Mark F. Pittenger

Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects

BACKGROUND A novel therapeutic option for the treatment of acute myocardial infarction involves the use of mesenchymal stem cells (MSCs). The purpose of this study was to investigate whether implantation of autologous MSCs results in sustained engraftment, myogenic differentiation, and improved cardiac function in a swine myocardial infarct model. METHODS MSCs were isolated and expanded from bone marrow aspirates of 14 domestic swine. A 60-minute left anterior descending artery occlusion was used to produce anterior wall infarction. Piezoelectric crystals were placed within the ischemic region for measurement of regional wall thickness and contractile function. Two weeks later animals autologous, Di-I-labeled MSCs (6 x 10(7)) were implanted into the infarct by direct injection. Hemodynamic and functional measurements were obtained weekly until the time of sacrifice. Immunohistochemistry was used to assess MSC engraftment and myogenic differentiation. RESULTS Microscopic analysis showed robust engraftment of MSCs in all treated animals. Expression of muscle-specific proteins was seen as early as 2 weeks and could be identified in all animals at sacrifice. The degree of contractile dysfunction was significantly attenuated at 4 weeks in animals implanted with MSCs (5.4% +/- 2.2% versus -3.37% +/- 2.7% in control). In addition, the extent of wall thinning after myocardial infarction was markedly reduced in treated animals. CONCLUSIONS Mesenchymal stem cells are capable of engraftment in host myocardium, demonstrate expression of muscle specific proteins, and may attenuate contractile dysfunction and pathologic thinning in this model of left ventricular wall infarction. MSC cardiomyoplasty may have significant clinical potential in attenuating the pathology associated with myocardial infarction.

In Vivo Magnetic Resonance Imaging of Mesenchymal Stem Cells in Myocardial Infarction

Background—We investigated the potential of magnetic resonance imaging (MRI) to track magnetically labeled mesenchymal stem cells (MR-MSCs) in a swine myocardial infarction (MI) model. Methods and Results—Adult farm pigs (n=5) were subjected to closed-chest experimental MI. MR-MSCs (2.8 to 16×107 cells) were injected intramyocardially under x-ray fluoroscopy. MRIs were obtained on a 1.5T MR scanner to demonstrate the location of the MR-MSCs and were correlated with histology. Contrast-enhanced MRI demonstrated successful injection in the infarct and serial MSC tracking was demonstrated in two animals. Conclusion—MRI tracking of MSCs is feasible and represents a preferred method for studying the engraftment of MSCs in MI.

Dynamic Imaging of Allogeneic Mesenchymal Stem Cells Trafficking to Myocardial Infarction

Background—Recent results from animal studies suggest that stem cells may be able to home to sites of myocardial injury to assist in tissue regeneration. However, the histological interpretation of postmortem tissue, on which many of these studies are based, has recently been widely debated. Methods and Results—With the use of the high sensitivity of a combined single-photon emission CT (SPECT)/CT scanner, the in vivo trafficking of allogeneic mesenchymal stem cells (MSCs) colabeled with a radiotracer and MR contrast agent to acute myocardial infarction was dynamically determined. Redistribution of the labeled MSCs after intravenous injection from initial localization in the lungs to nontarget organs such as the liver, kidney, and spleen was observed within 24 to 48 hours after injection. Focal and diffuse uptake of MSCs in the infarcted myocardium was already visible in SPECT/CT images in the first 24 hours after injection and persisted until 7 days after injection and was validated by tissue counts of radioactivity. In contrast, MRI was unable to demonstrate targeted cardiac localization of MSCs in part because of the lower sensitivity of MRI. Conclusions—Noninvasive radionuclide imaging is well suited to dynamically track the biodistribution and trafficking of mesenchymal stem cells to both target and nontarget organs.

