Stem Cell and Gene Therapy for Cardiovascular Disease

435–436.

Part I

Stem Cells

Outline

Chapter 1 Introduction and Overview of Stem Cells

Chapter 2 Pathological Assessment of Experimental Models of Stem Cell and Other Regenerative Therapies

Chapter 3 Large Animal Models for Cardiac Cell Therapy

Section I Body’s Native Repair Mechanisms

Section II Autologous Bone Marrow

Section III Autologous Adipose Derived Regenerative Cells

Section IV Allogeneic Alternatives to Autologous Bone Marrow: The MSC

Section V Cardiac Progenitor Cells

Section VI Other Allogeneic Sources of Stem Cells

Section VII Genetic Engineering/Cell Transformation

Section VIII Methods of Delivery

Chapter 1

Introduction and Overview of Stem Cells

Leslie W. Miller¹ and Emerson C. Perin²,    ¹Visiting Research and Clinical Consultant, Texas Heart Institute, Houston, TX, USA,    ²Research in Cardiovascular Medicine, Stem Cell Center, Texas Heart Institute, Houston, TX, USA

One of the most exciting advances in medicine today has been the use of stem cell and gene therapy for repair in all forms of acute and chronic illness. Stem cells have been shown to be the primary mechanism for tissue repair everywhere in the body, and not limited to cardiovascular disease. Some have even opined that diseases like atherosclerosis represent a failure of stem cells. However, because cardiovascular disease has become the leading cause of death and morbidity in the world, it is the area that has seen the greatest evaluation by clinical trials of cell therapy in what is known as regenerative medicine. This introductory chapter is designed as a guide to understanding all the new sources and types of stem cells now in clinical trials, as well as the surface markers that help identify each type of cell.

Keywords

Stem cells; surface markers; stem cell sources and types

One of the most exciting advances in medicine today has been the use of stem cell and gene therapy for repair in both acute and chronic illness. Stem cells have been shown to be the primary mechanism for tissue repair everywhere in the body, and not limited to cardiovascular disease. Some even believe that diseases such as atherosclerosis represent a failure of stem cells. However, because cardiovascular disease has become the leading cause of death and morbidity in the world [1–3], this area has seen the greatest evaluation by clinical trials of cell therapy in the field known as regenerative medicine. This introductory chapter is designed as a guide to understanding the new sources and types of stem cells now being examined in clinical trials, as well as the surface markers that help identify each type of cell.

What is Regenerative Medicine?

Regenerative medicine can be defined as the use of stem cell or gene therapy to repair or recover damaged organs, tissues, or vessels. Stem cells have been the predominant therapy used to date, as they are the most immediately mobilized response to acute injury, but the use of targeted gene therapy is also being explored vigorously (see Part II on Gene Therapy). There are many examples in nature of tissue regeneration, including the newt, which can regenerate an entire limb within 6 weeks of amputation, and the zebrafish, which can restore amputation of the distal cardiac apex within 10–14 days [4]. The liver is the human organ most capable of regeneration [5].

Attempts to induce the heart to regenerate run contrary to the long-held tenant that the heart is a postmitotic, terminally differentiated organ [6,7]. However, programmed cell death, or apoptosis, occurs in the heart as in every other organ and tissue. Estimates of the loss, and therefore turnover, of the heart during a lifetime range between 50% and 3–4 fold [8]. Millions of people are living beyond 80 years of age without evidence of cardiac dysfunction, which by definition, requires the capacity of the myocardium to regenerate.

Many important lessons have been learned over the past decade, but it has been more challenging than anticipated to identify the mechanisms that will enable us to achieve significant regeneration of new functional cardiomyocytes and vasculature. Our understanding of the mechanisms involved in cell therapy has evolved as has our understanding of stem cell biology, including identification of factors such as chemokines, cytokines, adhesion molecules, and matrix metalloproteins that are involved in tissue repair (see Chapter 39). This knowledge has led to an increasing number of clinical trials that have also helped to move the field forward. However, questions remain including identification of the optimal cell type and number, the method and timing of delivery, and ways to potentially precondition cells or target tissue to enhance responses. In addition, extensive research is now focused on ways to enhance cell retention in the target tissue and to gain a better understanding of the importance of the extracellular matrix in this process (see Part II on Tissue Engineering). Here, we provide an introduction to the terminology and basics of stem cells used to drive native tissue repair and regeneration and a firm foundation of the current status of knowledge in this field.

