From cardiogenesis to cardiac regeneration : focus on epicardium-derived cells Winter, E.M.

From cardiogenesis to cardiac regeneration : focus on epicardium-derived cells

Winter, E.M.

Citation

Winter, E. M. (2009, October 15). From cardiogenesis to cardiac regeneration : focus on epicardium-derived cells. Retrieved from https://hdl.handle.net/1887/14054

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden

Downloaded from: https://hdl.handle.net/1887/14054

Note: To cite this publication please use the final published version (if

applicable).

General Introduction

1

Background

Embryonic heart development Cardiac regeneration therapy 1. Embryonic versus adult cells

2. Adult cells harvested from extracardiac structures 3. Adult cells derived from the heart

Functional assessment of the infarcted mouse heart Chapter Outline

General

Introduction

12

|

General Introduction

Background

The epidemic proportions of cardiovascular disease, with coronary artery occlusions and its consequences ranking first in the mortality list of the Western world

, warrant the development of more appropriate treatment modalities. Promising results of recent innovative stem cell studies direct ischemic heart disease research into an entirely novel field. To accurately design such new experiments, and to fully understand the implication of the outcome, knowledge about embryonic development of the heart might be of benefit. Cardiogenesis and cardiac regeneration therapies will be discussed, emphasizing the outer layer of the heart, the epicardium, which might have promising potential in the adult setting.

Embryonic heart development

Early in gestation, two crescent-shaped cardiogenic plates arise from the anterior splanchnic mesoderm and fuse to form the primitive single-chambered heart tube

(Figure 1a, b). This straight tube undergoes a complex process of looping and segmentation, orchestrated by a cascade of genes that are important for left-right programming and cardiomyocyte differentiation, before a four- chambered heart is formed. During this process various cells from the so-called second heart field (SHF) add to and grow into the primitive heart, which was initially formed solely by derivatives of the first heart-forming field (FHF)

. Within the cardiac crescent the splanchnic mesoderm destined to form either of these two heart fields can already be distinguished, with the precursors of the SHF located at the centre (Figure 1a). The contribution of the SHF is crucial for proper cardiogenesis. The FHF-derived structure does not contain the entire assembly of necessary elements to build a complete heart.

Initially, before cell populations from the SHF are added, the primitive heart tube consists of only endocardium and myocardium with a cardiac jelly in between (Figure 1c). During the looping of the heart this cardiac jelly becomes unevenly distributed, resulting in large protrusions at the sites of the atrioventricular (AV) junction and outflow tract (OFT). The initially acellular structures become invaded by mesenchymal cells derived from the endocardium as a result of epithelial-mesenchymal transformation (EMT)

. These early cushions will give rise to the cardiac valves in the mature heart, but are also instrumental in cardiac septum-formation. At the moment that the cushions become colonized, SHF-derived cells and cardiac neural crest cells (CNCs) start to populate the outflow and inflow portions of the heart (Figure 1d). The CNCs play likewise an important role in cardiac septation and in cushion development

. Since the majority of the CNCs is destined for apoptosis

, their contribution is suggested to be mainly instructive, rather than constructive.

The SHF contribution is both instructive as well as constructive. The SHF is divided in an anterior heart field (AHF), which adds to the OFT or arterial pole

, and a posterior field (PHF), from which the derivatives are located at the inflow tract (IFT) or venous pole (Figure 1d)

. Besides a considerable contribution to the IFT myocardium

, the PHF delivers the epicardial progenitors to the heart by forming the pro-epicardial organ (PEO)

.

The PEO consists of mesothelial protrusions and villi in the splanchnic mesoderm located near the venous pole of the heart (Figure 1d). During the looping stages of cardiac development, cells cross the coelomic cavity as free-floating cell aggregates (mammals and fish)

or guided by tissue bridges (avians)

, although a combination is also suggested

. At first, the epicardial (progenitor) cells attach to the naked heart tube at the dorsal side of the atrioventricular sulcus. From that point, they spread over the heart to cover it with its epicardial layer

.

