**7. Generative and re-generative time windows: Definition and meaning**

As we saw earlier, *generation* and *re-generation* are capabilities shared by the same deputy cells named *stem cells*, the only ones capable of differentiating into all cell types that make up a complex organism. The number of stem cells is limited as well as the number of generations, and their *potency* is maximal during the pre-natal life in the *embryonic stem cells*, and progres‐ sively decreases in post-natal life remaining confined within the *adult stem cells*. These processes have very tight *time constraints* to allow the *harmonious development* and *mainte‐ nance* of a very complicated structure such as a higher vertebrate; thus generative and regenerative processes are only possible at the opening of a *specific time-window* that coordinates them through the activation of specific gene sequences. In this respect, the evolution towards multicellularity is a form of adaptation to the environment that involves a high cost in terms of biological resources to handle *differentiation, specialization*, *establishment of functional hierar‐ chies* and development of a *fully developed complex organisms* (Figure 11)*.*

It is therefore evident that the availability of resources for other forms of adaptation in *adult life* such as the *re-generation* process for *maintenance* or *repair* is limited, especially if we take into account the complex organisms. These *time-windows* have very different characteristics, the *generative time-window* close formally when the organism reaches its full development, although with large time differences between the various tissues. On the other hand, the *regenerative time-window* differ depending on the tissue and on the functional hierarchy that it covers; more precisely, in *permanent tissues*, the re-generative time window is *formally closed* and the cells that compose them cannot divide after generation; in *stable tissues* the window *can be re-opened* in the event of a *limited damage* to allow the *repair*; finally, in *labile tissues*, the window *remains open* almost throughout the life cycle for maintenance and repair (Figure 12).

It should be recalled, therefore, that the experiment of life on earth includes the *possibility of damages* caused by *unfavorable interactions* with the environment and, above all, that there are also some solutions to fix them (Ciulla et al., 2011). These solutions range from the optimal one which consists, obviously, in the re-generation, to that of lower efficacy such as the replacement Adaptation and Evolution in a Gravitational Environment — A Theoretical Framework… http://dx.doi.org/10.5772/55931 665

The number of stem cells and their replication cycles are limited; their potency is maximal during pre-natal life (embry‐ onic stem cells) and progressively reduces in post-natal life (adult stem cells). Generative and re-generative processes are possible only at the opening of a specific time-window (Figure 12). The potency of the cells is inversely proportion‐ al the degree of cellular specialization, that is thus maximal at monopotency.

1988). Therefore, even if gravity is a rather constant parameter on earth, it conditions the development of living organisms, before and after birth, with an increasing impact depending on having a blood circulation and, at the same time, on the propensity to postural change, and

Finally, several findings suggest that gravity continues to play a decisive role *during aging*, since the reduction of loading conditions that characterizes the more sedentary life typical of the elderly has important effects on the organism that resemble, in many respects, to what happens in microgravity environment during prolonged space flight (Vernikos and Schneider, 2010). In particular, the reduction of motion and acceleration typical of *aging* and of the *prolonged space flight*, can decondition the cardiovascular reflexes, altering the control of blood pressure with *orthostatic hypotension*, and lead to a reduction of muscle mass and the loss of

**7. Generative and re-generative time windows: Definition and meaning**

As we saw earlier, *generation* and *re-generation* are capabilities shared by the same deputy cells named *stem cells*, the only ones capable of differentiating into all cell types that make up a complex organism. The number of stem cells is limited as well as the number of generations, and their *potency* is maximal during the pre-natal life in the *embryonic stem cells*, and progres‐ sively decreases in post-natal life remaining confined within the *adult stem cells*. These processes have very tight *time constraints* to allow the *harmonious development* and *mainte‐ nance* of a very complicated structure such as a higher vertebrate; thus generative and regenerative processes are only possible at the opening of a *specific time-window* that coordinates them through the activation of specific gene sequences. In this respect, the evolution towards multicellularity is a form of adaptation to the environment that involves a high cost in terms of biological resources to handle *differentiation, specialization*, *establishment of functional hierar‐*