What Defines a Stem Cell?

A stem cell can be defined by two specific characteristics:

1. Endless self-renewal, with no limitations or maximum number of replications.

2. Capability to differentiate into any tissue type and cell line in the body. The bone marrow provides two primary types of stem cells: those that will form all of the hematopoietic cells in the body, including red blood cells, white blood cells, platelets, lymphocytes, macrophages, etc. [9], and those from ectoderm, endoderm, and mesoderm origins that can differentiate into all tissue types in the body. Most stem cells have a predominant form(s) of cell that they differentiate into, which helps define them in the laboratory. For example, mesenchymal stem cells are characterized by differentiating into bone, cartilage, and adipose tissue [10,11], but they can also differentiate into functioning cardiomyocytes, blood vessels, and even neuronal tissue. This differentiation capacity is true of mesenchymal cells whether they originate from the bone marrow, adipose tissue, umbilical cord, or even placenta.

Types of Stem Cells

Stem cells may be classified in many different ways. The most traditional classification of stem cells is based on their plasticity or developmental versatility. They may thus be classified as totipotent (can give origin to an entire organism), pluripotent (can give rise to all tissues), and multipotent (can give rise to a limited range of cells within a tissue).

Stem cells may also be classified according to their origin.

Embryonic: These pluripotent cells may carry a high risk of development of unusual tumors called teratomas [12]. Research on embryonic stem cells is currently limited to basic science studies to help understand mechanisms of stem cell function. There are no clinical cardiac trials of embryonic stem cells. Their use has been associated with a significant controversy because of the moral and ethical issues surrounding their use.

Adult stem cells: These are multipotent cells that are undifferentiated cells in a differentiated tissue. They are used in widespread clinical investigations.

Stem cells are commonly classified according to cell surface markers. Many surface markers help identify individual types of stem cells. This feature aids in separating specific cell types by flow cytometry and other methods to obtain pure cell cultures. However, not every stem cell has a specific set of surface markers. For example, mesenchymal stem cells may have several different surface markers rather than one defining characteristic set of markers [13]; however, their unique feature of adherence to plastic surfaces helps define this cell type. Table 1.1 provides a list of the common surface markers that help define a specific type of stem cell.

Table 1.1

Surface Markers Used to Identify Stem Cells in Bone Marrow and Blood

BMPR, bone morphogenetic protein receptor; EPC, endothelial progenitor cell; HSC, hematopoietic stem cell; MSC, mesenchymal stem cell; WBC, white blood cell; Lin, lineage surface antigen; Sca-1, stem cell antigen 1; Stro-1, stromal cell antigen 1.

Stem Cell Sources

Another common way to classify stem cells is according to their source. Although stem cells were originally thought to be restricted primarily to adult bone marrow and embryos, the number of cell sources (from different tissues) has expanded significantly.

Bone Marrow

The bone marrow, although no longer the sole source of adult stem cells, is by far the greatest reservoir of stem cells. Its composition includes largely mononuclear cells, of which 95% are hematopoietic precursors, and an array of natural killer–type cells, as well as T and B lymphocytes [7,14,15]. In addition, other types of stem and progenitor cells, including endothelial progenitor cells (EPCs) [16], make up 1–2% of the total cell composition, and mesenchymal stromal cells (MSCs) constitute only 0.01–0.2%. Clinical trials have examined the use of the entire bone marrow mononuclear cell (BMMNC) population, as well as selected cell populations such as CD34+ EPCs, CD133+ smooth muscle progenitors, or, more recently, MSCs [17,18].

One of the lessons learned from the use of autologous bone marrow is of particular importance for treating patients with cardiovascular disease, who are often over the age of 60 years—the number and functional capacity of bone marrow cells decreases with each decade of life [19]. This is due in part to the well-documented reduction in telomere length of both hematopoietic and other stem cells with advancing age [20–22] (see Chapter 5), which may limit the capacity for these cells to respond as effectively as needed for optimal tissue repair. Clearly, there is often a disparity between chronologic and physiologic age, and no absolute age should be considered an exclusion for autologous cell therapy.

The bone marrow is the source of several other cells that have been developed for clinical use, including the multipotent adult progenitor cell (MAPC) [23] as well as the derivation into cardiac MSCs [24] (see below for details). Another fraction of cells from the bone marrow is characterized by the presence of an enzyme (aldehyde dehydrogenase; ALDH) and not by cell surface markers; this population is highly enriched for mesenchymal and EPC activities [25,26].

