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Utility of Dual-modality Bioluminescence and MRI in Monitoring Stem Cell Survival and Impact on Post Myocardial Infarct Remodeling

Posted Dec 31, 2010 Updated Apr 7, 2023
By Hualei Zhang BS , Hui Qiao MD PhD , Ashley Bakken BS , Fabao Gao MD PhD , Bin Huang MD , Yingqiu Y. Liu MS , Wafik El-Deiry MD PhD , Victor A. Ferrari MD , Rong Zhou PhD
12 min read

Rationale and Objectives

Firefly luciferase (Fluc) reporter gene is an authentic marker for surviving stem cells. However, it is unable to visualize the intramyocardial delivery of stem cells or their impact on cardiac function. The investigators demonstrate that bioluminescence imaging (BLI) combined with magnetic resonance imaging (MRI) allows better assessment of cell delivery and the impact on post–myocardial infarction remodeling.

Materials and Methods

Murine embryonic stem cells (0.3 million) were double-labeled with Fluc and superparamagnetic iron oxide particles and injected into the infarct border zone of athymic rat hearts. BLI and MRI were performed serially up to 2 months after injection, followed by immunohistochemistry.

Results

Dual-modality imaging was able to verify the initial intramyocardial delivery of the cells and their survival status. Over time, BLI signal increased in seven of nine hearts and disappeared in the other two hearts. The divergence of BLI signal over time was supported by MRI findings. Left ventricular ejection fraction and fractional shortening estimated by MRI suggested that cell engraftment mediated a positive impact on post–myocardial infarction remodeling. Two months after intramyocardial injection, superparamagnetic iron oxide–associated signals facilitated the localization of the injection site.

Conclusions

Dual-modality imaging has the unique ability to monitor cell delivery, survival status, graft morphology, and impact on post–myocardial infarction remodeling.

Magnetic resonance imaging (MRI)–based tracking of stem cells grafted in the heart is especially challenging in rodent models. Cardiac and respiration motion introduce significant artifacts, particularly in the setting of their high heart rates. Electrocardiographically gated MRI has been successful in visualizing superparamagnetic iron oxide (SPIO) particle–labeled stem cells grafted in the myocardium of mouse and rat models . However, later studies revealed that the SPIO-MRI method was insensitive to monitor cell survival over time . In comparison, approaches using reporter genes to “label” stem cells have shown advantages in tracking cell survival and proliferation in the heart . Compared to other reporter genes, the firefly luciferase (Fluc) reporter combined with bioluminescence imaging (BLI) provides a sensitive, simple, and inexpensive method for stem cell tracking in small animals. Although it is highly sensitive, BLI is poor in resolving the signal in a tomographic fashion. In the past, BLI and MRI were examined for monitoring stem cell interventions in rodent models of myocardial infarction (MI) . However, the unique strength of combining the two modalities to track the same population of stem cells over time has not been demonstrated.

To visualize the stem cells by both BLI and MRI, we used a double-labeling strategy in which Fluc-expressing murine embryonic stem cells (ESCs) were labeled with SPIO particles. Murine ESCs were rejected in immune competent allogeneic mice . Therefore, immune-deficient or syngenic animals must be used to achieve long-term monitoring of ESC survival by BLI. The above consideration combined with our intention to generate a reperfused MI model led us to choose athymic nude rats, which are suitable for demonstrating the utility of dual-modality imaging. Indeed, the MI model in nude rats has been used to receive stem cells of human and mouse origin in many previous studies .

Materials and methods

ESCs Stably Expressing Fluc Reporter Gene and Labeled with SPIO Particles

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Surgical Procedures to Induce MI and Intramyocardial Injection of Stem Cells

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Experimental Groups

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Table 1

Reasons for Exclusion and Final Group Size

Group Total Animals Used Death After Surgery Improper Injection of Cells ∗ Infarct Size < 10% or >30% of LV Mass Final Group Size Control 10 1 NA 2 7 ESC treated 24 4 6 5 9

ESC, embryonic stem cell; LV, left ventricular; NA, not applicable.

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In Vivo BLI and MRI

Imaging Schedule

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BLI

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MRI

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Analyses of Bioluminescence and Magnetic Resonance Images

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Histologic Analysis to Identify Grafted Cells, Macrophages, and Cardiac Differentiation

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Statistical Analysis

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Results

Viability and Labeling Efficiency and Iron Content

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Both MRI and BLI Are Necessary to Confirm Intramyocardial Delivery and Survival of Stem Cells

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Figure 1, Distributions of grafted cells relative to infarct region revealed by magnetic resonance imaging (MRI) of the embryonic stem cell–treated group at day 1 (a–c) and improper cell delivery revealed by bioluminescence imaging (BLI) and MRI (d–e) . One short-axis slice is displayed for each heart. Infarcted region is marked between red lines. Superparamagnetic iron oxide–induced hypointense region is indicated by yellow arrows. MRI revealed the exact location of grafted cells: in the center of the infarct (a) , inside the infarct region but close to anterior border (b) , or in the lateral border next to the infarct region (c) . The heart shown in (a) was from the nongraft subgroup, while the hearts in (b) and (c) were from the engraftment subgroup. Improper cell delivery led to BLI signal from thoracic region and right upper abdomen (d) (color scale in photons per second) and an absence of hypointense MRI signal in the myocardial wall (e) . LV, left ventricle; RV, right ventricle.