It is therefore evident that the availability of resources for other forms of adaptation in *adult life* such as the *re-generation* process for *maintenance* or *repair* is limited, especially if we take into account the complex organisms. These *time-windows* have very different characteristics, the *generative time-window* close formally when the organism reaches its full development, although with large time differences between the various tissues. On the other hand, the *regenerative time-window* differ depending on the tissue and on the functional hierarchy that it covers; more precisely, in *permanent tissues*, the re-generative time window is *formally closed* and the cells that compose them cannot divide after generation; in *stable tissues* the window *can be re-opened* in the event of a *limited damage* to allow the *repair*; finally, in *labile tissues*, the window *remains open* almost throughout the life cycle for maintenance and repair (Figure 12). It should be recalled, therefore, that the experiment of life on earth includes the *possibility of damages* caused by *unfavorable interactions* with the environment and, above all, that there are also some solutions to fix them (Ciulla et al., 2011). These solutions range from the optimal one which consists, obviously, in the re-generation, to that of lower efficacy such as the replacement

*chies* and development of a *fully developed complex organisms* (Figure 11)*.*

both of these characteristics are typical of higher vertebrates.

calcium from the bones.

664 Regenerative Medicine and Tissue Engineering

**Figure 11.** Relationship between potency and specialization of cells

of damaged tissue with *scar tissue,* a process known as *scarring*. Thus, the primary roles of *adult stem cells* in a living organism is to support the *functional reserve* by *maintaining* and, eventual‐ ly, *repairing* the tissue in which they are found, according to a specific re-generative time window. Unfortunately there is a very small number of adult stem cells in each tissue, with large numerical differences between a tissue and another, and, therefore, the *re-generative potential* is unevenly distributed and, in any case, is limited. It should be remarked that in the *vascular system* a vast reservoir of adult stem cells is maintained for *renewal processes* including

Master plan of re-generative time-windows in the three main tissues, according to the cell replication capabilities dur‐ ing human life. The white arrows indicate the opening and the closing of each window.

**Figure 12.** Re-generative time-windows

the continuous production of the endothelial cells lining the vessel wall and of the circulating blood elements.

In complex organisms the functional hierarchy is paramount in allocating this functional reserve where it is needed establishing the priorities in the *distribution of the available resour‐ ces*. In mammals many adult tissues contain populations of adult stem cells that have the capacity for *renewal after disease* or *aging* but some *highly specialized tissues* -such as heart and brain- have a very limited re-generative potential. At this regard, we cannot exclude that the extreme structural and functional specialization reached by some tissues is a further and, possibly, inherent limit to re-generation.

Thus, returning to a broader view, it is a matter of fact that in the complex interplay between organization of the living matter, natural selection and adaptation, mammals have evolved with limited or no re-generative capabilities of the heart after birth and the reasons for this apparent flaw have to be sought in organization and allocation of resources in a hierarchically structured multi-cellular organism with an evolved system of transport and communication, such as the circulation of blood.

Focusing on *adaptive resource allocation*, it is widely acknowledged that during the post-natal development of the heart in higher vertebrates there exists a limited generative time-window. In figure 13 we show the upper limit of such time-window for different animal species.

During the post-natal cardiac development exists a limited time-window of potential tissue regeneration; the graph shows the lower (yellow) and the upper (green) length of this period. The data were taken from the following referen‐ ces: (Li et al., 1997; Brodsky et al., 1988; Hoerter et al., 1981; Clubb and Bishop, 1984; Burrell et al., 2003; Morgan and Beinlich, 1997; Bishop and Hine, 1975). Data collected by courtesy of Paola Nicolini, MD.

**Figure 13.** Limited generative time-window of the heart in higher vertebrates

the continuous production of the endothelial cells lining the vessel wall and of the circulating

Master plan of re-generative time-windows in the three main tissues, according to the cell replication capabilities dur‐

ing human life. The white arrows indicate the opening and the closing of each window.

In complex organisms the functional hierarchy is paramount in allocating this functional reserve where it is needed establishing the priorities in the *distribution of the available resour‐ ces*. In mammals many adult tissues contain populations of adult stem cells that have the capacity for *renewal after disease* or *aging* but some *highly specialized tissues* -such as heart and brain- have a very limited re-generative potential. At this regard, we cannot exclude that the extreme structural and functional specialization reached by some tissues is a further and,

Thus, returning to a broader view, it is a matter of fact that in the complex interplay between organization of the living matter, natural selection and adaptation, mammals have evolved with limited or no re-generative capabilities of the heart after birth and the reasons for this apparent flaw have to be sought in organization and allocation of resources in a hierarchically structured multi-cellular organism with an evolved system of transport and communication,

blood elements.

**Figure 12.** Re-generative time-windows

666 Regenerative Medicine and Tissue Engineering

possibly, inherent limit to re-generation.

such as the circulation of blood.