Adipose Tissue

Perhaps, one of the most unexpected sources of stem cells is adipose tissue. Several studies have shown that there are 500–2000 times more stem cells per gram of tissue in abdominal adipose tissue than there are in bone marrow in age-adjusted comparisons [27,28]. While abdominal tissue tends to be the most common repository of adipose tissue in the body, particularly with advancing age, other adipose sources from which stem cells have been derived include the buttocks, thighs, and even the lateral knee ligaments [29]. Harvesting of adipose tissue for stem cells and the addition of the enzyme collagenase yields what is known as stromal vascular fraction (SVF), which is a heterogeneous mix of cells that includes mesenchymal, hematopoietic, and EPCs. The SVF can be processed and made available for clinical application in as little as 2 h. In contrast, a component of the SVF may be cultured over time and used as a single cell type (e.g., adipose-derived mesenchymal cells) [28]. There are few side-by-side comparisons of bone marrow cells and adipose tissue-derived cells, but adipose stem cells respond to the same signals as bone marrow cells, such as stromal cell–derived factor-1, and seem to be potentially as potent and effective as bone marrow stem cells. Adipose stem cells have been studied in several clinical trials for cardiovascular diseases and in an even larger number for other conditions including orthopedic, autoimmune, and neurologic indications (see Chapter 10).

Heart—Cardiac Resident Progenitor Cells

As discussed in several subsequent chapters, transplanted stem cells rarely remain in situ for more than a few days, suggesting that their major mechanism of action seems to be the release of trophic factors, which, via paracrine mechanisms, activate native repair mechanisms [30,31]. One of the most interesting observations in the field of regenerative medicine is the presence of lineage-specific progenitor cells in every organ of the body. Cardiac progenitor cells are by definition not true stem cells, as they have only one committed fate—cardiomyocytes. In fact, two types of cardiac-specific progenitor cells have been identified and isolated from the heart: c-kit+ cells [32], which are seemingly restricted to the right atrium, and cardiospheres [33], which have been obtained from biopsy specimens of the right ventricle [10]. These two cell types do not appear to share surface markers or lineage. Both have shown significant potency in preclinical and pilot clinical trials [34,35] and are now being studied in phase 2 clinical trials. Future trials are needed to demonstrate if this lineage specificity provides superior outcomes compared to other types of stem cells.

Somatic (Skin) Cells

One of the newest and technologically most sophisticated sources of stem cells has been created by genetic engineering or transformation [36]. This approach began with the seminal observations by Yamanaka [37] that all somatic cells, including skin fibroblasts, contain a full genome and can be transformed with the addition of as few as four transcription factors to derive what has become known as an inducible pluripotent (iPS) cells [38,39]. This genetically transformed cell has the same robust capability of differentiating into any cell type in the body seen with embryonic cells. These iPS cells may potentially become an important source of stem cells for clinical applications, but is currently the furthest from clinical trials (see Chapters 20 and 21 for details on this cell). Cardiac-specific MSCs have been derived from these cells in a process known as cardiopoiesis, and these cells are now being studied in clinical trials [40].

Umbilical Cord and Placenta

Because of the inherent decrease in the number and function of stem cells with age, much research has been focused on a new source of stem cells at the opposite stage of life—those from the umbilical cord [41], placenta [42], and amniotic fluid (see Chapter 18). These tissues are an easily accessible source as they are typically discarded at the time of delivery. There has been over 20 years of experience using umbilical cord blood for allogeneic bone marrow transplantation, and there is extensive experience with long-term storage of the umbilical cord (see Chapter 18). Many families have stored the umbilical cord for the unlikely event that their child may need a bone marrow transplant, in which they would be able to use their own cells derived from the frozen umbilical cord. The umbilical cord has been stored for over a million people and remains viable upon thawing after being frozen at −160° for decades. The myriad indications for stem cell therapy greatly expand the potential use of a patient’s stored umbilical cord cells.

Moreover, only a portion of the umbilical cord is necessary to obtain the large number of cells needed for use. Half of the cord can now be dedicated for use as a potential source of donor stem cells, including the mesenchymal-type stem cell fraction, using either the Wharton’s jelly, or the more selective dissection to harvest only perivascular MSCs. In fact, umbilical cords in most cord banks are being utilized more commonly for obtaining donor stem cells than for storing for the individual.

