/  Part III.6 – Pathology Assessment for Regenerative Therapy



Pathology Assessment for Regenerative Therapy

Stefanie Dimmeler PhD, Annarosa Leri MD, Marcello Rota PhD, Marion Muhly-Reinholz MTA, Andreas M Zeiher MD and Piero Anversa MD

A. Characteristics of the Normal Myocardium

The morphology of the normal myocardium involves three principal stages of organization: tissue, cellular and subcellular. The proportion and distribution of cardiomyocytes, vascular structures, connective tissue cells, collagen framework, and other extracellular constituents comprise the architecture of the tissue level; the quantities and configurational properties of the cytoplasmic organelles characterize the subcellular level. It is at the intermediate cellular level, however, that the fundamental mechanisms of cell renewal and cellular hypertrophy are defined. Endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts can increase in number and volume, but they constitute only a small fraction of the myocardium, so that their changes have only modest effects on cardiac anatomy. Conversely, cardiomyocytes represent the major portion of the tissue, and modifications in their number, size, and shape have important consequences for ventricular dimension (1, 2).

For the purpose of this chapter, we will focus on the physiological composition of the myocardium in the adult rodent heart, because mice and rats continue to represent the most common laboratory animals used in the analysis of spontaneous or cell-induced tissue regeneration. At 3-4 months of age, the rat left ventricle (LV) contains ~23 x 106 cardiomyocytes with nearly 95% of the cells being binucleated and ~5% mononucleated; binucleated myocytes are ~20,000 μm3 in volume and mononucleated myocytes ~10,000 μm3 in volume. A similar proportion of mononucleated and binucleated myocytes is present in the mouse LV, which is composed of 3.2 x 106 parenchymal cells (3-5). Cell size is comparable in both species, and the difference in myocardial mass is dictated by the larger number of cardiomyocytes in the rat heart. In the mouse and rat heart, the myocyte compartment comprises ~80-85% of the tissue, while the volume percent of capillary lumen varies from 8% to 9%; capillary numerical density averages ~3,500 capillaries/mm2 of myocardium, and the capillary-to-myocyte ratio ranges from 0.8 to 0.9, indicating that an almost equal number of myocyte and capillary profiles are present in cross or longitudinal sections of the myocardium (5, 6). The other interstitium occupies a small fraction of the parenchyma with fibrillar collagen accounting for ~2% of the tissue (2, 7). These simple quantitative parameters are critical for the understanding of the cellular mechanisms implicated in the myocardial response to infarction experimentally, in the presence and absence of stem cell therapy.

B. Myocardial Infarction

It is common practice to measure infarct size histologically by computing the fractional volume of damaged tissue within the ventricle, acutely, subacutely, and chronically after permanent coronary artery ligation or transient ischemia followed by reperfusion. Similarly, estimations of infarct size continue to be based on the evaluation of the proportion of endocardial circumference or endocardial surface bordering the infarcted portion of the ventricle. These relative measurements of infarct size may be only partially correct early after the ischemic event when modest changes have occurred in both the quantity of the infarcted tissue and the remaining intact myocardium. However, important structural modifications supervene throughout the healing process, making this methodology highly unreliable. These histometric techniques, in fact, contain various sources of error, dependent upon the progressive reduction of necrotic tissue with time, scar formation with thinning of the wall, dilation of the ventricular chamber (Fig. 1), and the unknown amount of hypertrophic growth and regeneration of the spared myocardium (8). These are all dynamic processes that progressively change the proportion between viable and nonviable tissue in the injured heart (2,9-12).

figure-1Figure 1. Myocardial infarction. Cross-section of a large myocardial infarction in a mouse heart 8 days after permanent coronary artery occlusion. The infarcted area (MI) included between the two arrowheads is characterized by significant thinning of the wall. Cavitary dilation is also apparent.

Myocyte hypertrophy and regeneration develops in the stressed spared myocardium, and shrinkage of the necrotic zone progresses with time, leading to an underestimation of the extent of myocardial infarction and to an overestimation of the initial volume of ventricular mass destined to survive the ischemic event. These volume changes impose the implementation of a morphometric methodology, which is independent from such unknown factors, providing a consistent reference point for comparison short-, mid-, and long-term after the ischemic event. The most logical approach involves the quantitative estimation of the number of myocytes lost and remaining after infarction (2,9,13-19). Moreover, there is a direct relationship between the amount of scar being formed and the quantity of myocyte death. By this analysis, absolute infarct size, i.e., percentage of myocytes lost, can be accurately measured at any point in time throughout the evolution of the process, offering a reliable parameter for comparison. Similarly, the number and size of the spared myocytes give a true measurement of the myocardial response to infarction directly correlated with the level of depression or recovery of ventricular performance in the damaged heart. The determinations at the myocyte level can be easily integrated with the characteristics of the coronary microcirculation defining the structural variables that condition tissue oxygenation and myocyte contractility.