Dual-Modality Monitoring of Targeted Intraarterial Delivery of Mesenchymal Stem Cells After Transient Ischemia

Background and Purpose— In animal models of stroke, functional improvement has been obtained after stem cell transplantation. Successful therapy depends largely on achieving a robust and targeted cell engraftment, with intraarterial (IA) injection being a potentially attractive route of administration. We assessed the suitability of laser Doppler flow (LDF) signal measurements and magnetic resonance (MR) imaging for noninvasive dual monitoring of targeted IA cell delivery. Methods— Transient cerebral ischemia was induced in adult Wistar rats (n=25) followed by IA or intravenous (IV) injection of mesenchymal stem cells (MSCs) labeled with superparamagnetic iron oxide. Cell infusion was monitored in real time with transcranial laser Doppler flowmetry while cellular delivery was assessed with MRI in vivo (4.7T) and ex vivo (9.4T). Results— Successful delivery of magnetically labeled MSCs could be readily visualized with MRI after IA but not IV injection. IA stem cell injection during acute stroke resulted in a high variability of cerebral engraftment. The amount of LDF reduction during cell infusion (up to 80%) was found to correlate well with the degree of intracerebral engraftment, with low LDF values being associated with significant morbidity. Conclusions— High cerebral engraftment rates are associated with impeded cerebral blood flow. Noninvasive dual-modality imaging enables monitoring of targeted cell delivery, and through interactive adjustment may improve the safety and efficacy of stem cell therapy.

Adipogenic differentiation of human mesenchymal stem cells

Mesenchymal stem cells have the capability to differentiate into a number of cell types including adipocytes. The adipocytic phenotype is characterized by intracellular accumulation of lipid droplets as well as transcription of adipocyte-specific genes. This paper details a basic protocol for adipogenic induction of bone marrow and adipose tissue-derived stem cells, as well as protocols for staining lipid accumulation and the transcriptional analysis of PPAR-γ and aP2 by real-time RT-PCR.

Human mesenchymal stem cells: progenitor cells for cartilage, bone, fat and stroma.

Bone marrow provides the rich milieu necessary to maintain myeloid and lymphoid progenitor cells throughout the life of an organism. At least two stem cell populations have been identified in marrow, the hematopoietic stem cell (HSC) and the mesenchymal stem cell (MSC). The HSC has been characterized in many ways, but much remains to be learned about its intrinsic potential and interactions with other cells of the marrow environment. We have studied the human stem cell population for mesenchymal tissues that resides in adult bone marrow. These MSCs potentially have the ability to differentiate to all mesenchymal cell types, including osteocytic, chondrocytic, adipocytic, myocytic, tenocytic, and also dermal and stromal lineages (1, 2). We have sought to understand the potential role(s) that MSCs play in healthy individuals and their response to trauma, disease or aging.

111In oxine labelled mesenchymal stem cell SPECT after intravenous administration in myocardial infarction

Mesenchymal stem cells (MSCs) have shown therapeutic potential if successfully delivered to the intended site of myocardial infarction. The purpose of this pilot study was to test the feasibility of 111In oxine labelling of MSCs and single photon emission computed tomography (SPECT) imaging after intravenous administration in a porcine model of myocardial infarction. Adult farm pigs (n = 2) were subjected to closed chest experimental myocardial infarction. 111In oxine labelled MSCs (1×107 to 2×107 cells) were infused intravenously, and SPECT imaging was performed initially and on days 1, 2, 7 and 14. High quality SPECT images were obtained through 2 weeks of imaging. High initial MSC localization occurred in the lungs and slow progressive accumulation occurred in the liver, spleen and bone marrow. Renal activity was mild and persistent throughout imaging. No appreciable accumulation occurred in the myocardium. It is concluded that 111In oxine radiolabelling of MSCs is feasible, and in vivo imaging with SPECT provides a non-invasive method for sequentially monitoring cell trafficking with good spatial resolution. Because intravenous administration of MSCs results in significant lung activity that obscures the assessment of myocardial cell trafficking, alternative routes of administration should be investigated for this application.

