Evaluation of the autologous bone marrow mononuclear therapy and functional restoration in the scarred myocardium by imaging analysis.

A 62-year-old male patient with previous history of myocardial infarction, akinetic myocardial segments, and an ejection fraction of 31% with the NYHA class III category was selected for the autologous bone marrow (ABM)-derived mononuclear cell fraction injection during CABG surgery. Nitrate augmented myocardial tracer uptake was imaged by ECG gated SPECT pre- and 1 year post-ABM therapy, using radiotracer Tc99m Sestamibi. The baseline gated SPECT demonstrated full thickness infarct in 40% area of LAD territory. Bone marrow aspirate of 20.0 ml from sternum yielding a mono nuclear cell fraction of 4.5 × 10(7) cells/ml was suspended in 2.0 ml of sterile normal saline to be injected at eight sites of the injured myocardium. There were no apparent side effects due to the procedure, i.e., life threatening events, major bleeds, reaction, or shock. The case was followed at the end of 1, 3, 6 months by ECG and Holter monitor and ECG gated SPECT at the end of 12 months. The gated SPECT images demonstrated mild but definitely improved tracer uptake within part of the infarcted segments along with improvement in ejection fraction to 45%, and a clinical change in the NYHA Class to II. Cell-based therapy may offer benefits of induction of normal tissue microenvironment.

INTRODUCTION

Cardiac remodeling using adult stem cell therapy in clinical trials of post-myocardial infarction (MI) injury, as the therapeutic strategy during cardiac surgery is entering clinical practice.[1] The experimental and clinical models in various studies have yielded equivocal results. At present, this treatment is highly promising to become a standard treatment due to its effectiveness. The variables are confounding in these studies. It is yet to be determined the appropriate cell dosing, cell type, timing of the therapy, choice of cases, and the degree of stem cell approach for treating these otherwise nonamenable to therapeutic option cases.[2] Here we report adult stem cell therapy for myocardial revascularization and repair during CABG surgery. It is also important to determine objectively the functional improvement in these cases due to stem/progenitor cell therapy alone. Molecular imaging is rapidly taking the center stage in determining the effects of the stem cell therapy in vivo and the long-term follow-up.[3] Molecular imaging itself is currently evolving with focus on the multimodal approach for addressing the issues of stem cell homing, proliferation, and differentiation.[4] It has been, therefore, aimed at, to assess the improvement in the LV function and the global ejection fraction, using autologous bone marrow mononuclear fraction as an adjunct to CABG surgery, using noninvasive molecular imaging, 2D Echo along with the changes in the NYHA class changes in a case of scarred myocardium due to severe myocardial infarction.

MATERIALS AND METHODS

The study has been IRB/IEC approved. High risk video consent was obtained. The case has been recruited into the study which is registered with ICMR with universal trial number: TEMP UTRN 042233121-1506201022763720.

Screening procedures

The case was screened at the baseline for the extent of scarred myocardium using ECG gated SPECT imaging of myocardium after rest injection of Tc99m Sestamibi augmented with sublingual nitrate preparation. NYHA class was determined based on the questionnaire of NYHA and was classified under category III. The cardiac MRI and SPECT images were addressed at the baseline and the ejection fraction was estimated along with the 2D-echocardiogram. Akinetic and dyskinetic segments were assessed.

Bone marrow aspiration

Bone marrow (20.0 ml) from sternum during coronary bypass surgery (CABG) from 62-year-old male was aspirated under general anesthesia (GA).

Mononuclear fraction isolation

The bone marrow was equally diluted with normal saline, and the mononuclear fraction was isolated using the clinical grade Ficoll gradient and centrifugation method (Sigma, USA). The cells were counted in a coulter counter and viability was checked using the trypan blue dye exclusion method. The sample was assessed for CD34+/CD45+ cells (0.7%) using FACS Calibur prior to dispatch of cells to the theater before the completion of the CABG surgery. The cells thus isolated were suspended in 2.0 ml of normal saline.

Stem cell therapy

The cell suspension was injected circumferentially at eight sites of 0.1 ml volume of cell suspension each in this case, surrounding the scarred myocardium.

Follow-up

The case was followed up periodically at 1, 3, 6 months using ECG, Holter monitor and ECG gated SPECT at the end of 1 year. The parameters assessed included the global ejection fraction, increased perfusion using SPECT image analysis, end-diastolic volume (EDV), end-systolic volume (ESV), echocardiography and changes in the NYHA class category and angina status.

