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Cell fate decision processes by immature precursor cells and stem cells are of fundamental importance in developmental biology. While much is known about the mechanism and regulation of such decisions in genetically tractable invertebrate organisms such as Drosophila and C.elegans, relatively little information is available in the mammalian system. At its heart a stem cell fate decision process involves symmetric or asymmetric cell division [1]. In general, all cell fate decisions are governed by a combination of mechanisms intrinsic to the immature precursor, and signals originating from the microenvironment. There are numerous examples of a stochastic and/or instructive nature inherent in the mechanisms that regulate cell fate choices [2]. It has also been suggested that these decisions are mediated by a combination of both.
In the mammal, an attractive system to analyze developmental cell fate decisions is hematopoiesis. At the center of all hematopoietic systems is a small population of stem cells, which collectively give rise to at least eight distinct lineages of mature blood cells. Because mature blood cell populations have finite half-lives, the stem cells must produce large numbers of functional blood cells in a continuous manner throughout life. This requires that the stem cell population be continuously present in the adult hematopoietic tissues. The hallmark feature of hematopoietic stem cells is therefore the ability to balance a self-renewal decision with a decision to commit to differentiation pathways. Stem cells have been described in other tissues that require continuous replenishment, or which have the ability to regenerate after extensive injury. Good examples of these are the skin, the intestinal epithelium, the liver, and the muscle [3]. Recently, it has been shown that tissues, such as the central nervous system, previously thought to be produced only during fetal development, can also be replenished from an adult stem cell compartment [4] mammalian hematopoietic stem cell system is one of the most accessible stem cell systems for analysis. At least four decades of studies in rodents and in humans have defined the hallmark properties of this cell population. The most primitive stem cell population can only be measured by transplantation into an ablated recipient. In the mouse this is feasible in an experimental setting, while in the human, such transplantation is performed therapeutically. The first hallmark property of the hematopoietic stem cell is multipotentiality. It has been shown that single stem cells can give rise to all myeloid, erythroid, thrombocytic, dendritic, and lymphoid lineages [5]. In fact, the entire murine blood cell system can be produced by single transplanted stem cells. These cells can maintain a normal, lifelong functioning hematopoietic system. This latter feature also demonstrates a high degree of self-renewal potential; the second hallmark property of hematopoietic stem cells. The ability of single stem cells to generate an intact blood cell system demonstrates a third characteristic property of stem cells, as well; the enormous proliferative ability necessary for the production of an entire hematopoietic system. A fourth property of stem cells is their rare and infrequent nature; about one in ten thousand to one in a hundred thousand total bone marrow cells [6]. The bone marrow is the principal location of stem cells in the adult, while the fetal liver serves this function during development. In the bone marrow of the mouse there are on the order of several thousand total stem cells. The fetal liver is the principal site of stem cell expansion, yielding the entire complement necessary for adult life [7]. Currently, the site of stem cell origin during development is controversial. Some have suggested that stem cells originate in the extraembryonic yolk sac [8] while others point to the aorta, gonad, mesonephric region [9] and its precursor the para-aortic splanchnopleura [10] as the site where definitive stem cells first originate. More recently it has been suggested that both sites are capable of generating long-term repopulating stem cells [11]. An additional property of stem cells is their relative quiescence in adult tissue [12]. During fetal development, the stem cell population is in active cell cycle, and the adult stem cell population can be induced to rapid proliferation during situations of hematologic stress [13]. Taken together, the hallmark features of stem cells necessitate tightly controlled regulatory processes that must be sufficiently flexible to respond to situations of need.
All of the above properties of stem cells were determined retroactively. That is, by analyzing mature cell populations from mice transplanted with whole hematopoietic tissue containing some complement of genetically marked stem cells. In order to begin analyses of stem cell regulatory mechanisms it was necessary to develop technologies to define the stem cell prospectively; that is, prior to transplantation. In other words, it was necessary to purify the stem cell population from a given tissue. Numerous strategies to purify stem cells have been described [14]. In general, these rely on the judicious application of relatively non-specific cell surface markers or physical properties as positive and negative parameters for cell fractionation. It has been feasible to obtain populations of stem cells that are several thousand fold enriched for transplantable activity. It should be noted that given the necessity of an in vivo assay to measure stem cell activity, it is very difficult to estimate the absolute degree of stem cell homogeneity attained in any purification strategy. Quantitatively accurate estimates of relative enrichment can be obtained by careful design of the transplantation experiment. One such design is competitive repopulation as demonstrated in the tranplantations activity graphs (1, 2) above using the Ly5 congenic system [15]. Regardless of these limitations, the ability to purify stem cells elevated a biological activity to the status of a physically defined cell. Thus, it became possible to directly address the biological properties of these cells.
