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
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General Discussion
9
Introduction
Cellular treatment of the infarcted heart
1. The adult heart
2. The developing heart as basis for the design of cellular cardiac regeneration research 3. Mesodermal derivatives as equipment for cardiac regeneration
A. Non-heart originated mesenchymal stem cells B1. Heart originated epicardium-derived cells (EPDCs)
- Generation of EPDCs by adult epicardium - Effect of EPDC transplantation on cardiac function - Differentiation of transplanted EPDCs in vivo - Paracrine mechanism of transplanted EPDCs
B2. Heart originated cardiomyocyte progenitor cells C. Which one to choose?
4. Combining important building stones to treat the infarcted heart
Functional evaluation of the murine heart
Conclusions
General
Discussion
Introduction
In this thesis the potential of several cell types with regard to cardiac regeneration was studied.
It was demonstrated that adult epicardium-derived cells (EPDCs), as well as systemically derived mesenchymal stem cells (MSCs), improved cardiac function after myocardial infarction (MI). Co- transplantation of EPDCs and cardiomyocyte progenitor cells (CMPCs), which are complementary in light of cardiac development, was of synergetic profit for the infarcted heart. Moreover, several points of attention concerning functional analysis of the mouse heart were addressed. In this chapter the experimental data on cell types derived from the heart and the bone marrow will be discussed first, with a focus on their developmental origin. In this context future perspectives will be described.
Second, the different techniques available for functional assessment of the mouse heart will be pointed out.
Cellular treatment of the infarcted heart 1. The adult heart
The dogma that the heart is a terminally differentiated organ lacking any regenerative capacity has recently been questioned 1-7. It might indeed be considered remarkable that ventricular cardiomyocytes, contracting 60 times per minute, have a life-span of approximately 75 years, corresponding to that of an individual. However, the scarcity of primary cardiomyocyte tumors supports the old paradigm of the absence of regenerative growth in this organ 8.
It was calculated for the rat heart, that, if cardiomyocyte renewal was absent, spontaneous cell death would have resulted in atrophy of the organ within 5 months (approximately 94.000 cardiomyocytes are lost during a 24 hour period, and the heart consists of 13 million cardiomyocytes), suggesting at least little regeneration capacity 6. Besides this indirect argument, observations in human tissue pointed at some reproductive properties of the adult myocardium. Recently, Bergmann et al elegantly identified the age of cardiomyocytes in humans by using integration of carbon-14, which was generated by nuclear bomb tests during the Cold War 7. It was calculated that around 50% of cardiomyocytes are renewed during a normal life span. Other studies revealed that mitotic cardiomyocytes could be detected in left ventricular (LV) biopsies of human adults 2,3. Interestingly, with 0.08% mitosis in the borderzone, and 0.03% mitosis in the remote area of the ventricular infarcted tissue, proliferation was increased (70x and 24x, respectively) in infarcted myocardium when compared to normal healthy tissue 2. It has not yet been revealed whether the amplifying cardiomyocytes originated from resident cardiac stem cells, from circulating stem cells that have homed to the heart, or both 6. With regard to the latter, studies with cardiac tissue from sex- mismatched heart transplantation patients detected cardiomyocyte and endothelial progenitor cells (as demonstrated by expression of stem cell markers c-kit, MDR1 or stem cell antigen-1 [sca-1] besides cell type specific markers) from the recipients within the donor hearts, suggesting that cells from the systemic circulation invade and populate the heart 5.
However, the incidence of cardiac chimerism appears to be highly variable, with some studies reporting considerable amounts of recipient-derived cardiomyocytes 5,9,10 whereas others observed none at all 11-13. It remains to be investigated whether the discordance is due to methodological difficulties in Y-chromosome identification 12,14, such as misinterpreting the Y-chromosome of leukocytes for that of cardiomyocytes 15, and to what extent cell fusion may occur 16,17. Moreover, regarding the findings of replicating cardiomyocytes, it must be taken into account that replication of DNA does not necessarily mean that karyokinesis takes places, which itself does not inevitably connote cytokinesis 18, as indicated by the high number of multinucleated cardiomyocytes in human adults 19.