RESULTS

A 62-year-old male patient with previous history of myocardial infarction, akinetic myocardial segments and an ejection fraction of <31% with the NYHA class III category was selected for the autologous bone marrow-derived mononuclear cell fraction injection during CABG surgery. Myocardial SPECT demonstrated full thickness infarct in 40% area of LAD territory. Bone marrow aspirate (20.0 ml) from sternum has yielded a mono nuclear cell fraction of 4.50 × 107 cells/ml with a positive 0.7% of CD45+/CD34+ cells. The cells were suspended in 2.0 ml of sterile normal saline to be injected at eight sites of the injured myocardium. There were no apparent side effects due to the procedure, i.e., life threatening events, major bleeds, reaction or shock. The case was followed at the end of 1, 3, 6 and 12 months. At the end of 1 year period, improved tracer uptake could be demonstrated by SPECT within part of the infarcted segments along with improvement in ejection fraction to 45% and clinical change in the NYHA Class to II [Figure 1].

Figure 1

Nitrate augmented myocardial tracer uptake was imaged by ECG gated SPECT using radiotracer Tc99m Sestamibi which revealed full thickness infarct in 40% area of LAD territory. At the end of 1-year period, improved tracer uptake could be demonstrated by SPECT within part of the infracted segments along with improvement in ejection fraction to 45%.

Ejection fraction improved from 31% to 45% as demonstrated by the % gated SPECT analysis of the hemodynamic data. LV end-diastolic volume per ml was 184 ml prior to the therapy followed by 208 ml post-1 year. Stroke volume index improved from 33 ml/m2 to 50 ml/ m2 . Similarly, LV end-systolic volume was 127 ml prior and 120 ml post-therapy. The infarct region as severe perfusion defect measured as % area under LAD territory has shown an improvement from 40% to 30%. The NYHA class demonstrated changes from Class III to Class II [Table 1].

Table 1

Cardiac function analysis at 1 year follow-up

DISCUSSION

The crucial issues that need consideration, while using stem cell therapy as a treatment option either as standalone therapy or synergistic in nature, are the cell type, cell number, therapeutic time window, route of delivery and critical evaluation of therapeutic benefit.[5-8] As reported by the Strauer et al., it is thought that the use of bone marrow mononuclear fraction would include the hematopoietic and nonhematopoietic cells that would be actively harnessing the tissue microenvironment to a desired protection level.[9] The preclinical models have demonstrated that hematopoietic fraction could differentiate to lineages that could regenerate the damaged tissue due to in vivo differentiation to cardiomyocytes using host niche (microenvironment).[10] The nonhematopoietic component owing to its immunomodulatory properties and release of trophic factors is assumed that it could induce the remodeling of the injured myocardium in a synergistic way to accelerate the repair process and regenerate a viable tissue and thereby increase the LV function.[11] There are preclinical reports suggesting the route of delivery as other important criteria for the accessibility of the stem cells. It was observed that the i.v. infusion of stem cells would be of little value as they would primarily go to liver, spleen and the residual fraction reaching the injured myocardium may not be adequate and hence, the controversial results of lack of improvement in some studies. other studies have demonstrated good results using combination of routes, i.e., intracoronary and intramyocardial for the maximum homing of cells for tissue repair.[12] However, other studies did not corroborate similar findings owing to complex variables and confounding factors. In order to maximize the availability of viable cell to the scarred tissue, direct small volume injections at number of sites surrounding the injured myocardium was administered to the case under discussion. Reports from Lunde and Aakhus indicated the long-term improvement in acute myocardial infarction (AMI) using intracoronary mBMC treatment.[13] The optimum cell dose for the stem treatment is yet to be determined. However, the dose that we adopted in this case was as per our previous publication, where the improvement of LV function was dose-dependent.[14] Meluzín et al. reported that mBMC transplants demonstrate regional myocardial function of the infarcted wall in a dose-dependent manner.[15] In an another study of dilated cardiomyopathy, our results corroborated with the group on the improved ejection fraction with increased CD34+/CD45+ cells.[15] Dose-dependent improvement is a significant observation from different groups necessitating a need to arrive at an algorithm based on variable factors. And a large cohort of the sample size from all over world can alone circumvent the dose criteria. The current follow-up criteria from each study group thus far have singularly focused on the functional improvement. With the availability of noninvasive technology, it is now possible to address the structural stratification vis-à-vis the functional improvement.[1617] The reported case has significantly demonstrated the increased perfusion and the improved viable myocardium on the rest MPI gated SPECT images due to stem cells although it may be argued that CABG is responsible for the LV function improvement. The gated SPECT images demonstrate the nitrate augmented myocardial tracer uptake using radiotracer Tc99m Sestamibi which revealed improved tracer uptake within part of the infarcted segments along with improvement in ejection fraction to 45%. It is pertinent to the stem cell therapy to optimize the therapeutic window time as this may limit the performance of the cells in the acute stage with inflammatory responses and the implications thereafter in the AMI.[18] With the advances in the imaging technology, it is important to address the stem cell tracking for its homing, proliferation, and differentiation potential to be able to provide tangible solution to the critical factors and the benefit of the stem cell therapy in modern medicine.