Similar research is ongoing with other tissues discarded with no risk to the mother such as the placenta and amniotic fluid. All of these cells obtained immediately postdelivery are the most potent by functional assays and have normal telomeres, and therefore theoretically have a great capacity as stem cells for therapy. Clinical trials are under way using umbilical cells for multiple indications.

Endometrial Tissue

Another new source of stems cells is the endometrial tissue derived from menstruation [43]. The cyclic development and buildup of intense vascular tissue in the inside lining of the uterus results in menstruation and the monthly sloughing of that tissue; this scenario clearly suggests a renewable, pro-angiogenic type of stem cell. Preclinical testing has verified the pro-angiogenic potential and benefits of these menstrual cells, which are now being tested in clinical trials for different forms of ischemic vascular disease.

Dental Pulp

The newest source of stem cells is dental pulp [44,45]. For this process, the teeth are degraded by enzymes to allow extraction of the cells, which can then be expanded in culture.

Other Classifications of Stem Cells

Autologous versus Allogeneic Cells

One of the most transformative advances in the field of stem cell therapy is the ability to use allogeneic cells (see Chapter 13) [29]. Initially, autologous cells were used in stem cell therapy to avoid the recognition and elimination of foreign cells or tissue by the body’s effective immune response. Because of this precise recognition of non-self, it was thought that the cardiac injection of cells from an allogeneic donor would result in severe inflammation and rejection of the cells with no benefit and the potential for measureable damage without the use of potent immunosuppressive drugs.

However, MSCs show little to no measurable expression of transplant human leukocyte antigen (HLA) class I molecules, very limited expression of class II HLA antigens, and no expression of co-stimulatory molecules, which are needed for the host to mount an effective immune response [46,47]. As such, MSCs are often referred to as immune privileged, meaning that cells could be transplanted from any unrelated host without the risk of significant rejection or alloreactivity (see Chapters 15 and 16). Extensive preclinical and clinical studies have shown that MSCs are immune evasive and are therefore safe to administer allogeneically. The only evidence of alloreactivity to date in clinical trials has been a very low (5%) frequency of donor-specific alloantibodies in patients treated with MSCs. These antibody titers were low, usually transient, and not considered clinically significant. These findings provided the basis for initiation of a pivotal phase 3 trial of allogeneic MSC therapy in patients with heart failure.

The major advantage of allogeneic therapy is the ability to harvest cells from a carefully screened young, healthy donor and then expand the cell population in culture. Using a healthy, young donor overcomes the issue of cell dysfunction associated with comorbidities and age when autologous therapy is used. In addition, allogeneic cells add a uniform potency to the cell product across multiple doses. In this fashion, an allogeneic cell preparation allows for a readily available, off-the-shelf product. The cells can be stored at −160° for an extended period, shipped to any site, thawed over a period of minutes, and then delivered to a patient.

Multipotent Adult Progenitor Cells

MAPCs are a newly identified cell type that has the capability to differentiate into all cell lines (much like embryonic cells) but is derived from adult cells [23]. This use of MAPCs has been validated in preclinical studies and is being examined in clinical trials, initially in stroke patients. Phase 2 trials of MAPCs will soon be conducted for acute myocardial infarction and other cardiovascular indications.

Cardiopoiesis-Derived MSCs

Recently, bone marrow–derived MSCs have been driven toward a cardiac-specific phenotype by a process known as cardiopoiesis, which involves exposing cells to several transcription factors similar to that used in the development of iPS cells above. Based on extensive preclinical data and encouraging pilot clinical trials [40], these cells are now being tested in phase 3 trials in Europe and the United States.

Mechanisms of Action of Stem Cells

There has been a substantial evolution in the understanding of the mechanisms by which stem cells exert their beneficial effects in cardiovascular diseases [4–50]. The initial concept was that regardless of the type or location of injury, stem cells were mobilized from the bone marrow and differentiated into the specific cell type needed in response to cues from local signaling molecules. This led to early clinical trials of the direct delivery of a bolus of exogenous BMMNCs [51] or to the use of skeletal myoblasts, since the heart is a skeletal muscle [52], with the thought that the cells would differentiate into functioning cardiomyocytes. However, the concept of direct differentiation of transplanted cells was refuted, and, in fact, there is very little evidence of true myogenesis with any type of stem cell investigated to date (see Chapter 4).