The general objective for exploring the regeneration capacity of the heart is to discover a way to repair the injured organ. Not only the endogenous capacity is investigated, also transplantation of exogenously harvested cells that have cardiomyocyte differentiation potential, is extensively studied.
However, those supposed immature cardiomyocyte progenitors will need to be directed. Moreover, even if endogenous cardiomyocyte renewal takes place, regeneration capacity appears still very modest, requiring thorough protection and support of the existing tissue. Therefore, to mend the broken heart, the presence of assistive non-cardiomyocyte cells must at least be as essential as the recruitment of premature cardiomyocytes.
2. The developing heart as basis for the design of cellular cardiac regeneration research It is difficult to decide in which direction one should focus research to discover cells expected to be appropriate for cardiac repair. Knowledge about cardiac development might give some indications, assuming that mimicking the original build-up of the heart for the embryo seems most probable to be successful for the adult setting as well. Starting with the process of embryonic heart formation in mind, one could search in the adult heart for remnants of developmental key players.
As described in the General Introduction (Chapter 1) two heart fields can be distinguished, the first heart field (FHF) and second heart field (SHF). The FHF mainly contributes to the formation of the LV, the atrioventricular canal and part of the atria, whereas the SHF adds to a more diverse collection of cardiac structures, including the right ventricle (RV), outflow tract (OFT) and atria 20-25. Primitive remnants of the FHF have not yet been reported in the adult heart, but this might be due to the lack of a specific marker. Recently, islet1 (isl1) positive cells, which might be considered as representatives of the SHF 23,26, have been observed in mouse, rat and human tissue samples collected early after birth
24. Their distribution throughout the heart matched the defined contribution of SHF precursors during embryogenesis 24, suggesting them to be indeed remnants of developmental SHF progenitors. They could be isolated, expanded and differentiated in vitro into cardiomyocytes. Of note, no isl1 positive cells were detected in the fully developed mature heart of adults, because of which they are not yet applicable for cardiac regeneration therapy.
In the adult heart some potent primitive cardiac progenitors are described, having the potential to form cardiomyocytes, but their developmental origin and their relation to each other remain unclear. They were characterized based on the expression of stem cell markers c-kit 27, sca-1 28-
30, their potential to exclude dyes (side population or SP cells) 31, and their adherence properties (cardiospheres) 32. Although surface marker profiles were distinct 33,34, it can be suggested that these cells represent different subsets of one and the same progenitor pool 35, especially since it would be highly remarkable for an organ known for its restricted regeneration capacity to harbor several different stem or progenitor populations. They might represent remainders of the embryonic FHF or SHF in the adult heart, which suggests that they could be of high potential for regeneration therapy. However, it is not clear to which extent c-kit, sca-1 and isl1 are linked to each other. Wu et al and Moretti et al supposed that, during early cellular differentiation, c-kit is expressed prior to isl1 and Nkx2.5 36,37. Expression of c-kit in the most primitive cells is followed by isl1 and low amounts of Nkx2.5, after which cells are further differentiated by losing isl1 and c-kit expression and obtaining expression of mature cardiomyocyte or smooth muscle cell markers 36,37. If the FHF and SHF have one common progenitor 20,23,25, which is still not definitely known, c-kit might qualify as a marker. Tbx18 38 and isl1 23,39, which have occasionally been detected in FHF regions, might also denote this population.
However, it needs to be investigated which populations detected in the adult heart represent primitive multipotent remnants of early development and in which way these are related to each other. If further elucidated, knowledge about cardiogenesis and cardiac repair could be combined, which might result in a big step forward for cellular therapy of the infarcted heart.