Since generation and re-generation of the heart rely on the same physiological reserve consisting in proliferating cardiomyocytes, is logical to assume that the generative and regenerative time-windows coincide, but the questions on re-generative potential still open are:


In humans what appears to be a flaw may not have been a problem for thousands of years until, in the twentieth century, the increase in *life expectancy* (Burger et al., 2012) has given rise to diseases generally less frequent in the first three or four decades of life. Therefore, the significant increase in *morbidity* and *mortality* related to cardiovascular disease, seen mainly in Western countries in the last years (Smil, 1989; Ramsden et al., 2009), has brought to the foreground the problem of *cardiac damage* and of its repair. According to this broader view of the problem, the limited regenerative capacity should be read in the context of the processes associated with aging (Frangogiannis, 2012). At this point it should be emphasized that in Western societies cardiovascular disease have a cost and this cost is fully or partly covered by the community; therefore, as economic resources are finite and health care costs are program‐ med at the political level on the basis of emerging issues, the new opportunities arising from research on adult stem cells have represented in recent years an attractive location for public and private funds. As in the overall budget military spending represents an item in competition with those of health, research on adult stem cells has the great merit to redirect spending towards scientific research in the biomedical field.

#### **2.** *What is the physiological significance of this post-natal re-generative time-window?*

Some studies have tried to characterize the post-natal re-generative window by using its potential to repair a myocardial damage. Among them, one of the most impressive for the scientific community was conducted a few years ago on the zebrafish with the amputation of a limited portion of the cardiac apex (Poss et al., 2002). The results were in line with the expectations since this kind of injury is coherent with the pathophysiology of teleost fish and urodele amphibians which are known to regenerate body parts, and the resected ventricle was regenerated (Poss et al., 2002; Neff et al., 1996). More recently, a similar experiment was conducted on mice achieving a similar regenerative result (Porrello et al., 2011) even if the resection of about 15% of the ventricular myocardium has a poor clinical applicability to humans. Unfortunately the goal of both these studies, was not to elucidate the physiological significance of this re-generative time-window but, rather, to evaluate its potential under *extreme experimental conditions* far from a clinical setting, even if hypothetical.

In this regard, the limited duration of the re-generative cardiac time-window and its placement between fetal and postnatal life clearly argues for its physiological function linked to the development of the cardiovascular system in a gravitational environment whose impact grows, indeed, after the birth. The plasticity of the heart is therefore required in mammals to support the new load conditions: birth is an event that carries great challenges for the heart, not only because there is a change from the fetal to the neonatal circulation which imposes haemodynamic stresses on the ventricles but also because it is marked by a transition from a microgravitational to a gravitational environment, with gravity playing a key role in the maturation of the cardiovascular system (Magrini et al., 1989).

Thus it cannot be excluded that the use of this post-natal potential for *re-generative purposes* that go beyond the physiology, such as the repair of a myocardial damage, would interfere with normal cardiac development.

Aboutthemechanismbywhichmyocytesproliferate,theproliferationofcardiomyocytesimplies their dedifferentiation and the previous cited study (Porrello et al., 2011) provided evidence of sarcomeric disassembly, but this is somewhat far from a biological point of view. Indeed, with the sole exception of cancer stem cells it is generally accepted that cell division does not require the cell to move backwards along its differentiation pathway. Other authors (Bersell et al., 2009) suggestthat sarcomericdisassemblyoccurs in*differentiated*cardiomyocytesandis theprocessby which the cell reorganises its contractile apparatus to avoid interference with karyo- and cytokinesis. But the most likely hypothesis is that the replicating cells belong to an earlier stage of differentiation since possibly are adult cardiac stem cells committed to the myogenic line‐ age. There is convincing data that in vitro *c-kit*+ stem cells show an immature morphological and functional phenotype, including sarcomeric disassembly (Beltrami et al., 2003).

to diseases generally less frequent in the first three or four decades of life. Therefore, the significant increase in *morbidity* and *mortality* related to cardiovascular disease, seen mainly in Western countries in the last years (Smil, 1989; Ramsden et al., 2009), has brought to the foreground the problem of *cardiac damage* and of its repair. According to this broader view of the problem, the limited regenerative capacity should be read in the context of the processes associated with aging (Frangogiannis, 2012). At this point it should be emphasized that in Western societies cardiovascular disease have a cost and this cost is fully or partly covered by the community; therefore, as economic resources are finite and health care costs are program‐ med at the political level on the basis of emerging issues, the new opportunities arising from research on adult stem cells have represented in recent years an attractive location for public and private funds. As in the overall budget military spending represents an item in competition with those of health, research on adult stem cells has the great merit to redirect spending

**2.** *What is the physiological significance of this post-natal re-generative time-window?*

*extreme experimental conditions* far from a clinical setting, even if hypothetical.

maturation of the cardiovascular system (Magrini et al., 1989).

with normal cardiac development.