Major efforts using purified stem cells have focused on developing ex vivo culture systems designed to recapitulate their in vivo behavior. Of primary interest are systems that may promote the self-renewal process; thus permitting stem cell expansion. Not only would such an ability have profound implications for clinical transplantation, it would facilitate more complex therapeutic approaches, such as gene therapy. Generally there are two basic variations on in vitro stem cell culture systems: 1) those relying on defined cocktails of known cytokines, and 2) those supported by an adherent stromal cell layer often in the absence of exogenous cytokines. In can be argued that the latter more closely resemble the in vivo situation where stem cells have been shown to co-localize to certain regions of the bone marrow, presumably in close apposition to supportive microenvironments [16]. These stem cell supportive hematopoietic inductive microenvironments constitute stem cell niches [17].
Before discussing the stromal cell supported ex vivo culture systems, it is worth summarizing the collective results using defined cytokine cocktails in stromal-free cultures. The reason for this is that these systems are more defined, and in principle, could provide efficient pluripotent stem cell expansion conditions. Moreover, there are numerous identified (and cloned) cytokines, growth factors, and chemokines along with their cognate receptors. Despite extensive efforts by numerous investigators, it has not been possible to promote significant in vitro expansion of the self-renewing stem cell with any cytokine combination. Numerous groups have reported significant expansion of more committed progenitor cell populations [18]. From a clinical point of view, such populations may be very useful, especially in situations where the rapid production of mature blood cells is required. Clearly, in many cases stem cells respond to mixtures of cytokines by entering into active cell cycle, and producing large populations of progeny [19]. The vast majority of the progeny is more committed in its differentiation status, and has lost the ability to function as stem cells upon transplantation . It is also possible that these cells retain stem cell properties, but have lost the ability to home to the correct microenvironment after transplantation. A third possibility is that the engraftment capabilities of stem cells vary with the progression of the cell cycle; with the relatively quiescent cells retaining the bulk of the in vivo functional ability [20]. Collectively, these are interesting observations given the large numbers of available cytokines and other molecules, and the fact that both ES and neural stem cells can be dramatically expanded without loss of developmental potential. For murine Embryonic Stem (ES) cells this is mediated by Leukemia Inhibitory Factor (LIF) [21] while neural stem cells can be expanded in Epidermal Growth Factor (EGF) or basic Fibroblast Growth Factor (bFGF) [22]. One interpretation of these largely negative and discouraging results is that the correct hematopoietic stem cell "expansion factor(s)" has not yet been identified. There is some merit to this argument because if stem cells are rare, it is possible that the microenvironmental cells that may be producing such a factor may be equally rare (see below). Alternatively, it may be that the expansion of stem cells will not be mediated by any simple factor combination. Rather, it could be that such expansion is a function of many microenvironmental signals, none of which alone or in simple combinations is sufficient. If this is the case, the regulation of hematopoietic stem cells is radically different than in the ES, neural, and possibly other stem cell systems. A reason for this may be evolutionary. Given the remarkable daily production rate of mature blood cell populations, all originating from a small compartment of stem cells, it may be too "dangerous" to relegate the hematopoietic stem cell proliferative control to simple mechanisms. Simply stated, the simpler the regulatory mechanism, the greater the probability of malfunction. In tissues such as the nervous system the generation of new neurons proceeds at a slow rate. Therefore, a simple mechanism of regulating proliferation and cell fate choices in these may be acceptable in terms of risk. In the hematopoietic system a breach of normal stem cell regulation could yield leukemic states, or other systemic abnormalities. This is particularly true if normal hematopoiesis originates from few stem cells clones.
