Assessment and optimization of cell engraftment after transplantation into the heart

Abstract

Myocardial regeneration using stem and progenitor cell transplantation in the injured heart has recently become a major goal in the treatment of cardiac disease. Experimental studies and clinical applications have generally been encouraging, although the functional benefits that have been attained clinically are modest and inconsistent. Low cell retention and engraftment after myocardial delivery is a key factor limiting the successful application of cell therapy, irrespective of the type of cell or the delivery method. To improve engraftment, accurate methods for tracking cell fate and quantifying cell survival need to be applied. Several laboratory techniques (histological methods, real-time quantitative polymerase chain reaction, radiolabeling) have provided invaluable information about cell engraftment. In vivo imaging (nuclear medicine modalities, bioluminescence, and MRI) has the potential to provide quantitative information noninvasively, enabling longitudinal assessment of cell fate. In the present review, we present several available methods for assessing cell engraftment, and we critically discuss their strengths and limitations. In addition to providing insights about the mechanisms mediating cell loss after transplantation, these methods can evaluate techniques for augmenting engraftment, such as tissue engineering approaches, preconditioning, and genetic modification, allowing optimization of cell therapies.

Figure 1

Immunocytochemistry images of eGFP overexpressing cardiac derived stem cells transplanted in the hearts of infarcted rats, at 21 days post cell injection. A: eGFP+ cells (green-yellow arrow), B: cardiac Troponin I+ cells (cardiomyocytes-red-white arrow), at the infarct border zone, C: nuclei (stained with Hoechst 33342-blue), d: merged image. eGFP does not co-localize with cardiac TropI, indicating that cells have not differentiated yet.

Figure 2

Immunocytochemistry images of human cardiac derived stem cells transplanted in the hearts of infarcted immunocompromised (SCID) mice, at 6 weeks post cell injection. A: white light image, B: nuclei (blue) and human nuclear specific antigen (green), C: merged image of cardiac troponin I (red) and human specific nuclear antigen (green), indicating in vivo differentiation of the CDCs into cardiomyocytes.

Figure 3

Fluorescent in situ hybridization (FISH) image of human cardiac derived stem cells injected into infarcted SCID mice, at 4 days post injection. Human specific sequences (A:red dots and B: white dots) as well mouse specific sequences (A:green dots and C:white dots) were used as targets in the nuclei (A:blue). Red arrows in panel A point to the nuclei containing human sequences (human cardiac derived cells). In panel B, red arrows point to the area corresponding to the human nuclei in A. Green arrows in panel A point to the nuclei containing mouse sequences (mouse cardiomyocytes). In panel C, green arrows point to the area corresponding to the mouse nuclei.

Figure 4

A: Standard curve correlating ex vivo luciferase activity in rat heart homogenates with known cell numbers of luciferase-expressing cardiac derived stem cells, indicating the excellent sensitivity and linearity of the assay. B: Absolute quantification of CDC number at 24hrs after direct intramyocardial injection, in 3 rats, using ex vivo luciferase assay. C: A quantitative real time PCR standard curve used for ex vivo quantification of male cell numbers injected in female hearts. A genomic sequence of the SRY gene is used as target. The curve shows excellent sensitivity of the assay, high efficiency of the reaction and linearity of the relationship between SRY gene copies and cell numbers. D: amplification plots of the reactions corresponding to the points of the standard curve. Data were analyzed with the SDS 2.1 software, by Applied Biosystems.

Figure 5

Engraftment of cardiac derived stem cells in a syngeneic rat, myocardial infarction model, assessed by in vivo Bioluminescence Imaging (A: Day 1, B: Day 2, C: Day 8 post cell injection).

Figure 6

Validation of engraftment results obtained by Bioluminescence Imaging (A), using quantitative real time PCR as the gold standard (B). Both techniques yield similar cell survival patterns, although BLI results have greater variability.

Figure 7

Positron Emission Tomography (PET) images of directly radiolabeled with ¹⁸FDG cardiac derived stem cells (red) injected in the infarct area of rats, at 1hr post cell injection. ¹³NH₃ (green) was used as perfusion tracer to delineate normally perfused myocardium. In vivo PET images are co-registered with CT images, for better anatomic detail and attenuation correction. (A: transverse image orientation, B: coronal, C: sagittal).

Figure 8

Single Photon Emission Computed Tomography (SPECT) images of human sodium iodide symporter (hNIS) overexpressing cardiac derived stem cells (red) injected in the infarct area of rats, at one day post cell injection. ^99mTc-pertechnetate (^99mTc-red) was used as tracer for the hNIS+ cells. 201 Thalium (²⁰¹Tl-green) was used as perfusion tracer to delineate normally perfused myocardium. Images were obtained in a hybrid SPECT/CT scanner. (A: transverse image orientation, B: coronal).