From the developmental point of view, one can search for primitive transformed remains in the adult heart like described above. Another interesting approach would be to seek in the adult heart for differentiated adult cells that have the potential to revive their embryonic program by dedifferentiation or redifferentiation, supposed that this predetermination might indeed be of function. Of note, during embryonic heart formation, maturated epicardial cells, which are a subset of the SHF, have the capacity to generate undifferentiated primitive mesenchymal cells, the EPDCs. Translating this into the adult setting, it can be stated that the adult epicardium would be a promising target to study for its potential to revive its embryonic program. Previous in vitro experiments have shown that adult EPDCs can indeed be harvested from adult epicardial explants 40. Adult EPDCs are of therapeutic interest because their embryonic counterparts are crucial contributors to proper development. During normal cardiogenesis, embryonic EPDCs generate non-cardiomyocyte cells including fibroblasts 41,42, while they support accurate formation of the ventricular wall (reviewed in 42). Adult EPDCs would thus, as was mentioned before, qualify for the group of ‘assistive’ cells.
Moreover, cardiac fibroblasts can themselves be considered an attractive therapeutic target 43 since cardiomyocytes are structurally and functionally highly dependent on their surrounding fibroblasts (reviewed in 43,44), which constitute up to 70% of the myocardial wall 45-47. The origin of cardiac fibroblasts has not yet been elucidated, with endocardial cushion cells 44,48 and systemically derived mesodermal cells 44,49,50 being possible suppliers besides EPDCs.
With the mesoderm being the major source of the entire heart, including the FHF and SHF, we explored various mesodermal populations. First, the non-cardiac derived MSCs 51, which have the advantage of being primitive multipotent cells, but the disadvantage of being ontogenetically only sparsely related to the heart, were investigated with regard to cardiac repair. Second, the above described adult EPDCs, harvested from adult cardiac specimens 40, were studied for their contribution to cardiac healing. Adult EPDCs are not multipotent, but their embryonic equivalents are strongly associated with cardiac building, considering their crucial instructive role in cardiogenesis. Third, another population of cells, the sca-1 positive CMPCs, which can be isolated from the adult heart
30,52,53 and which do have the potential to generate new cardiomyocytes 30,53, was explored mainly with
regard to a supposed synergistic effect with the supportive EPDCs.
3. Mesodermal derivatives as equipment for cardiac regeneration A. Non-heart originated mesenchymal stem cells
MSCs form a population of multipotent self-renewing cells, located in the bone marrow stroma 51. They can differentiate into a variety of mesodermal cell types, including cartilage, bone, tendon, ligament, muscle, fat, marrow stroma and several other connective tissue fibroblasts 51,54. In vitro 55, as well as in vivo, they can give rise to cardiomyocytes, not only in the healthy subject 56, but also in the ischemic heart after myocardial infarction (MI) 57,58. It is, however, debated whether this differentiation is responsible for the observed functional improvement of MSC transplantation into the heart as was initially suggested 57-60. We demonstrated in this thesis, like others 59,60, that injection of MSCs into the infarcted heart resulted in a functional improvement although no cardiomyocytes were generated by the engrafted MSCs. They expressed α-smooth muscle actin (α-SMA), characteristic for myofibroblasts 61, but no smooth muscle myosin, which is highly specific for smooth muscle cells
62. Moreover, their shape was elongated and they were located in between the cardiomyocytes and not integrated in the vessel lining as would be expected for smooth muscle or endothelial cells. The engrafted MSCs can thus be considered as having acquired a myofibroblast phenotype. This is not necessarily remarkable regarding their origin and their natural fate 54. In our opinion, the physical contribution of the engrafted myofibroblast-like MSCs can not explain the benefit, since engrafted cell numbers are relatively low if compared to the entire ischemic area. Altogether we propose that paracrine signaling of the MSCs is responsible for the functional improvement in LV function. A contribution of released angiogenic factors is expected 59, since vascular density in the infarcted area
was clearly increased by the MSCs at 2 weeks after MI. However, the exact mechanism of this so- called paracrine effect needs to be further elucidated. We especially question whether the suggested paracrine mechanism is specific for MSCs, and, if so, whether particular aspects of the MSCs suits their application for a constrained type of ischemic damage.
We demonstrated in our experimental study with MSCs from ischemic heart disease (IHD) patients, that indeed MSCs derived from the patient itself are applicable for cellular regeneration studies.