Not all cytokine supported hematopoietic stem cell culture efforts have been without reward. In fact, it is known that three classes of cytokines (and their cognate receptors) are likely to play important roles in the in vivo regulation of stem cells. These are the gp130 mediated signaling molecules (IL-6, IL-11, and LIF), Thrombopoietin, as well as the ligands (SCF, FLT3L) for the receptor tyrosine kinases c-Kit and Flk2/Flt3 [23]. For the latter two categories such functions have been confirmed by genetics [24]. As an aside, it has been shown that gp130 signaling through the action of LIF and the downstream activation of STAT3 is the relevant mechanism for ES cell expansion [25]. It is intriguing why hematopoietic stem cells, while responsive to this signaling pathway, do not undergo self-renewal expansion. It is also interesting that hematopoietic stem cells require combinations of multiple factors for entry into active cell cycle. Culture systems supported by combinations of factors from the above three categories have been shown to maintain stem cell activity for short in vitro time intervals. In all cases the in vivo activity of the cultured stem cells is maintained at near input levels with no, or very little quantitative expansion. In some cases, it has been shown that the stem cells undergo limited division, although this division does not appear to yield increased numbers of functional stem cells. A particular kind of in vitro blast cell colony can also be grown in combinations of the above cytokines. These colonies have been shown to retain most, if not all, hematolymphoid potentials after replating [26]. These efforts have suggested that the activation of the Notch [27], Wnt [28] and other developmental signaling pathways [29] may play a positive role in hematopoietic stem cell expansion. In all of these cases, the in vitro data show at best, only modest increases in net stem cell activity after culture. In addition, it is possible that the chemokine SDF-1 and its receptor CXCR-4 [30] play a role in stem cell maintenance; although it is likely that this role is to promote the homing of stem cells. Significantly, the in vivo involvement of this signaling pathway in stem cell biology has been genetically confirmed [31]. In summary, the efforts utilizing defined cytokines or other molecules to mimic the in vivo regulation of the hematopoietic stem cell compartment have yielded modest success. They have also highlighted the general paucity of definitive information that describes the regulation of stem cells in vivo. A reasonable conclusion from the collective studies is that hematopoietic stem cells will be regulated by many synergistic pathways, each of which contributes its own small share.
In parallel with efforts to culture stem cells with defined cytokines were efforts, beginning with the Dexter culture system, that sought too recapitulate in vitro the normal and complex environment of the bone marrow. These cultures amounted to a "bone in a flask", where the entire cellular contents of the bone marrow was allowed to "set-up-shop" in a tissue culture flask [32]. While enormously complex and heterogeneous in cell content, these cultures were in fact capable of supporting in vitro hematopoiesis for very long time intervals. This alone suggested very strongly that the stem cell compartment is capable of self-renewal under these in vitro conditions. A direct proof of such self-renew activity was provided using clonotypic retroviral markers [33]. In short, the Dexter culture system provided a starting point to define the relevant cellular component that might correspond to the stem cell in vivo niche.
The "dissection" of the Dexter culture system required not only purified stem cells, but also purified cellular components of the stromal microenvironment. Because of a lack of cell markers, the most feasible avenue to obtain "purified" stromal elements, is to convert the stromal cell population in to a series of immortalized clonal cell lines. In some cases, these cell lines were produced using conditional immortalizing agents such as SV40 T-Antigen [34], its temperature sensitive variants [35], and human papilloma virus E6/E7 genes [36]. The hope was that the problem of stem cell regulation, while not yet converted into a set of defined factors, could be recast in terms of a purified stem cell and a single supportive cell line. The underlying hypothesis was that a single type of cell in the hematopoietic microenvironment can support the balance of self-renewal and differentiation, or possibly promote undifferentiated stem cell expansion. In addition, it was argued that because stem cells are rare, then their supportive microenvironments might be composed of equally rare cell types.
Over time numerous stromal cell lines have been produced, in most cases from adult bone marrow. Many of these cell lines are capable of supporting in vitro hematopoiesis for prolonged time periods in the presence or absence of exogenously added defined cytokines [37]. In some cases a single cell line could support both myelopoiesis and B-lymphopoiesis depending on the exact culture conditions [38]. The development of supportive cell lines led to the development of quantitative in vitro assay systems for primitive hematopoietic stem and progenitor cells. To an extent, these assay systems are surrogates to in vivo transplantation as a measure of stem cell activity. Although there appears to be a close correspondence to the content of in vivo reconstituting stem cells and in vitro culture initiating cells, it is unclear if in fact the same cell can function in these two contexts. The nature of the in vitro surrogate assay system is complex. In general, the longer it takes a single deposited cell to make a distinctive "cobblestone" colony in co-culture with a stromal cell line, the more primitive the input cell is [39]. This is due to the largely quiescent nature of the most primitive cells, and the likely stochastic nature of their entry into cell cycle. The most common long-term stromal supported assay system is the Long Term Culture-Initiating Cell (LTC-IC) [40] and the related Extended (E) LTC-IC assays [41]. Both of these assay systems have been widely utilized to measure primitive human stem and progenitor cells. In comparison with xenogeneic in vivo systems to measure human cells, the ELTC-IC seems to be more appropriate as a measure of primitive cells. It has also been possible to devise modifications of these assays to allow the development of myeloid and lymphoid progeny from the same precursor cell [42]. Results from our own efforts that relate the in vivo and in vitro assay systems will be discussed in a subsequent section.