Figure 2, Diverged time courses of bioluminescence imaging (BLI) signal intensity in the embryonic stem cell (ESC)–treated group. Bioluminescence images from a heart in the nongraft subgroup (a–c) and one in the engraftment subgroup (d–f) at day 1 (D1), day 7 (D7), and week 4 (W4). The color scales are different for images at day 1, day 7, and week 4, representing an order of magnitude increase in BLI signal intensity. (g) Total flux (photons per second) quantified from the engraftment subgroup ( n = 7) and nongraft subgroup ( n = 2). The nongraft subgroup and two animals from the engraftment subgroup contributed to the last time point (2 months) on the curve. Error bars represent values of standard deviation, and for clarity, only the positive or negative portion is shown.

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Two Outcomes From the ESC-treated Group Revealed by Imaging and Confirmed by Histology

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Table 2

Myocardial Wall Thickness in ESC-treated and Control Groups at Week 4

Wall Thickness at Week 4 Septal Anterior Lateral Posterior ESC treated Nongraft ( n = 2) 1.3 ± 0.2 1.5 ± 0.1 1.5 ± 0.2 1.5 ± 0.3 Engraftment ( n = 7) 1.3 ± 0.1 1.7 ± 0.2 † 2.2 ± 0.9 † 1.6 ± 0.2 † Controls ( n = 7) 1.3 ± 0.1 1.4 ± 0.2 1.2 ± 0.3 † 1.2 ± 0.1 Wall thickness at day 1 ( n = 16) ∗ 1.4 ± 0.2 1.4 ± 0.3 1.5 ± 0.2 1.3 ± 0.3

Three short-axis slices (3 mm apart) with the middle slice at the midventricular level were used. Diastolic wall thickness was measured in the septal, anterior, lateral, and posterior quadrants using a line connecting the centroid of the left ventricular wall and the vertex of the epicardium border.

ESC, embryonic stem cell.

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Figure 3, Changes of hypointense volume (a) and contrast (b) from day 1 (D1) to week 4 (W4) in embryonic stem cell–treated hearts. Open circles: from hearts in the engraftment subgroup ( n = 7). Triangles: from hearts in the nongraft subgroup ( n = 2). Solid circles: average ( n = 9). (a) P < .05 when the average at week 4 and day 1 was compared. (b) No statistical difference between day 1 and week 4 for either group ( P > .40). The formulas to calculate hypointense volume and contrast are found in “Materials and Methods.”

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Figure 4, Histologic analyses of hearts from the nongraft subgroup (a–c) and the engraftment subgroup (d–g) . Firefly luciferase (Fluc; green) and Prussian blue (PB; blue particles) double staining revealed a few double-positive cells (arrows in a) in the heart, but no substantial graft was formed. Superparamagnetic iron oxide (SPIO)–containing macrophages were identified by cluster of differentiation 68 (red) and PB double staining (arrows in b and e). Combination of PB and Masson’s trichrome staining (c) revealed SPIO-containing cells in the scar tissue. The majority of cells inside the teratoma were Fluc positive but PB negative (d) . The dashed line in (e) marks the boundary of a cardiac teratoma. (a–c) , (d) , and (e) were adjacent sections. Hematoxylin and eosin staining of the teratoma in which tissue from three germ layers was identified (f) : squamous cells with keratin pearl originated from ectoderm (small arrowhead), osteoid of mesoderm origin (large arrowhead), and columnar gland of endoderm origin (arrow). Cardiomyocytes inside the teratoma were identified by striations positive for sarcomeric α-actinin (brown color in g). The images in (d) to (g) were obtained from hearts harvested 4 weeks after the injection of cells. The scale bar is 50 μm in all panels except (f) .

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Figure 5, Fractional shortening of the myocardial wall of the embryonic stem cell–treated and control groups. Hearts at day 1 from the (a) engraftment subgroup, (b) nongraft subgroup, and (c) control group. (d–f) Corresponding hearts at week 4. The anterior (border 1 [b1]) and posterior (border 2 [b2]) borders of the infarct were defined on images as follows: at day 1, b1 and b2 are placed at the boundary of the infarct defined on delayed hyperenhancement images (a–c) . At week 4, boundaries of the graft were defined as b1 and b2 in the engraftment subgroup (d) ; borders defined in (b) were transferred to images of the corresponding location for the nongraft hearts (e) ; boundaries of the dyskinetic region identified on cine images were defined as b1 and b2 in the control hearts (f) . Arrows point to the region where myocardial wall became thin. Fractional shortening was measured from b1, b2, and septum (remote) on magnetic resonance images at day 1 and week 4 (g) . # P < .05 compared to the septum (remote region) at day 1; ∗ P < .05 comparing day 1 and week 4. Statistical analysis was not applied in nongraft subgroup because of small size ( n = 2).

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Global and Regional Cardiac Function

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Table 3

Global LV Function at Day 1 and Week 4 in the ESC-treated and Control Groups

Day 1 Week 4 Group EDV (μl) ESV (μl) LVEF (%) EDV (μl) ESV (μl) LVEF (%) ESC treated ( n = 9) 232 ± 61 72 ± 50 69 ± 13 276 ± 55 ∗ 66 ± 34 ∗ 76 ± 11 Control ( n = 7) 227 ± 69 89 ± 26 62 ± 11 412 ± 111 † 181 ± 105 59 ± 18

EDV, end-diastolic volume; ESV, end-systolic volume; LVEF, left ventricular ejection fraction.

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Discussion

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Conclusions

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Academic Radiology, Volume 18, Issue 1
Journals General Radiology
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