Others 57,59,60,63 have mentioned the advantage of using autologous cells, but they used MSCs from healthy donors. They did not take into account that capacities of MSCs from IHD patients might be reduced (reviewed in 64). Since the mechanism of action has not yet been revealed, it is difficult to speculate on the negative effect of various risk factors on the function of MSCs when transplanted into the ischemic heart. These issues must be considered during new study design. Moreover, we suggest that in the near future basic studies are performed in which MSCs from healthy donors are compared to MSCs from patients with various risk factors. Part of the required research is currently ongoing in the first clinical trial with MSCs, in which autologous MSCs from IHD patients are studied (see http://clinicaltrials.gov/ct2/show/NCT00114452 for trial design).
The major focus of current clinical research is the bone marrow, from which bone marrow mononuclear cells are most frequently used 65,66. MSCs are a rare subset of the mononuclear cells (<0.1%), which, in contrast to some of their parenchymal counterparts (hematopoietic stem cells [HSCs] or endothelial progenitor cells [EPCs]), do not express the surface proteins needed to surpass the bone marrow barrier. MSCs can therefore not enter the circulation, and they must inevitably be harvested from the bone marrow itself. However, bone marrow aspiration is only little compelling, since it can be performed under local anesthesia, making MSCs relatively easy to acquire. The results of the above described clinical trial are awaited eagerly.
B1. Heart originated epicardium-derived cells Generation of EPDCs by adult epicardium
Based on the extensive physical and directing role of embryonic EPDCs in embryonic heart formation
42, we studied in this thesis the therapeutic potential of adult EPDCs in the adult infarcted heart.
While much is known about embryonic epicardium and its important derivatives (EPDCs) 42, the adult epicardium has received only little attention. We previously demonstrated in vitro 40, as was later confirmed for murine tissue 67, that human adult epicardial explants generate cobble stone derivatives. As they spontaneously undergo epithelial-mesenchymal transformation (EMT) and acquire a spindle shape morphology 40 they become qualified as adult EPDCs 41. We used those cultured human adult EPDCs for our cardiac regeneration experiments, as discussed further below.
By viral labeling experiments we demonstrated in this thesis that EPDCs might also be generated in vivo from the adult epicardium in case of MI. We had labeled the epicardial layer by injecting a fluorescent lentivirus into the pericardial cavity of hearts with and without MI. Whereas normal hearts showed labeling of the outer squamous layer only, the hearts with MI contained reporter cells scattered through the infarcted ventricular wall. These data suggest for the first time that EPDCs are generated in vivo in the adult heart. We speculate that endogenous EPDC formation is required for homeostasis of the adult heart. In line with their role during heart formation, we suppose that EPDCs are continuously generated and that they contribute to maintenance of the vasculature and the compact ventricular wall. Future research must unravel the function of adult epicardium in the healthy, but also in the diseased (ischemic) heart.
Effect of EPDC transplantation on cardiac function
It was demonstrated in this thesis that the adult epicardium is indeed a promising subject with
regard to cardiac regeneration therapy. Transplantation of exogenous adult human EPDCs into the ischemic mouse myocardium improved LV ejection fraction (EF) both at 2 and 6 weeks after MI, as was detected by magnetic resonance imaging (MRI) assessment. Moreover, adverse remodeling was attenuated by EPDC injection. End-systolic volume (ESV) and end-diastolic volume (EDV) were significantly smaller in the EPDC-transplanted group than in the control group, up to 6 weeks after MI. We demonstrated that the beneficial outcome was stable, since it was sustained until a definitive scar had been formed, at 6 weeks after MI 68, qualifying EPDCs derived from the adult as promising candidates for cellular therapy. A previous study notified that functional improvement by embryonic stem cells (ESCs) might be transient, with increased LV function at 4 weeks after MI which had disappeared at week 12 69. Of note, healing is just completed by week 4 after MI, while both week 6, our endpoint, and week 12, their endpoint, represent a fully mature scar 68. We therefore expect that the positive outcome for EPDCs is maintained if follow-up is extended.