A number of years ago we reasoned that it would be fruitful to generate immortalized stromal cell lines from the murine fetal liver. The fetal liver is a major (perhaps the only) site of stem cell expansion during normal development. Therefore, this may be an appropriate source for stromal cells that will promote the in vitro expansion of stem cells. We generated over two hundred clonal cell lines using temperature sensitive SV-40 T-Antigen. The individual lines were screened for their abilities to support long-term in vitro hematopoiesis originating from partially enriched primitive stem cells. The lines were grouped into categories based on their abilities to support hematopoiesis for different periods of time. The rationale was that the long-term supporting cell lines were more likely to act on more primitive stem cells, because long-term hematopoiesis can only originate from these cells. The number of cell lines that were capable of long-term maintenance of hematopoiesis was small, around two percent. This was consistent with our initial hypothesis that stem cell microenvironments may be rare. A subset of cell lines representative of the various temporal categories was tested for their abilities to maintain in vivo reconstituting stem cells for prolonged, three weeks, in vitro time intervals. These studies, although carried-out with whole bone marrow as the source of stem cells, demonstrated a wide heterogeneity in the ability of individual cell lines to support long-term repopulating stem cell activity [43]. The collective data do suggest that individual cell lines act directly on stem cells and support the maintenance of their in vivo potentials for prolonged in vitro time intervals. It should be noted that few, if any, cytokine combinations can support even in vitro hematopoiesis for prolonged time periods. It seems clear that the stromal microenvironment provides a qualitatively and/or quantitatively different type of support to stem and progenitor cells.
We pursued more extensive characterization of a small subset of stromal cell lines. It was of paramount importance to demonstrate that at least some of the lines could support highly purified stem cells. If so, then it would be very likely that the cell lines act directly on stem cells, and therefore, that the entire collection of relevant molecular signals is expressed by an individual cell line. Indeed, we identified two cell lines, AFT024 and 2012 that can maintain the transplantable biological activity present in highly purified fetal liver and adult bone marrow stem cell populations [44]. Numerous other cell lines such as 2018 and BFC012 had little, if any, supportive ability. Most of our efforts focused on the AFT024 cell line because the stem cells maintained in co-culture with this cell line retained in vivo repopulating ability, both qualitatively and quantitatively. This was observed even when one hundred or fewer cells were used to initiate the cultures. It is of interest that the recovered stem cell activity is quntitatively the same as the input; with no net expansion or depletion. The cultured stem cells contributed to all mature cell lineages, and functioned in secondary transplant recipients to an extent indistinguishable from their freshly purified, non-cultured counterparts. Based on our extensive analyses of AFT024 (see biological data section), we have suggested that this cell line represents an in vitro correlate of a stem cell niche. As mentioned above, the entire panel of stromal cell lines was heterogeneous with respect to their stem cell supporting abilities. This is in spite of the fact that they were all isolated from the same developmental source, and all are of the same genetic background.
At this point it may be useful to consider some properties that define a stem cell niche a priori. The primary characteristic of a stem cell niche is the ability to maintain a compartment of stem cells in an undifferentiated state. This involves not only the prevention or overriding of commitment and differentiation promoting signals, but also maintenance of stem cell viability. An effective stem cell niche must also physically retain at least some stem cells in proximity to itself. Given that upon transplantation, stem cells appear to "home" to particular microenvironments, a stem cell niche may also be active in this process. Finally, a stem cell niche must know "when to let go"; that is when to allow a stem cell to embark on pathways of commitment and differentiation. Ultimately, the function of an effective stem cell niche is to provide the appropriate cellular milieu where proper numbers of undifferentiated stem cells are maintained in balance with those destined for active hematopoiesis. As discussed below, a closer investigation of the stem cell/AFT024 co-culture system has provided insights into how such a cellular milieu may function.
What is the nature of the stem cell supportive ability of AFT024? It is possible that the cultured stem cells are simply maintained in a quiescent state for the overall four to six week culture period. Alternatively, it is possible that the culture system promotes a balance of self-renewal and commitment to differentiation. We have addressed this issue. It is clear that while the net in vivo reconstituting activity in the AFT024/stem cell co-cultures remains at input levels, other primitive progenitor cell populations are greatly expanded. These include: 1) cells capable of initiating secondary cobblestone colonies after replating on AFT024 2) cells capable of initiating long-term B-lymphopoiesis after replating in Whitlock-Witte conditions and 3) cells capable of generating HPP-CFC-Mix colonies in semisolid media . We have also observed that at early times of co-culture the in vivo repopulating activity actually decreases, only to re-bound to input levels after four weeks. It seems clear that the stem cells in the AFT024 co-cultures are proliferating to give rise to expanded populations of committed progenitors and a regenerated population of self-renewing or in vivo repopulating stem cells. In other words, the AFT024 microenvironment facilitates the balance of self-renewal and differentiation.