By applying this methodology, it has been possible to demonstrate that an approximate 60% shrinkage of the necrotic myocardium occurs with healing. Additionally, by comparing the histometric method with the morphometric method, the former technique results in an underestimation of real infarct size of nearly 70% (2,9). These striking differences cannot be ignored, strongly suggesting that more sophisticated methodologies have to be introduced in the characterization of the infarcted heart.

In summary, the morphometric approach discussed above enables the evaluation of (a) real infarct size, (b) magnitude of normal and induced tissue growth occurring in the spared myocardium, (c) extent of myocyte and capillary adaptation, and (d) residual deficits in capillary and myocyte growth. These four aspects are fundamental for the recognition of the potential beneficial effects of cell therapy on acute myocardial infarction and chronic ischemic cardiomyopathy.

C. Myocardial Fibrosis in Post-Infarction Ischemic Cardiomyopathy

There are no good animal models that mimic the complexity of post-infarction ischemic cardiomyopathy (IC) in humans. Myocardial fibrosis, in its different aspects, is commonly found in the decompensated heart of patients affected by IC; however, it is almost impossible to reproduce this condition experimentally leaving unanswered some critical questions concerning the mechanisms involved in the disease pathology of the disease. Before discussing the methodologies required for a correct analysis of myocardial fibrosis, some comments on the characteristics of human IC may be helpful (20).

Ischemic cardiomyopathy is initiated by primary events in the coronary circulation that lead to myocyte loss, scarring, and ventricular failure. Cell loss occurs as a result of narrowing and/or occlusion of coronary arteries by atherosclerosis, spasm of major or intramural arterial branches of the coronary vasculature, or alterations of the microcirculation, which, alone or in combination, produce varying degrees of ischemia and tissue injury (21-27). In the most common manifestation, in which scattered foci of replacement fibrosis are found in conjunction with a healed infarct, the question remains concerning the pathophysiological significance of these two types of myocardial lesions. It has not been established as yet, whether the segmental loss of myocardium associated with coronary artery occlusion and myocardial infarction is the principal determinant of myocyte cell loss, wall thinning, ventricular dilation, and worsening of the hemodynamic performance with time, or whether the multiple isolated sites of tissue injury in the noninfarcted portions of the wall are major factors in the progression of IC (Fig. 2).

figure-2Figure 2. Ventricular remodeling after myocardial infarction. Large transverse myocardial sections of paraffin embedded tissue illustrating a healed myocardial infarct with thinning of the wall (left panel) and multiple sites of replacement fibrosis, in the noninfarcted viable tissue (right panel).

Segmental myocardial scarring, replacement fibrosis, and interstitial fibrosis together contribute to the accumulation of collagen in the ventricular myocardium with IC. The volume percent of myocardial scarring associated with infarct healing, 9%, was less than that dictated by replacement fibrosis in the nondamaged myocardium, 14% while nterstitial fibrosis comprised 6% of the tissue (20). When these forms of myocardial fibrosis are combined, 28% of the ventricular wall with IC is replaced by fibrotic tissue. These values represent dramatic changes with respect to the structure of the intact heart.

Experimentally, chronic myocardial infarction in rodents is characterized by widening of the interstitial space and collagen deposition in the spared portion of the wall. However, the magnitude of the process is far from being comparable to that seen in humans (28-30), and small foci of replacement fibrosis are typically absent. The latter form of collagen accumulation is observed with prolonged coronary artery narrowing (31,32), myocardial aging (33,34), and angiotensin II dependent systemic hypertension (35,36).