To investigate whether the EPDCs improved LV performance by exerting a real gain of function after initial loss, or whether they were responsible for a continuous preservation, we performed short- term transplantation experiments, with a read-out at day 2, 4 and 7 after MI. Results revealed that, in accordance with the suggested paracrine effect from the long-term EPDC studies, the engrafted exogenous EPDCs were responsible for a preservation of function, rather than a recovery of previous deterioration. Moreover, a significant difference between ESV and EDV, in favor of the EPDC group, was already present at 2 days after MI. We suppose that this latter finding implicates that EPDCs beneficially affect the ischemic host myocardium by a very early paracrine signaling, since tissue regeneration can not be accomplished within 2 days. It might also explain part of the reported benefit at 2 and 6 weeks, since, considering the Frank-Starling mechanism 70, a small but early decrease in LV volumes might result in large functional profit on the long term. But ongoing paracrine signaling of the engrafted EPDCs was also present, as we showed persistent increased DNA-damage repair in the ischemic host tissue of the EPDC group at 14 days after MI (discussed below).
Differentiation of transplanted EPDCs in vivo
Apparently, the signals within the injured area were appropriate for the human adult EPDCs to engraft, since the cells were almost always detected within the injured LV wall, and only very scarcely in the healthy area. They were located in between and directed in line with the host tissue cells.
Supporting their paracrine signaling as explanation for the functional improvement, the exogenous adult EPDCs did not express any cardiomyocyte markers. They acquired α-SMA expression within a few days of engraftment, as wells as discoidin domain receptor 2 (DDR2), which is a fibroblast marker 44,71. Together with their elongated shape, the protein expression characterized them as myofibroblasts 44,72. This was to be expected since embryonic EPDCs differentiate into interstitial and adventitial fibroblasts, besides smooth muscle cells 41,73-77. Regarding the debate about the endothelial fate of embryonic EPDCs 73,75,77-80, it must be notified that the adult exogenous EPDCs were never detected within the vessel lining, strongly challenging endothelial differentiation. In accordance with the in vivo constraints of adult EPDCs, they could not give rise to endothelial cells in vitro 40 . Although the engrafted EPDCs were reported to express von Willebrand factor (vWF) at 2 weeks after transplantation, this was not detected at the earlier time points (2, 4, 7 days) or at 6 weeks after injection into the heart. Of note, the α-SMA expression was also lost within 6 weeks, suggesting that the engrafted cells can switch their protein expression, probably adapting to the requirements of the cardiac environment.
Paracrine mechanisms of transplanted EPDCs
It was demonstrated within this thesis that paracrine signaling of the engrafted EPDCs on the surrounding host tissue largely explained the functional improvement after MI. A distinctive early and late effect, detected by day 2 and 14 respectively, could be distinguished. The latter included
sustained enhanced DNA-damage repair activity by the host tissue cells in the injured zone.
Proliferating cell nuclear antigen (PCNA) protein expression, as marker for DNA-damage repair 81,82, was much higher in the host tissue of the EPDC group at day 14 than in the control group. The observed difference was not yet detected in the first 7 days after MI. During the first week the PCNA protein was abundantly present in the injured area of both groups. Another target of the late paracrine effect was the vasculature. At day 14 vascular density was markedly increased in the infarcted area and borderzone of the EPDC group compared to the control group. A physical contribution of human EPDCs to the vasculature was excluded because the vessels consisted of murine endothelial cells only.
The difference in favor of the EPDC group was not yet present in the first week after MI, with many small vessels located in the infiltrate of each group. We suppose that the engrafted EPDCs instigated improved and/or extended natural DNA-damage repair, which resulted in augmented survival of the vasculature that normally occurs within the infiltrate early during cardiac healing. In fact, decreased tissue death further substantiated by better oxygen supply, can also account for the augmented LV wall thickness observed in the EPDC group. Overall, these data suggest ongoing signaling from the engrafted EPDCs, which is different from the demonstrated early paracrine effect (discussed below).