Other theories of mechanisms were that transplanted stem cells would fuse to native heart cells [53] or that transplanted stem cells would serve primarily as a signal for substantial mobilization of stem cells from the host bone marrow into the circulation and then into the area of injury. The latter theory has some supporting evidence but is not believed to be the primary mechanism of cell therapy.

Most current evidence suggests that the transplantation of millions of exogenous stem cells results in the release of a bolus of several important trophic factors, which then secondarily stimulate native tissue repair by resident cardiac progenitor cells in what is called the paracrine mechanism [30,31]. This theory was clearly demonstrated by Dzau et al. [31], who showed that the supernatant from the medium of stem cells placed in culture for several weeks was as effective as the transplantation of the cells alone (see Chapter 4). Thus, it is the secretions of the stem cells that produce the benefits, not the cells themselves. This concept is supported by the observation that transplanted cells remain in situ for only a matter of days and are not present long enough to induce other mechanisms.

A provocative hypothesis comes from the observation that nearly half or more of the heart is turned over during one’s lifetime. Since many live to advanced age without an intercurrent cardiac injury to trigger the regeneration needed to maintain normal cardiac function, there must be a native regenerative capacity. The ability of fibroblasts to differentiate into iPS cells by addition of transcription factors as well as the ability to direct MSCs to differentiate into cardiac cells raises speculation as to whether the conversion of fibroblasts to pluripotent cells or cardiac cells can take place in situ. This hypothesis is now being evaluated.

While true stem cells are capable of differentiating into all types of tissue, it is clear that certain stem cells are destined to become specific types of cardiac tissue. For example, the EPC, also known as CD34+ cells, has a distinct pro-angiogenic role to drive new blood vessel formation [54,55], whereas mesenchymal-type stem cells are somewhat more effective as a myogenic stimulus and source [56]. It is clear that there is cross talk and interdependence between the various cells types in the cardiovascular system, but knowledge of these differences allows the selection of a particular type of cell that may be more effective with variable types of cardiovascular disease.

Endogenous versus Exogenous Tissue Repair

Many factors may limit the body’s native repair mechanisms, including the extent of the acute injury, the presence of comorbidities, and perhaps most significantly, the potential adverse impact of increasing age with regard to the both the number of stem cells available and their functional capacity. Native, or endogenous, tissue repair includes activation of local repair cells in the area of tissue injury (e.g., myocardial infarction) and also mobilization from other peripheral niches, or storage sites for these cells, with the bone marrow being the largest reservoir. These repair cells are mobilized in response to an array of chemoattractant factors called chemokines and adhesion molecules, growth factors, cytokines, and other molecules (see Chapter 39).

The acuteness and extent of tissue injury, such as acute myocardial infarction or stroke, may limit the capacity of native repair processes to respond quickly enough or adequately enough to recover lost organ or tissue function. In addition, patients with cardiovascular disease commonly have comorbidities such as diabetes and are older, both of which adversely affect the number and functional capacity of native stem cells. Thus, there is a significant focus on the use of delivering a large number of exogenous stem cells to enhance and drive native repair mechanisms. Exogenous stem cell therapy may include cells from the host bone marrow or adipose tissue. These cells are obtained autologously and as such are safe because they do not cause rejection or adverse effects.

Functional Assays

Until recently, we have used cells in clinical trials without having the ability to identify potentially suboptimal cells for reasons such as age and/or comorbidities [57]. However, several assays and biomarkers are now available that might help to define a signature of responders and to select patients who have the best chance of benefiting from treatment with a particular cell type (e.g., autologous bone marrow vs allogeneic MSCs).

Dimmeler and Zeiher have utilized an Stromal Derived Factor (SDF) mobilization assay to retrospectively analyze patients treated with BMMNCs for acute myocardial infarction [58]. On average, these patients had received cell therapy 5 years previously, which allowed sufficient longitudinal follow-up to assess long-term impact. SDF has been shown to be a key chemokine in stem cell mobilization and trafficking postinjury. This assay could be applied to all patients undergoing a bone marrow aspiration and would allow an estimate of functionality of the cells before their use in a given patient. The time required for the assay is being shortened (<24 h) and will allow fairly rapid decision making (see Chapter 7). Other assays are also being developed to examine cell potency in vitro.