Although there is general agreement that myocyte loss is the etiological factor of replacement fibrosis in the ventricular wall, less clear is the mechanism responsible for activation of the cardiac interstitium, resulting in the accumulation of fibrillar collagen between myocytes. Claims have been made that hormonal and/or hemodynamic overloads (37,38) may trigger fibroblast proliferation and collagen neosynthesis in the myocardium, independently from myocytolytic necrosis and muscle cell loss. However, death of individual myocytes by the activation of the necrotic pathway occurs in apparently healthy individuals (39-41), and in rodents in the absence of pathologic stresses (15,42), raising the possibility that this scattered form of cell death may stimulate discrete healing promoting the expansion of the interstitium. Moreover, cell apoptosis is commonly seen physiologically and it increases dramatically with myocardial aging and chronic heart failure (43-45) representing an important variable of the cumulative loss of cardiomyocytes during the lifespan of the organ in animals and humans (15,46-49). Thus, the ischemic cardiomyopathic process may be initiated by coronary occlusion and myocardial infarction, but its evolution is controlled partly by a number of interrelated events occurring in the noninfarcted myocardium.

Several protocols have been used for the identification and quantitative evaluation of myocardial scarring, replacement fibrosis, and interstitial fibrosis. Trichrome and picrosirius red staining have frequently being used to label collagen, but the level of resolution of the light microscope may interfere with an accurate measurement of fibrillar collagen inter-dispersed between cardiomyocytes. The most accurate evaluation of collagen content within the myocardium is obtained by combining immunolabeling of collagen type 1 and type 3 with confocal microscopy. This strategy provides an accurate recognition of the distribution of fibrillar collagen as well as a clear definition of the boundary of the sites of replacement fibrosis within the myocardium. Segmental myocardial scarring associated with myocardial infarction can also be clearly defined by this state of the art methodology (48,50-52).

D. Stem Cells and Cardiac Repair

To properly appreciate the role that endogenous or exogenous stem cells have in myocardial regeneration and the methodology required for its identification, it is important to briefly discuss the controversy in the field that continues to divide the scientific community. The acceptance of the shift in paradigm of myocardial biology promoted by the discovery of c-kit-positive cardiac stem cells (CSCs) and the subsequent identification of various other stem cell populations (e.g. Is1t+, Sca-1+ cells, cardiosphere-derived cells, cardiac side population cells, mesenchymal stromal cells), has been problematic (Fig. 3) (5,50,53-59). The initial enthusiasm triggered by the identification of endogenous CSCs was followed by a wave of skepticism about the possibility that myocyte regeneration is a critical determinant of cardiac homeostasis and pathology. This position led to opposing studies published in high impact factor journals (60), with accompanying editorials that neglected CSC function, emphasizing anachronistically the limited nature of myocyte renewal in the human heart (61-64).

figure-3Figure 3. Cardiac progenitor cell classes. Schematic representation of distinct CPC categories in the embryonic-fetal and adult heart.

In 2003, one year after the identification of CSCs (53), the ability of the heart for proliferative growth was defined as “on shadings between none and almost none” (65), whether it derives from activation of resident CSCs or hematopoietic stem cells (HSCs). More recently, however, cardiomyocyte proliferation gained new and increasing attention. After being controversially debated for almost two decades, recent lineage tracing experiments support the occurrence of a very high regeneration of cardiomyocytes in early postnatal mice (66) and in young humans (67). Of note, growth factors and small non-coding RNAs were suggested to stimulate the endogenous cardiomyocyte proliferative activity (68,69).

Myocyte apoptosis occurs in the normal human heart (48), and it increases markedly with chronic heart failure (CHF) (43-45). Moreover, myocyte necrosis, documented directly by alterations in the cell membrane (70), or indirectly by plasma levels of troponin T (40), is present in apparently healthy individuals and in patients with stable coronary artery disease (39,41). In the latter case, this biomarker represents an independent prognostic indicator of the incidence of cardiovascular events and CHF, suggesting that ongoing myocyte necrosis accompanies the natural evolution of the disease, and its magnitude has consequences on clinical outcome. Myocyte death has to be accompanied by myocyte regeneration for the heart to continue to exist (71,72). The simple concept of a requisite equilibrium between myocyte death and renewal (cardiac cellular homeostasis) has been ignored and inconsistencies have been overlooked to perpetuate the old notion of myocardial biology. Findings supporting the view of high levels of myocyte formation in humans (48,49) were criticized and negative results considered valid (73-76), neglecting the principle that new cells are required to offset the cumulative effect of myocyte death. Similarly, results showing the potential contribution of HSCs to cardiomyogenesis (13,14) were questioned and negative data (77,78) were accepted as further demonstration of the absence of myocyte renewal in the adult heart. With extreme care, new data on HSC differentiation (79) were ignored and the ingrained paradigm purporting the lack of myocyte formation in the adult heart perpetuated (64).