The early paracrine effect of EPDC-transplantation included a temporal shift forward in the upregulation of Wilms’ Tumor 1 (WT1) protein expression in the injured zone. WT1 is a nuclear transcription factor involved in development of several mesodermal organs 83. During cardiogenesis, WT1 expression is present in undifferentiated EPDCs and it is required for proper functioning, with artificially induced absence of the protein in the primitive EPDCs leading to myocardial thinning
84,85. One study has investigated its expression in the adult heart, and reported that it becomes
upregulated after MI 86, but its function during cardiac healing has not yet been revealed 86. We observed that in the control group this protein was expressed by epicardial, interstitial and vascular host tissue cells of the infarcted area from day 4 after MI. In contrast, when exogenous EPDCs were transplanted, WT1 was detected at day 2 after MI already. Because this time point coincided with the moment that first functional improvement in the EPDC-transplanted group was detected, it is strongly suggested that WT1 upregulation is protective for ischemia. This earlier protein expression in EPDC-transplanted hearts might indicate accelerated generation of WT1 positive primitive EPDCs by the endogenous epicardial layer. But it might also denote advanced renewed protein expression by interstitial or vascular cells already present within the infarcted area 86. WT1 is known to inhibit apoptosis 83,87, to stimulate robust capillary development 85,88, and to increase proliferation 85-87,89,90. Since we did not detect differences between groups with regard to these processes, WT1 expression itself is not suggested to be responsible for the functional improvement. Although a contribution of WT1 positive cells from the systemic circulation can not be excluded 90-92, we suppose that the accelerated WT1 expression in the EPDC group indicated renewed endogenous EPDC formation. The cubic shape of the host tissue epicardial cells covering the infarcted area, as opposed to the normal squamous morphology, supports the latter since a cubic form indicates EMT. Moreover, as discussed above, virus-mediated tracing experiments described in this thesis suggested that endogenous epicardium indeed generates new EPDCs by EMT in case of MI. It might be speculated that these endogenous- and exogenous-derived EPDCs present in the ischemic ventricle act in a synergistic manner during cardiac healing.
The altered WT1 expression might as well be representative for a series of other proteins that are modified in their expression either forward or backward in time as a result of the exogenous EPDCs.
We expect that the conceptually new finding of a timed balance-shift of natural processes by donor cell transplantation holds great promise for the development of new targets. Secure observation and documentation of the normal healing process, with a focus on the proteins that are strongly associated with proper functioning of the cell type that is injected (e.g. WT1 with regard to EPDCs), could reveal new insights.
B2. Heart originated cardiomyocyte progenitor cells
As demonstrated by Goumans et al, the adult human heart as well as the fetal heart harbor CMPCs that can differentiate in vitro without the requirement of being co-cultured with neonatal cardiomyocytes 30,53, like necessary for other human 32 and murine 24,93 cardiac progenitor cell
populations. The CMPCs could be isolated easily 30,53 from dissociated human adult cardiac specimens through clonal expansion and through magnetic cell sorting by means of their recognition of the murine sca-1 antibody 28,29,94,95. When cultured with 5’-azacytidine, a cytosine analogue that causes demethylation of DNA, and tissue growth factor-β (TGF-β), adult CMPCs differentiated very efficiently (80-90%) into spontaneous beating cardiomyocytes, which showed cross-striation, gap-junctional metabolic and electrical communication, and action potentials that resembled those of ventricular cells 30,53. The differentiation potential, together with a high nucleus-to-cytoplasm ratio, telomerase activity, expression of isl1 protein (although cytoplasmatic), the early cardiac transcription factor GATA-4 protein, but not of markers of differentiated cardiomyocytes, qualified the adult CMPCs as true cardiac progenitors 30. These CMPCs are suggested to originate in the SHF, since they are located mainly in the atria 52. As addressed below, we studied this cell population mainly with regard to their complementary effect on EPDCs. It was investigated whether the ‘constructive’ (i.e. with the capacity to generate cardiomyocytes) and ‘instructive’ (i.e. having a paracrine effect on the surrounding host tissue) properties of the SHF-derived adult CMPCs and EPDCs, respectively, could synergistically improve cardiac function after MI.
