groups at baseline (BSL), before the 1^st treatment (Pre-Rx) (i.e., 30 days after MI), 35 days after the 1^st treatment (1^st Rx), 35 days after 2^nd treatment (2^nd Rx), and 35 days after the 3^rd treatment (3^rd Rx); B. Cumulative changes in IWTd and IW ThF vs. pretreatment (Pre-Rx) values. C. Akinetic endocardial length (akinetic length [AL]) before the 1^st treatment (Pre-Rx) (i.e., 30 days after MI), 35 days after the 1^st treatment (1^st Rx), 35 days after 2^nd treatment (2^nd Rx), and 35 days after the 3^rd treatment (3^rd Rx); D. Cumulative changes in AL from pretreatment (Pre-Rx). Data are means ± SEM.

Figure 3. Echocardiographic assessment of regional LV function

A. End-diastolic thickness of the infarcted LV wall (IWTd), thickening fraction in the infarcted LV wall (IW ThF), end-diastolic thickness of the posterior (noninfarcted) LV wall (PWTd), and thickening fraction in the posterior (non-infarcted) wall (PW ThF) in the vehicle, single-dose, and multiple-dose groups at baseline (BSL), before the 1^st treatment (Pre-Rx) (i.e., 30 days after MI), 35 days after the 1^st treatment (1^st Rx), 35 days after 2^nd treatment (2^nd Rx), and 35 days after the 3^rd treatment (3^rd Rx); B. Cumulative changes in IWTd and IW ThF vs. pretreatment (Pre-Rx) values. C. Akinetic endocardial length (akinetic length [AL]) before the 1^st treatment (Pre-Rx) (i.e., 30 days after MI), 35 days after the 1^st treatment (1^st Rx), 35 days after 2^nd treatment (2^nd Rx), and 35 days after the 3^rd treatment (3^rd Rx); D. Cumulative changes in AL from pretreatment (Pre-Rx). Data are means ± SEM.

Figure 4. Echocardiographic assessment of global LV function

A. Line plots showing the time-course of LV EF in individual animals (black lines) and average values of LV EF in the vehicle, single-dose, and multiple-dose groups (colored lines); B. Bar graph illustrating LV EF at baseline (BSL), before the 1^st treatment (Pre-Rx) (i.e., 30 days after MI), 35 days after the 1^st treatment (1^st Rx), 35 days after 2^nd treatment (2^nd Rx), and 35 days after the 3^rd treatment (3^rd Rx); C. Changes in LV EF (absolute units) at 35 days after the 1^st, 2^nd, and 3^rd treatment vs. the respective pretreatment values; D. Cumulative changes in LV EF (absolute units) vs. pretreatment (Pre-Rx) values. Data are means ± SEM.

Figure 5. Hemodynamic assessment of LV function

Hemodynamic studies were performed with a Millar conductance catheter at 35 days after the 3^rd treatment, just before euthanasia. A. Representative pressure-volume loops recorded during preload manipulation by brief inferior vena cava occlusions. B. Quantitative analysis of hemodynamic variables. Data are means ± SEM.

Figure 6. Morphometric analysis (A and B) and myocardial collagen content (C and D)

A. Representative Masson trichrome-stained myocardial sections. Scar tissue and viable myocardium are identified in blue and red, respectively. B. Quantitative analysis of LV morphometric parameters. The size of the risk region, left ventricle (LV), viable myocardium, and scar was calculated in mg. The risk region comprises both the border zones and the scarred region. C. Representative microscopic images of an LV section stained with picrosirius red; images were acquired with transmission light (left) or polarized light (right). D. Quantitative analysis of polarized light microscopic images showing collagen content per mm² in the risk and noninfarcted regions. Data are means ± SEM. Bar is 2 mm.

Figure 7. Analysis of myocyte cross-sectional area and myocyte density

Myocyte cross-sectional area and myocyte density were determined in rat hearts stained with WGA (green) and α-SA (red). A. Representative epifluorescent microscopic image, sequentially acquired from 18 fields of the infarcted region of a vehicle-treated rat at a magnification of x300. Myocytes with round nuclei and clearly defined sarcolemmal borders were selected for analysis of cross-section area. B and C. Quantitative analyses of myocyte cross-sectional area (B) and myocyte density (C) per mm². WGA binds to the myocyte membrane, thereby facilitating the evaluation of myocyte cross-sectional area and density. The risk region comprises both the border zones and the infarcted region. Data are means ± SEM. Bar is 100 µm.

Figure 8. Proliferation of CPCs and mature myocytes

Rats received BrdU infusion for 35 days after the 1^st CPC administration and IdU infusion for 35 days after the 3^rd CPC administration. A and B. Representative confocal microscopic images acquired from the infarcted region (A) and the border zone (B) of a vehicle-treated rat. Positivity for BrdU is shown in green and for IdU in red. Myocytes were stained with an anti-cTnI antibody (white) and nuclei with DAPI (blue). In B, the yellow arrowhead indicates an IdU^POS mature myocyte, whereas the yellow asterisk shows a BrdU^POS/IdU^POS mature myocyte. C–N. Quantitative analysis of the number of BrdU^POS, IdU^POS, and BrdU/IdU^POS cells expressed as a percent of total nuclei (C–F) and the number of BrdU^POS, IdU^POS, and BrdU/IdU^POS myoctes expressed as a percent of total nuclei (G–J) and as a percent of total myocytes (K–N). The region at risk comprises both the border zones and the scarred region. Data are means ± SEM. Bar is 10 µm.

Figure 9. Analysis of Y-Chromosome^POS cells

A–B. Representative confocal microscopic images acquired from the infarcted region (A) and border zone (B). Green arrowheads indicate Y-chromosome fluorescent signals (green/cyan) in nuclei. Note a cluster of five Y-chromosome^POS nuclei in A (top left), suggesting Y-chromosome^POS cell division. Yellow arrowheads indicate BrdU^POS mature myocytes, whereas the yellow asterisk shows a Y-chromosome^POS/BrdU^POS/IdU^POS mature myocyte (B). BrdU is shown in white, IdU in red, and nuclei are stained with DAPI in blue. Myocardial morphology was examined with the confocal transmitted light channel’s detector (ChD) in which the pseudocolor selected for the myocardial background in the ChD channel was gray white. C–K. Quantitative analysis of the number of Y-chromosome^POS, BrdU^POS, and IdU^POS cells. The risk region comprises both the border zones and the infarcted region. Data are means ± SEM. Bar is 10 µm.