Some studies have tried to characterize the post-natal re-generative window by using its potential to repair a myocardial damage. Among them, one of the most impressive for the scientific community was conducted a few years ago on the zebrafish with the amputation of a limited portion of the cardiac apex (Poss et al., 2002). The results were in line with the expectations since this kind of injury is coherent with the pathophysiology of teleost fish and urodele amphibians which are known to regenerate body parts, and the resected ventricle was regenerated (Poss et al., 2002; Neff et al., 1996). More recently, a similar experiment was conducted on mice achieving a similar regenerative result (Porrello et al., 2011) even if the resection of about 15% of the ventricular myocardium has a poor clinical applicability to humans. Unfortunately the goal of both these studies, was not to elucidate the physiological significance of this re-generative time-window but, rather, to evaluate its potential under

In this regard, the limited duration of the re-generative cardiac time-window and its placement between fetal and postnatal life clearly argues for its physiological function linked to the development of the cardiovascular system in a gravitational environment whose impact grows, indeed, after the birth. The plasticity of the heart is therefore required in mammals to support the new load conditions: birth is an event that carries great challenges for the heart, not only because there is a change from the fetal to the neonatal circulation which imposes haemodynamic stresses on the ventricles but also because it is marked by a transition from a microgravitational to a gravitational environment, with gravity playing a key role in the

Thus it cannot be excluded that the use of this post-natal potential for *re-generative purposes* that go beyond the physiology, such as the repair of a myocardial damage, would interfere

Aboutthemechanismbywhichmyocytesproliferate,theproliferationofcardiomyocytesimplies their dedifferentiation and the previous cited study (Porrello et al., 2011) provided evidence of sarcomeric disassembly, but this is somewhat far from a biological point of view. Indeed, with

towards scientific research in the biomedical field.

668 Regenerative Medicine and Tissue Engineering

Finally, as it is known that the physiological processes taking place during postnatal cardiac growth lie along a continuum in time (Ahuja et al., 2007) it is reasonable to suppose that the proliferative response to amputation, in terms of intensity and duration, would also exhibit a temporal gradient. Unfortunately it is not possible to track its dynamics since none of these studies investigate multiple time-points and neglect the intermediate time-window. In future studies aimed to the characterization of this re-generative time-window valuable information might be obtained by exploring earlier time-points around the time of birth, possibly including the late prenatal period. Moreover, considering that physiological reserves are finite, to exclude that the use of these resources does not interfere with cardiac development, both the contractility and the residual proliferative potential of the heart should be evaluated carefully.

#### **3.** *What are the clinical implications of this last re-generative chance for the heart?*

As we have seen, this time-window that appears to be the last post-natal re-generative "chance" of the heart argues for a function that seems to be closely linked to the development of the cardiovascular system in a gravitational environment and, in particular, to support the new load conditions at birth. Thus it is difficult hypothesize its role in a specific clinical scenario. Assuming a clinical role for this time-window restricted to the neonatal period, it is possible to take into consideration neonatal acute myocardial infarction (AMI) as clinical scenario, but AMI is rare, usually due to congenital heart disease, paradoxical coronary artery throm‐ boembolism or perinatal asphyxia in premature newborns with severe respiratory distress (Poonai et al., 2009), and is often extensive (Fesslova et al., 2010; Abdurrahman et al., 1999; Iannone et al., 1975) and with an extremely high mortality rate, approaching 90% (Poonai et al., 2009). This, of course, raises questions about the possibility to use the re-generative resources for reparative purposes in this context. On the other hand, if applied to the clinical setting of adult AMI, it would be more appropriate since, according to recent epidemiological data, the last few decades have witnessed a marked decrease in infarction severity, with a growing predominance of non-ST elevation AMI (NSTEMI) over transmural AMI (Roger et al., 2010). Unfortunately this time-window is almost completely closed soon after birth in mammals, therefore his only possible clinical use in adults requires the exact knowledge of the mechanisms that control its opening and closing in order to re-open it when necessary as to repair a myocardial damage. Finally, it is possible to speculate that further experiments in microgravity environment, such as prolonged space flight, can provide further information on the functioning of the post-natal re-generative time-window of the heart.