It has only recently been discovered that the role of the outermost layer of the heart, the epicardium,

is much more extensive than being the ‘cover’ that enables friction-free movement in the pericardial cavity

. The derivatives of the epicardium, the epicardium-derived cells (EPDCs), contribute in both a constructive as well as an instructive way to normal cardiac development. EPDCs are generated soon after the heart has been enveloped entirely by epicardium, as a result of EMT

. From the subepicardial space, where the EPDCs are located at first, they migrate into the myocardium. They add to the AV cushions, the cardiac interstitium, and the fibrous heart skeleton by delivering fibroblasts

27-30

. EPDCs also stabilize the coronary arteries as smooth muscle cells and adventitial fibroblasts

26,28-31

. As regulators of cardiogenesis, EPDCs appear to be important for the formation of the compact

myocardium

and for proper ingrowth of coronary arteries into the aorta

. Cardiac looping

and development of the Purkinje fiber system

are also dependent on EPDC-generated signaling. It has not yet been investigated whether EPDCs remain situated in the cardiac interstitium or in the wall of coronary arteries once they have migrated there. Besides their ultimate destiny, their function in the adult setting remains to be studied since EPDCs hold great potential for application in cardiac regeneration therapy (discussed further below).

Cardiac regeneration therapy

Compared to the effective, mainly curative treatments of cardiac arrhythmias (catheter ablations, pacemakers and implantable defibrillators), cardiac valve dysfunction (artificial valves), and coronary obstructions (dotter procedures, stents and surgical bypasses), common therapy of the infarcted heart might be considered somewhat old-fashioned. Currently, general treatment aims at limiting the region of injury by reopening the blocked vessel, and at reducing workload of the remaining tissue with pharmaceutics. But real healing or revival of the dead tissue is not yet possible, and homograft transplantation is no first-line therapy because of the small size of donor organ pool, high risk of complications, and costs. Reasonably, the exciting data from the study by Orlic et al

which

Figure 1. Schematic picture illustrating consecutive stages of cardiac development. The cardiac crescent, which is derived from the splanchnic mesoderm (a) first forms the primary heart tube (PHT, pink, b) and atrioventricular endocardial cushions (EC, blue, d). The yellow area within the cardiogenic plates (a) and behind the PHT (c) depicts the second heart field (SHF) that in later stages adds myocardium to the developing heart (d). The subset of the SHF contributing to the arterial pole (AP) of the heart is called the anterior heart field (AHF), and the part that adds to the venous pole (VP) the posterior heart field (PHF, d). The pro-epicardial organ (PEO), which is a subpopulation of the SHF that can be distinguished as a lobulated protrusion at the VP of the heart delivers the epicardial cells to the heart (i.e. the epicardium, d). Cardiac neural crest cells (CNCs, dark blue cells) migrate from the neural tube into the heart, thereby passing through the SHF mesoderm (d). G: gut, C: coelomic cavity, Ep: epicardium.

Modified after Gittenberger-de Groot AC et al, Fetal Cardiology 3rd edition, in press.

14

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General Introduction

demonstrated that bone marrow-derived stem cells could regenerate the injured area and improve cardiac function, energized many cardiovascular scientists and instigated even more new research projects in this field.

1. Embryonic versus adult cells

A large variety of cell populations can be applied for cell-based therapy of the infarcted heart, each having its own profile of advantages, limitations and feasibility. Cell types used can be sorted along their developmental potential. The most potent cells in an organism regarding differentiation are the ‘totipotent’ blastomeres. They form the embryo and also the placenta. Later, during the blastocyst stage, the structure giving rise to the placenta (trophoblast) can be distinguished from the source of the three germ layers of the embryo, the inner cell mass. The cells from the inner cell mass are ‘pluripotent’ and are referred to as embryonic stem cells, with the capacity to self-renew, to differentiate into all lineages

, and to grow clonogenic

. Stem cells that reside within the adult somatic tissue are already committed to a specific lineage, and are only able to differentiate into a more selected variety of differentiation pathways, making them ‘multipotent’ stem or progenitor cells.