MR-trackable intramyocardial injection catheter

There is growing interest in delivering cellular agents to infarcted myocardium to prevent postinfarction left ventricular remodeling. MRI can be effectively used to differentiate infarcted from healthy myocardium. MR‐guided delivery of cellular agents/therapeutics is appealing because the therapeutics can be precisely targeted to the desired location within the infarct. In this study, a steerable intramyocardial injection catheter that can be actively tracked under MRI was developed and tested. The components of the catheter were arranged to form a loopless RF antenna receiver coil that enabled active tracking. Feasibility studies were performed in canine and porcine myocardial infarction models. Myocardial delayed‐enhancement (MDE) imaging identified the infarcted myocardium, and real‐time MRI was used to guide left ventricular catheterization from a carotid artery approach. The distal 35 cm of the catheter was seen under MRI with a bright signal at the distal tip of the catheter. The catheter was steered into position, the distal tip was apposed against the infarct, the needle was advanced, and a bolus of MR contrast agent and tissue marker dye was injected intramyocardially, as confirmed by imaging and postmortem histology. A pilot study involving intramyocardial delivery of magnetically labeled stem cells demonstrated the utility of the active injection catheter system. Magn Reson Med 51:1163–1172, 2004.

Mesenchymal Stem Cells

Many adult mammalian tissues maintain a healthy state by continuous renewal involving cell turnover. In response to trauma, disease or overuse, the body either repairs or regenerates the tissue.These two possibilities are distinguished in that regeneration results in new tissue that is indistinguishable from the original tissue in its structural organization, cellular content and function, whereas repair results in a high content of fibroblastic tissue, scar formation, limited structural organization and impaired function. Certain tissues, including skin, intestine, epithelium, and skeletal muscle have regenerative ability owing to resident progenitor cells. Other regenerating tissues, such as liver, have differentiated cells that retain the ability to de-differentiate and re-enter a proliferating growth phase before differentiating once again. Many types of blood cells originate from hematopoietic stem cells (HSCs) present in the sinusoids of bone marrow. In addition to progenitor cells resident in tissues, multipotent stem cells capable of connective tissue regeneration reside in bone marrow. The in vitro and in vivo study of these bone marrow-derived mesenchymal stem cells (MSCs) is important in developing a comprehensive understanding of the dynamic processes that occur in regenerating tissues and the roles that MSCs play. The characterization of proliferative fibroblastic marrow cells with the potential to differentiate has been explored from multiple species including mouse [1-7], guinea pig [8,9], rat [10-14], rabbit [15-20], dog [21-23], horse [24, 25] and man [26-35 and references therein], and several reviews have been published [49-55]. While many of these reports suggested the stem cell nature of the cells under study, the characterization was often incomplete.

Bone Marrow-Derived Stem Cell for Myocardial Regeneration: Preclinical Experience

Adult stem cells are found in many tissues and participate in adult growth as well as repair and regeneration of damaged tissue. Adult stem cells such as MSCs may be the cell of choice for tissue repair because the cellular and tissue environment in the adult is likely very different from the early embryo conditions that produce embryonic stem cells. Bone marrow provides an accessible and renewable source of adult mesenchymal stem cells that can be greatly expanded in culture and characterized. Culture-expanded and characterized MSCs have been tested for their ability to differentiate into several lineages in vitro and also tested in animal models for their ability to enhance tissue repair and undergo in vitro differentiation. One of their greatest attributes is their potential to supply growth factors and cytokines to repairing tissue. MSCs do not appear to be rejected by the immune system, allowing for large scale production, appropriate charaterization and testing, and the subsequent ready availability of allogeneic tissue repair enhancing cellular therapeutics. This provides for the further development of this new field and paves the way for the use of yet other stem cells. The potential to use MSCs to repair damaged cardiovascular tissue is very promising and moving forward quickly. The current results from many labs and early cardiac clinical studies suggest important therapeutic approaches will be forthcoming through the use of MSCs. Perhaps most importantly, the understanding of adult stem cells such as the MSCs will provide us with greater understanding of the role they play in human biology in the developing and aging man.