C. Which one to choose?
The results of the MSC and EPDC transplantation experiments described in this thesis reveal that both cell types are promising candidates for cellular cardiac regeneration therapy. Both were derived from IHD patients: the MSCs were harvested from bone marrow samples of patients with chronic myocardial ischemia 96, the EPDCs were isolated from atrial appendages which were acquired as left over material during coronary artery bypass graft (CABG) procedures. Both improved LV EF at day 14 after surgery, instigated through paracrine signaling. However, while ESV and EDV were significantly decreased by EPDC transplantation, adverse remodeling was not attenuated in the MSC-treated hearts. The early paracrine profit was detected for EPDCs only. It must be noted that the cell types were not compared within one study, and that the injected cell number was smaller for MSCs (2x105) than for EPDCs (4x105). But as recent studies denote that higher cell numbers and increased graft size do not result in enhanced functional improvement 69, we believe that the smaller transplant can not explain the absence of decreased volume expansion in MSC-treated hearts.
Considering MSCs and EPDCs, we assume that EPDCs are most suitable for therapeutic application.
This is supported by several of their unique properties. First they exert an early paracrine effect next to late ongoing stimulation of the surrounding host tissue, second they attenuate adverse remodeling besides increasing EF, and third the origin and natural niche of EPDCs are located within the organ of interest. The latter is of importance for further refining of their application by co- injecting complementary CMPCs, which themselves were demonstrated in this thesis to be almost equally effective with regard to cardiac repair when applied as isolated cell type. As discussed further below, our answer to the question which on to choose, would be: ‘a combination of EPDCs and CMPCs.’ With regard to feasibility though, MSCs are easier to harvest, since EPDCs, as well as CMPCs, must be derived from cardiac biopsies, and it takes time before they are sufficiently expanded. But if outcome is worth it, new research might find a solution to that disadvantage.
4. Combining important building stones to treat the infarcted heart
To mend a broken object, one must replace the materials that are lost. For the infarcted heart this obviously means that different cell types including cardiomyocytes and fibroblasts, as well as extracellular matrix must be provided. The heart might not optimally benefit from its transplant
if one cell type only is applied, especially since there is structural and functional interdependency between the various cell types in the heart 43,44. We demonstrated in this thesis that EPDC-
transplantation improves LV function and attenuates remodeling mainly through paracrine protection or stimulation of the surrounding host tissue, according to their function during embryonic heart formation 42,78,97,98. It was hypothesized that co-transplanting these ‘instructive’ EPDCs together with
‘constructive’ CMPCs, would further improve the reported profit after MI, supplying ontogenetically strongly related complementary cell types. Both EPDCs and CMPCs were harvested from human adult atrial appendages. It was shown in vitro that they mutually influence each other regarding proliferation, migration and growth factor mRNA-expression. Strikingly, in vivo application revealed that combined transplantation indeed further improved LV function after MI. EF was significantly higher in the co-transplantation group (CoT) than in single cell-type recipients, which themselves both demonstrated better cardiac function than vehicle controls. Moreover, EDV and ESV were further attenuated, and vascular density and wall thickness were increased in the CoT group. However, in contrast to our hypothesis, the engrafted adult human CMPCs had not differentiated into cardiomyocytes, implying that not only EPDCs, but also CMPCs acted through paracrine signaling.
Since graft volumes were comparable between the different groups, the detected synergistic benefit of EPDCs and CMPCs must be explained by distinct complementary paracrine effects.
We consider these results of high importance, as they suggest that each cell type might have its own unique effect which can add to the profit of another. If the molecular pathways underlying the reparative benefits of diverse cells used in the scientific world are elucidated, best combinations of cell types and proportions can be selected. We suppose that a cocktail of distinct ‘instructive’ and
‘constructive’ cells, possibly supplied with isolated (growth) factors or cytokines, or even seeded on specified scaffolds, holds the future for cardiac therapy.