In current basic fundamental animal research as well as in clinical experimental treatments mainly adult stem or progenitor cells are used, in comparison to the more scarce application of embryonic stem cells. Arguments for this imbalance could be that i) appropriate handling of embryonic stem cells is intricate, ii) usage of embryonic stem cells is ethically challenging, and iii) includes the risk of teratoma formation, iv) adult stem cells can be derived from the patient itself and thus rejection problems are obviated, v) some adult cells, like bone marrow-derived cells, are easily available in large amounts. Drawbacks of adult cells are, however, that possible genetic defects might impair function of the cells to be applied, that they can not be expanded unlimitedly, and that many types are not immediately available at the moment of e.g. acute infarction. Recently described induced pluripotent cells (iPS) might combine best properties of both. These are reprogrammed adult cells in which pluripotency can be induced in vitro by overexpressing a combination of specific transcription factors

43-46

. However, their feasibility in vivo has not yet been revealed, leaving them to be dreamed of for the

future.

2. Adult cells harvested from extracardiac structures

The most extensively studied adult stem cell populations at the moment are those derived from the bone marrow

. This is a rich source of various immature cells that are applied in basic and clinical experimental cardiac regeneration therapy, being hematopoietic stem cells (HSCs)

, a subset of these, the so-called side population cells

, mesenchymal stem cells (MSCs) or stromal cells

, multipotent adult progenitor cells (MAPCs)

, which are a subset of MSCs, and bone marrow-derived multipotent stem cells (BMSCs)

. MAPCs and BMSCs are able to differentiate into all three germ layers

a property which has always been considered to be exclusive for embryonic stem cells.

Commonly, especially in clinical trials, a mixture of these subpopulations is used, which are then referred to as bone marrow-derived mononuclear cells (BMCs)

. Bone marrow-derived cells do not necessarily need to be harvested from the bone marrow, some can also be isolated from the blood, like the circulating progenitor cells (CPCs)

which include the endothelial progenitor cells (EPCs)

63

. Furthermore, potential cardiotherapeutic progenitor cell populations can be derived from adipose tissue

, skeletal muscle

, placental cord blood

, and probably many other less obvious tissues.

3. Adult cells derived from the heart

It seems logical to expect cell populations derived from the target-organ being the most appropriate cell source for its own tissue regeneration, supposing these cells are present in the organ of interest.

With regard to the heart, the presumed cardiac derived progenitor cells would probably be committed

to the cardiac lineage, adapted to the specific environment and they might have been programmed to excrete factors of use for the heart. Research of the last decade focused to find progenitor cells in the adult heart revealed indeed several progenitor populations, although the heart has long been considered as an organ without regenerative potential. The heart was demonstrated to possess endogenous regenerative potential

and to harbor small multipotent cells that could differentiate into cardiomyocytes. These cells were recognized based on their expression of stem cell markers stem cell antigen-1 (sca-1)

(for human tissue referred to as cardiomyocyte progenitor cell or CMPC), c-kit

, or the primitive SHF marker islet1 (isl1)

. But they were also phenotyped by their expression profile in combination with adherence properties (cardiospheres)

, or their ability to efflux the Hoechst dye

. Of special interest are the CMPCs which can be isolated from human fetal and adult cardiac tissue, since they can differentiate into cardiomyocytes in vitro without the necessity of being co-cultured with neonatal cardiomyocytes

. The different multipotent residents described by various groups might, however, represent closely related subpopulations of one major cardiac stem cell pool. If these different cell types with cardiomyocyte differentiation potential characterize isolated populations resident in the heart, it is expected that ischemic injury would at least partly be mended by the organ itself. Although adult stem and progenitor cells from non-cardiac tissues are already widely tested for their therapeutic applicability, stem and progenitor cells resident in the heart itself are currently only of modest interest when evaluated for their usage in clinical studies.

Knowing that the adult heart harbors cardiomyocyte-precursors leads to the expectation that other lineage-destined ‘remnants’ of early development are also present in the postnatal heart, including EPDCs. As mentioned before, EPDCs are progenitors of the cardiac interstitial fibroblasts and medial and adventitial cells of the coronary arteries with extensive instructive properties during cardiogenesis. It might be speculated that adult EPDCs could recapitulate their embryonic properties, for example in case of ischemic injury. It is, however, not known whether EPDCs are indeed present and still potent in the adult heart.