The initial skepticism on the function of CSCs was followed by a second wave of criticisms, questioning the actual existence of c-kit-positive CSCs (61,62,76,80); if present, they could have, at most, a negligible or modest effect. For this reason, the use of cell therapy in patients with CHF was considered premature and potentially dangerous, until lineage tracing studies in mice were performed and the identification of “authentic” CSCs in small animals was definitely proven (64). According to this view, the answer to the human disease has to be found in transgenic mice and studies of the human heart are considered irrelevant to the recognition of novel approaches for the treatment of CHF. The devastating consequences of CHF and limitations in therapeutic options have prevented the detractors of myocardial regeneration from interfering with the implementation of cell therapy in the management of the human disease (81-83).

Stem Cell Fate

One of the most important questions posed by stem cell therapy concerns the destiny of the delivered cells. Fate mapping strategies, based on fluorescent reporter genes, are commonly used to track the origin of cells and their destiny in animals in which genetic manipulations are easily introduced. This approach would represent the ideal retrospective assay for the detection of a specialized progeny, since the expression of the fluorescent label can be placed under the control of promoters of genes coding for cardiomyocyte and vascular proteins. However, with lineage tracing studies, it is impossible to determine whether stem cells divide asymmetrically, i.e., self-renew, or whether the cell types of the tagged progeny derive from activation of an individual or several resident stem cells, i.e., unipotent or multipotent. Additionally, this genetic approach cannot be implemented in the characterization of human CSCs (72,84).

These limitations have been overcome by employing single cell-derived clonal CSCs infected with a lentiviral vector expressing enhanced green fluorescence protein (EGFP) (50,52). This strategy can provide indisputable evidence in favor of the ability of CSCs to self-renew and create myocardial tissue in vivo, following their delivery to animal models of the human disease. The most commonly introduced pathology is myocardial infarction or ischemia-reperfusion injury (50,52,85). To avoid rejection of the administered CSCs, immunodeficient mice and rats, or immunosuppressed mice, rats, dogs and pigs can be used, as repeatedly performed in several laboratories (18,50-52,57,86-91).

Three important requirements have to be met: (a) aliquots of clonal cells need to be analyzed by FACS to establish whether cell expansion preserved the undifferentiated state of clonal CSCs; (b) the telomere-telomerase axis has to be measured to determine whether this determinant of cellular growth reserve is largely maintained in clonal CSCs; and (c) the expression of the senescence-associated protein p16INK4a has to be assessed to evaluate whether only a small fraction of clonal CSCs has reached replicative senescence and growth arrest. Following this relatively simple characterization of clonal CSCs, the next critical issues concern the number of clonal CSCs to be delivered, the route of administration, and the mechanisms involved in their potential therapeutic effects.

Stem cell delivery

Experimentally, clonal and non-clonal CSCs have been predominantly injected intramyocardially in the region bordering acute or healed infarcts, although intracoronary delivery has been employed as well (18,50-52,57,85-87,91,92). The latter was consistent with the documentation by two-photon microscopy that c-kit-positive CSCs can traverse the vessel wall of the distal coronary microcirculation reaching areas of damaged myocardium (93). Whether a similar outcome applies to mesenchymal stromal cells (MSCs) isolated from the bone marrow, adipose tissue, or the epimyocardium (59,88,89,94-99) remains to be shown. However, c-kit-positive CSCs are significantly smaller than MSCs.

The adult myocardium is a rather compact structure with a limited interstitial compartment that precludes homing and engraftment of a large number of CSCs or MSCs, when cells are delivered directly into the muscle by needle injection. The use of millions of cells via this route is inappropriate; only a very small fraction of cells will ultimately survive within the narrow space that separates cardiomyocytes from the capillary network. It is common practice in our laboratory to make multiple injections, each of no more than 10,000 clonal CSCs. This amount takes into consideration the inevitable loss of cells during delivery and high beating rate of the mouse and rat heart with discharge of part of the cell solution. In larger animals, particularly in dogs, the intracoronary delivery of 5 x 106 c-kit-positive CSCs has been shown to be effective with no adverse events (51). Repeated intracoronary administration of 5 x 106 c-kit-positive CSCs have also been used successfully. However, no experimental protocol has been developed as yet to optimize the number of CSCs to be given either intramyocardially or via the coronary circulation. This remains an important unresolved issue, critical for testing the modality of intervention and the efficacy of different stem cell classes. Currently, the lack of this information precludes the comparison of therapeutic outcome among different laboratories; a variety of cell categories, prepared without a unified methodology, are administered in different quantities to distinct animal models raising more questions than providing answers to the complexity of cell therapy for human heart failure.