A detailed analysis has also been conducted on patients enrolled in the NIH-sponsored Cardiovascular Cell Therapy Research Network trials [59–61], which included the TIME [59] and LATE TIME [60] studies for acute myocardial infarction and the FOCUS-CCTRN [61] trial for chronic ischemic heart failure. Each of these trials tested the use of autologous BMMNCs. In the detailed analysis of all the trials, the investigators performed a multivariate analysis of parameters obtained on a battery of tests of the bone marrow aspirate in 75 of the 101 patients who showed a reduction in infarct size by quantitative MRI. They found a significant correlation of the number of CD31+ BMCs as well as faster BMC growth and endothelial colony forming units. These data will hopefully provide an eventual signature of responders to guide clinical trials (see Chapter 38). These data are now being validated and will in fact be used as part of the final screening criteria for patient enrollment in at least one phase 3 trial planned using BMMNCs for chronic ischemic heart failure. Comparing assays and results between studies will allow further refinement in defining a signature of responders, which could lead to better patient selection for specific cell trials, as well as an enhanced understanding of stem cell function.

Sex Differences

There is a growing body of evidence to suggest that there are sex-based differences in the functional capacity [62] and gene expression [63]. Stem cells from female donors appear to have some advantage. These preclinical observations will be further studied for clinical relevance.

Safety Profile

There have been no observed risks of using autologous cells in regenerative medicine studies for any type of disease in the body. In autologous studies, the patient’s own bone marrow cells are obtained, washed, and used to treat the individual. Despite significant clinical trial data demonstrating similar safety in autologous stem cells derived from adipose tissue, the FDA has been rigorous in restricting the use of autologous adipose cells outside of carefully controlled clinical trials. Similarly, allogeneic mesenchymal stem cells have a similar safety record to autologous stem cells; however, longer term follow-up is needed to ensure this safety.

One of the greatest concerns raised about stem cell therapy in the past was a perceived high risk of cancer. The concern is intuitively related to the stem cell theory of cancer (cancer being driven by a population of cancer stem cells) as well as the fact that there is a high risk of teratoma-type tumor formation seen exclusively with use of embryonic stem cells [12]. As a result, nearly all clinical stem cell trials to date have required careful screening of the potential recipient of the stem cell as well as long-term follow-up to document potential cancer development. The evidence to date appears to indicate that neither autologous nor allogeneic MSCs pose any additional risk of tumor formation above that expected for their age, gender, and other risk factors. Another concern related to the pro-angiogenic feature of stem cells was the potential progression of proliferative retinopathy, which is a frequent complication in patients with diabetes. This, too, has not been shown in patients with diabetes who have been treated with stem cells.

The delivery methods used in clinical trials of cell therapy have demonstrated safety across different platforms. There are nominal risks of cell delivery strategies associated with the invasive procedures overall. Also, there is an inherent risk of myocardial perforation when transendocardial injections are performed, but these have occurred at a very low frequency.

Summary

The field of regenerative medicine has moved forward rapidly from the previously held view that the bone marrow was the only repository of stem cells in the adult body. The recognition of an expanded number of sources of stem cells as well as cell types provides the potential to individualize cell therapy for treating specific forms of cardiovascular disease, as well as the potential for overlap by using combined cell strategies.

There have been enormous advances in nearly all aspects of regenerative medicine for cardiovascular disease over the past decade. Figure 1.1 captures many of the concepts described above. In particular, there is a much wider array of new sources of cells, types of cells—including allogeneic cells, and routes of delivery, all of which will lead to an improved understanding of how to harness the body’s native repair mechanisms. The first era of stem cell therapy has ended, and we are beginning a new era that promises to provide significant improvement in outcomes to reduce the morbidity and mortality associated with cardiovascular disease.

Figure 1.1 Overview of current understanding of newest sources and types of stem cells for cardiac indications, methods of delivery, methods to stimulate function, and mechanisms of clinical effects. Source: With permission [36].