In this thesis it is studied whether EPDCs could be derived from the adult epicardium

, and if these cells could be of benefit for cardiac repair of the adult ischemic heart. It is hypothesized that the adult EPDCs improve cardiac function after myocardial infarction through paracrine signaling like they demonstrate during embryogenesis. This presumed supportive and instructive effect of the adult EPDCs on the surrounding injured host tissue is also elucidated. It is tested whether their profit could be further increased by co-transplanting complementary CMPCs. Being mainly located in the atrium

, CMPCs are suggested to originate from the SHF like EPDCs, designating them as ontogenetically closely related. A further mesenchymal cell type is studied, the bone marrow-derived MSC. It is examined whether their potential is retained if they are derived from ischemic heart disease patients, as opposed to healthy donors.

Functional assessment of the infarcted mouse heart

The extensive interest in the cardiac stem-cell field, with the general aim to cure the adult heart by regenerative cellular therapies, has resulted in the emergence of numerous basic animal studies.

Although small rodents are phylogenetically less related to human than large mammals, mouse and rat are most commonly applied for basic animal studies, since their genetic background is well known and their usage is less complicated and expensive than that of larger animals. To evaluate the effect of the cell type of interest in vivo, i.e. to test whether it can stimulate cardiac performance, functional assessment is needed. This requires highly specialized techniques, in particular for the mouse, considering the extreme small size of the heart (7 mm) and high heart rate (~500 beats per minute).

Nonetheless, magnetic resonance imaging (MRI), echocardiography and invasive pressure-volume (PV)

loop measurements have become available for cardiac phenotyping of small animals.

16

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General Introduction

In this thesis several aspects of MRI, which is the method of choice for longitudinal stem cell

experiments

, are described. Additional applications of MRI, being infarct size measurements and

identification of transplanted cells in the living heart by usage of iron labeling, are discussed. Also,

MRI and PV loop measurements, which each address different aspects of left ventricular function, are

compared for their reliability in the infarcted mouse heart.

Chapter Outline

Chapter 1 provides background information regarding embryonic heart development and stem cell therapy for cardiac regeneration. Technical issues in functional basic experimental stem cell research are shortly introduced.

Chapter 2 describes the contribution of EPDCs to the developing heart. It speculates on the role of these cells in the adult heart, in specific their potential in the infarcted heart.

Chapter 3 translates the knowledge from the embryonic situation to the adult diseased heart, investigating whether EPDCs harvested from the adult heart can contribute to functional improvement after myocardial infarction.

Chapter 4 addresses potential underlying mechanisms that might explain the positive effect of adult EPDCs on the infarcted adult heart. It focuses on histological and functional differences between normal scar development and cardiac healing after adult EPDCs have been transplanted.

Chapter 5 reports on the interaction between CMPCs and EPDCs. Their mutual effect is studied parallel in the in vitro and in vivo situation, from which the latter tests the hypothesis that combined transplantation will be of a synergistic benefit for the infarcted heart.

Chapter 6 concerns the applicability of MSCs from ischemic heart disease patients to cure the infarcted heart. Engraftment and differentiation of transplanted MSCs are addressed, as well as their potential to improve LV function.

Chapter 7 compares two methods used for heart function determination of small laboratory animals.

Reliability of MRI and PV–loop measurements for assessment of cardiac function of the failing mouse heart is analyzed.

Chapter 8 describes methodological issues around iron-oxide labeling for tracing of stem cells in the mouse heart.

Chapter 9 provides a general discussion on the concept of using cardiogenesis as basis for cardiac

regeneration therapy. Application of adult EPDCs to improve cardiac function after myocardial

infarction is addressed, as are potential mechanisms which might explain the detected profit. Other

mesoderm-derived cell types are conferred, and technical issues in functional assessment of the

infarcted heart are placed in perspective.

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