Stem cell survival and engraftment

The fate of the delivered CSCs is dictated by their ability to incorporate within the recipient myocardium. These cells are easily recognizable by the expression of the stem cell surface antigen c-kit and green fluorescence. Relatively early in the process, ~12 hours after injection, CSCs seeded within the myocardium undergo apoptosis or integrate structurally with the surrounding cells; if CSCs fail to respond to the host microenvironment, the apoptotic rate increases progressively from 12, 24 to 48 hours leading to a complete disappearance of the implanted cells. Engraftment requires the synthesis of surface proteins that establish cell-to-cell contact and the interaction between cells and the extracellular matrix (79,100). Connexin 43, N-cadherin and E-cadherin are expressed in CSC homing to the myocardium. These proteins are found between CSCs and between CSCs and myocytes or fibroblasts, which function as supporting cells in the CSC niches (56); the injected CSCs have to form temporary niches within the unfamiliar myocardium to survive and grow in this unknown territory. Apoptosis is restricted to non-engrafted cells that failed to express connexin 43 and N-cadherin (100). This phenomenon is consistent with anoikis of the non-engrafted cells, which is one of the aspects of programmed cell death triggered by the lack of cell-to-cell contacts.

Importantly, a large proportion of engrafted CSCs enter the cell cycle as documented by BrdU labeling and expression of the cell cycle protein Ki-67. Phospho-H3 is also detected in dividing CSCs. Cell proliferation increases consistently with time, while cell apoptosis is gradually attenuated. Because of these two variables, ~25% of the injected endogenous or exogenous stem cells are present in the viable tissue of the border zone 2 days after infarction (79,100). Thus, CSCs survive and grow within the myocardium by forming junctional complexes between them and with resident myocytes and fibroblasts, crucial determinants of cell integration within the damaged myocardium.

CSCs and the cardiogenic fate

The high level of cell proliferation present in the engrafted CSCs typically distinguishes temporary niches from resident stem cell niches, which are composed predominantly of quiescent CSCs (56). The expression of connexin 43 accompanies the phenotypic conversion of primitive undifferentiated cells into a specialized cell progeny (Fig. 4). Markers of cardiomyocytes and vascular ECs and SMCs become apparent in the engrafted CSCs, suggesting that the myocardial microenvironment changes the fate of the delivered stem cells (50-54,57,79,86,87,92,100).

figure-4Figure 4. Differentiation of CPCs into cardiomyocytes. Schematic representation of the process of myocyte lineage commitment.

Recently, it has been shown that the commitment of CSCs to the myocyte lineage and the generation of functionally-competent adult cardiomyocytes is influenced by miR-499, which is barely detectable in primitive cells, but is highly expressed in post-mitotic cardiomyocytes (101). miR-499 traverses gap junction channels and may translocate in vivo from cardiomyocytes to structurally coupled CSCs favoring the activation of the differentiation program by downregulating the expression of the stemness genes Sox6 and Rod1. The mircrine mechanism of CSC lineage specification may apply equally to vascular ECs and SMCs.

CSC differentiation and/or paracrine effect

In the last several years, the possibility that cell therapy of the infarcted heart exerts its beneficial effects by the activation of resident stem/progenitor cells located in the surviving myocardium has been claimed (102). According to this hypothesis, the injected cells may contribute indirectly to cardiac regeneration by releasing a variety of peptides that exert a paracrine action on the myocardium and its resident progenitor cells. Alternatively, these cells may promote vasculogenesis through the recruitment of circulating bone marrow cells. However, with the exception of a few studies with bone marrow-derived cells and MSCs (103,104), this contention has predominantly been used to argue against HSC transdifferentiation (80). In an attempt to justify an improved cardiac performance in the absence of myocardial regeneration following the delivery of HSCs, the claim has been made that the injected cells release a variety of growth factors that, by unclear mechanisms, trigger an intrinsic positive response of the recipient myocardium.