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Chapter 2

Pathological Assessment of Experimental Models of Stem Cell and Other Regenerative Therapies

Deborah Vela¹ and L. Maximilian Buja¹,²,    ¹Cardiovascular Pathology Research Department, Texas Heart Institute, Houston, TX, USA,    ²Department of Pathology and Laboratory Medicine, the University of Texas Health Science Center at Houston, TX, USA

Pathologic assessment plays a crucial role in cardiac stem cell and gene therapy research by allowing researchers to evaluate the safety and effectiveness of such therapies in cardiac tissues. Knowing the cell labeling procedures that have been applied and the delivery route selected for the administration of the treatment is useful for planning a strategy for pathologic evaluation. The pathologic examination of the hearts can be tailored to specific delivery routes for best results. A thorough gross and microscopic pathological examination can yield invaluable information regarding the focal or remote effects of the intended therapy and the fate of the administered biological agent.

Keywords

Stem cells; regenerative therapies; tissue sampling; pathology; pathologic evaluation

Introduction

Pathologic assessment plays a crucial role in cardiac stem cell and gene therapy research. It allows researchers to evaluate the safety and effectiveness of such therapies in cardiac tissues. In this chapter, we will start with a brief description of some of the more commonly used labeling techniques as they have been applied to stem cell therapy research and a brief overview of delivery routes, followed by an elemental outline for pathologic assessment of hearts that have undergone cell, gene, or other regenerative therapies.

Preparation and Labeling

Commonly, the cell type selected for stem cell therapy is appropriately harvested, isolated, and expanded before it is labeled. Labeling procedures are of importance to the experimental design because they allow the fate of implanted stem cells to be tracked and monitored in order to confirm or rule out cell engraftment and potential trans-differentiation. We will briefly review several of the useful labeling procedures that are commonly used.

BrdU Incorporation

The thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) is used to identify cells undergoing DNA synthesis. Newly synthesized DNA thymidine is partly replaced by BrdU, which allows BrdU to serve as a proliferation marker. BrdU is generally used in cell culture settings [1,2] and can also be administered to live animals for acute (before euthanasia) or chronic administration.

Fluorescence Labeling and Dye Marking

Vital fluorescent dyes are used to mark cells temporarily. These dyes can be divided into two categories: nuclear and cytoplasmic.

Nuclear Dyes

Several DNA-binding dyes can be used to highlight the nucleus. These include Hoechst 33342, Hoechst 33258, 4′,6-diamidino-2-phenylindole (DAPI), mithramycin, propidium iodide, 7-amino-actinomycin D, SYTOX green, DRAQ5, and TO-PRO-3 iodide. They share many properties but vary in their excitation points. One of the most popular of these nuclear dyes is DAPI.

DAPI. The UV-excited fluorescent dye DAPI has been used as a cell marker since the 1980s [3]. Its high affinity for double-stranded DNA makes it an excellent nuclear labeling dye. It forms strong electrostatic interactions with adenine-thymine–rich regions of DNA. The fluorescence quantum yield of the free dye is very low, but the binding to DNA results in a highly fluorescent complex. DAPI is nontoxic and does not alter the ultrastructure of organelles. Its labeling efficiency is almost 100%, leaving virtually no DAPI fluorescence in the culture medium when used in in vitro experiments. Although DAPI is generally considered a reliable cell marker [4,5], false-negative results may potentially arise from DAPI dilution in proliferating cells, and false-positive results may arise if large numbers of marked cells die and DAPI is taken up by other cells [6].

Cytoplasmic Dyes

Fluorescent labels can be used to mark a variety of cytoplasmic structures and components, such as the cell membrane, organelles, and ions.

DiI. Cell membranes labeled with DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) emit an orange-red fluorescence. This is due to the insertion of the two long (C18 carbon) hydrocarbon chains of DiI into lipid bilayers. The popularity of this cross-linkable membrane dye is mostly due to its photostability and low toxicity, which allows long-term cell tracking both in vitro and in vivo [7].

Quantum Dots. Quantum dots are semiconductor nanocrystals (2–10 nm in diameter) that can be excited by light of a wide range of wavelengths, ranging from 525 to 800 nm. Quantum dots can emit different wavelengths of light, depending on their size and composition, making them capable of multiplex imaging.

These small particles are capable of entering stem cells through passive loading, receptor-mediated endocytosis, or transfection [8–10]; passive loading appears to be the most effective method. Quantum dots display intense fluorescence that is maintained under various biological conditions, including changes in intracellular pH, temperature, and metabolic activity. Additionally, they possess excellent photostability that can withstand extensive continuous illumination without photobleaching or degrading. Despite being passed on to daughter cells for several generations, they eventually become susceptible to dilution