Functional evaluation of the murine heart
Sophisticated and miniaturized methods are required to accurately study a presumed positive effect of cell transplantation on the infarcted mouse heart. Functional assessment is demanding because of the extreme small size of the heart (~100 mg and a long axis of ~7 mm) and because of the high contraction rate (~500 times per minute). Several methods have been adjusted for application in the mouse, including echocardiography, invasive pressure-volume (PV) -loop measurements by conductance catheter, and MRI. Of note, their suitability for the mouse heart under physiological conditions does not necessarily mean they are accurate in case of disturbed function.
Ultrasound echocardiography is the most widely used technique 99,100. It has the advantage of being non-invasive. Moreover, in comparison to the MRI, the equipment needed for echocardiography is quite easy to acquire and application is not as complicated. Employment is relatively subjective, and calculation of LV volumes is based on geometric assumptions, which makes the method less reliable if the shape of the LV is altered like after MI. Besides the commonly applied two-dimensional (2D) echocardiography, three-dimensional (3D) echocardiography has recently been developed for small animal application. It appears very accurate in cardiac phenotyping of healthy and chronically infarcted mouse hearts, even challenging the MRI as first method of choice 101,102.
We describe in this thesis a head-to-head comparison of two more objective methods, PV-loop measurements by conductance catheter and MRI. Both methods are reported to be accurate under physiologic conditions 102-104, but their wide usage in regenerative studies requires testing in ischemic mouse models 102,104-106. For the first, a miniature catheter is introduced via the carotid artery into the LV of the heart, where it simultaneously monitors LV pressures and volumes (computed from conductivity measurements). We demonstrated that both methods reliably assessed hemodynamics
and LV function of the failing mouse heart. Accuracy was maintained, although the extreme LV dilatation resulted in relative volumetric underestimation by PV-loop measurements. Which method is best suitable depends on the specific research question and experimental design. For advanced analysis of systolic of diastolic dysfunction, PV-loop must be chosen. In case of longitudinal examination, MRI is required.
MRI has several other unique valuable applications, like contrast-enhanced infarct size measurements
106 and the possibility to track iron-labeled stem cells 107-109. We showed in this thesis that both methods are easy to employ. We demonstrated that stem cell tracking by iron-loading of the cells must always be confirmed by histological evaluation though 110,111, since the method does not discern between dead and living iron-loaded cells. We suggest this problem must be overcome by infecting the cells with a lentiviral ferritin construct, instead of loading them with iron particles. The former will result in iron generation in living cells only 112.
We refined the MRI application further during the ongoing experiments described in this thesis.
Traditional MRI scanning requires respiratory and electrocardiogram (ECG) -leaded gating to acquire high-resolution images. In mice with a myocardial infarction cardiac triggering is highly complicated because the ECG signal is often disturbed, which results in decreased image quality. Recently, retrospective ‘wireless’ gating had become available, using a navigator slice to obtain information about cardiac and respiratory cycle, making ECG and respiratory triggering superfluous 113 The advantage of the ‘retrospective’ imaging above a traditional scanning method, especially for infarcted mouse hearts, is a higher image quality and a shorter scanning time. We demonstrated that besides the traditional ‘prospective’ scanning method as we applied for several studies in this thesis, the new
‘retrospective’ application could be very useful for cardiac regeneration studies. It must be combined with the newly developed IntraAngio tool (not yet commercially available) to enable analysis of image data with the specified software package. In general, because of its high reliability, its non- invasiveness, and the various extra applications as described above, we consider the MRI the first method of choice for functional assessment of the infarcted mouse heart.
Conclusions
In conclusion we demonstrated in this thesis that adult EPDCs are promising candidates for cellular cardiac regeneration therapy. We revealed that co-transplantation of complementary cell types like EPDCs and CMPCs could further increase the beneficial effect, which resulted from synergistic paracrine signaling. We encourage further research to i) use cardiogenesis as template for new research design, to ii) unravel the mechanisms underlying the benefit of cellular therapy, and, ultimately, to iii) invent the balanced cocktail of different cell types and growth factors, possibly seeded on a specific scaffold, that leads to the ideal cellular therapy.
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