It is rather surprising that, in the absence of actual supporting data, this phenomenon has been largely accepted. The documentation of a paracrine effect exerted by any cell type can easily be documented by measuring the formation of myocytes and coronary vessels by labeling protocols including BrdU or other thymidine analogs. Therefore, we tested whether the formation of myocytes and coronary vessels increases in the surviving myocardium after infarction in animals treated with HSCs or various classes of CSCs (16,79,92,105). Since BrdU was given to the animals throughout the experimental period, newly formed structures generated by endogenous mechanisms had to be positive for BrdU and negative for EGFP. Cell therapy had similar effects on the surviving myocardium. In all cases, the extent of myocyte and vessel formation was comparable to that measured in untreated infarcted animals (Fig. 5). Moreover, the degree of cell replication at sacrifice was determined by Ki-67 and MCM5 labeling. Again, comparable values were obtained in cell-treated and untreated infarcted hearts. Thus, our observations support the notion that CSCs or HSCs generate cardiomyocytes and coronary vessels restoring partly the structural and functional integrity of the infarcted heart.

figure-5Figure 5. Characteristics of the surviving myocaardium. Myocyte volume, arteriole and c apillary density, and proliferation of myocytes and ECs. SO indicates sham-operated; MI, untreated infarcted hearts; CPCs, infarcted hearts treated with CPCs.

Identification and Quantification of Stem/Progenitor Cell Mediated Cardiomyogenesis and Vasculogenesis

Several approaches can be used to ensure an accurate recognition and morphometric measurement of regenerated cardiomyocytes and coronary vasculature. The use of EGFP-tagged stem/progenitor cells from the heart or the bone marrow allows the identification of the derived progeny, since the reporter gene is transferred to the differentiated newly-formed cardiomyocytes and resistance arterioles and capillary microcirculation (16,50-53,57,79,92,105). To strengthen the protocol, male stem/progenitor cells may be delivered to female recipient animals so that the localization of the Y-chromosome by Q-FISH allows the distinction of regenerated male structures from resident female myocytes and coronary vessels (72).

Additional strategies can be implemented when human stem/progenitor cells are injected in immunodeficient or immunosuppressed animals. Under these conditions, human DNA sequences are easily detectable with an Alu probe, in combination with the distribution of the human X-chromosome and/or Y-chromosome (50,52,57,87). Myocardial structures carrying human sex chromosomes can be distinguished from myocardial structures labeled by mouse, rat or dog X-chromosome and Y-chromosome (50-52); this approach provides a reliable reference point for the separation of regenerated human myocardium from mouse, rat or dog host myocardium. Similarly, q-RT-PCR is commonly employed to detect the expression of human genes specific for sarcomeric proteins, and EC, and SMC proteins (50).

An important aspect related to immunolabeling of cardiomyocytes, ECs, and SMCs concerns the specificity of the fluorescent signal and its separation from tissue autofluorescence inherent to the fixation and processing of myocardial samples. This problem is overcome by introducing spectral analysis that discriminates these variables, supporting or questioning the validity of the protocol (51,52,86,87). Currently, confocal microscopes with incorporated spectral analysis are available facilitating the analysis of immunofluorescence.

E. Vascularization

The enhancement of blood supply either by stem/progenitor-cell mediated vasculogenesis or alternatively by angiogenesis (growth from existing capillaries) or atherogenesis (remodeling of pre-existing capillaries) is important for any type of cardiac repair. It is well established that capillary density in the infarct border zone determines infarct expansion (2). Moreover, recent studies additionally provide evidence in other organs such as lung and liver that endothelial-derived organ specific angiocrine signals mediate tissue regeneration (106,107).

Capillary density is measured by immunohistochemistry directed against endothelial markers such as PECAM (CD31), von Willebrand factor, or VE-cadherin or by binding of fluorescently labeled lectin to tissue sections (108-110). In addition, perfused vessels can be stained by i.v. infusing fluorescently labeled lectin (109) can be detected by counterstaining with smooth muscle actin (109) whereas pericyte recruitment can be detected by NG2 staining.

F. Summary and Conclusions

This chapter provided an overview of the pathology for regenerative therapy for the heart. The physiological cell and tissue mass composition of the mouse and rat heart were outlined. This background helped set the stage for understanding the pathological measurements of myocardial infarction and potential mechanisms for myocardial fibrosis post-infarction, including myocyte cell loss. Replacement of myocytes leads to the final sections of the chapter on stem cells. The sections on stem cells review current and past literature on different stem cell markers for cardiomyogenesis and vasculogenesis, engraftment and delivery.


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