Collectively, the primitive stem cells, and the various classes of committed progenitors present in the long-term AFT024 co-cultures represent the entire primitive portion of the hematopoietic hierarchy. Therefore, it is possible that individual stem cells can give rise to this hierarchy in culture with this cell line. If true, then the picture of a stem cell microenvironment that emerges is rather novel. Rather than having distinct microenvironmental cell types which mediate self-renewal and commitment, a single type of cell is sufficient to promote both processes, but in a balanced fashion. What this means in practical terms is that numerous primitive progenitors should be present in clones produced by single stem cells during co-culture with AFT024. In order to test this notion, we performed single cell deposition experiments with individual highly purified fetal and adult stem cells. The appearance of cobblestones in the AFT024 co-cultures was mapped weekly by microscopic inspection of the ninety-six well plates. We chose late-appearing and persistent colonies for a series of replating experiments designed to measure the entire range of myeloid and lymphoid progenitors. In three out of twenty cases, all hematolymphoid populations could be detected in cobblestone colonies derived from a single deposited stem cell. These data conclusively demonstrate that: 1) a cell with the entire complement of developmental potentials characteristic of an in vivo repopulating stem cell can proliferate in response to signals produced by AFT024, and 2) the AFT024 derived signals are acting directly on stem cells. Whether the cobblestone colonies contain actual repopulating stem cells remains an open question.
The proliferation of in vivo repopulating stem cells in AFT024 co-cultures was directly demonstrated using retroviral markers. Standard retroviral vectors require active cell division in order to integrate into the host chromosomal DNA. AFT024 co-cultures initiated with purified stem cells were pulsed at 2 different times during the culture period with different retroviral vector containing supernatants. The presence of a proviral marker in mature hematopoietic cell populations from mice engrafted with the cultured cells requires that the in vivo repopulating stem cells were actively dividing in the AFT024 co-cultures without losing their in vivo potentials. In preliminary experiments this was indeed the case. Importantly, the proviral markers were still present more than six months post-transplant, and in multiple hematopoietic tissues. In this experiment it was not possible to determine the integration site of the provirus. Therefore, no direct measure of multi-potentiality or in vitro self-renewal was provided. The latter process would be demonstrated by the detection of the same proviral integrant in more than one recipient of a single culture.
Taken together, our data support the hypothesis that stem cells are actively proliferating in the AFT024 co-cultures, and this is accompanied by a balance of self-renewal and commitment. As stated previously, cytokines can promote the proliferation of primitive stem cells; however this proliferation does not result in a balance of self-renewal and commitment. If stem cells are proliferating in response to AFT024, then why is there no net increase in transplantable stem cell activity? A seemingly inescapable answer to this question is that the more committed hematopoietic cells that are produced in the cultures may be inhibiting the generation of stem cells by feedback mechanisms. If this is true, then one role of the stem cell niche may be to provide an appropriate milieu where such "cross-talk" between the different compartments of the hematopoietic hierarchy can occur.
A number of laboratories have shown that AFT024 is a potent supporter of primitive human stem and progenitor cells. Therefore, the mechanisms that promote the balance of self-renewal and commitment are conserved between mouse and man. Given the central importance of the hematopoietic system and the functional similarities between murine and human stem and progenitor cells, this is not entirely surprising.
Recently, there has been much excitement concerning the potentially great plasticity of somatic tissue stem cells (for reviews see [45]) The traditional dogma is that tissue specific stem cells will be restricted in terms of differentiation potential to the cell types characteristic of their tissue of origin. In particular, it appears that bone marrow cells can give rise to liver, skeletal muscle, cardiac tissue, epithelial cells, and even neuronal tissues. In two cases a cell population enriched for primitive hematopoietic activity has been shown to give rise to liver [46]. While consistent with stem cell plasticity, these data do not prove such plasticity, because the enriched cell populations are still heterogeneous. Donor derived contribution to liver has also been observed in the setting of clinical hematopoietic transplantation. In one case it has been shown that the same cell can have hematopoietic and epithelial potentials [47]. Although it is still too early to conclude that such plasticity is a general and reproducible phenomenon, it seems clear that the bone marrow is a reservoir of progenitors for multiple tissues. Clinically, this is of great importance. In order for the plasticity phenomenon to be placed on a firm footing, precise clonal analyses will need to be performed [48]. With the exception of the one report mentioned above, to date this has not been possible. In the event that stem cell plasticity will be confirmed by other studies, it is almost necessarily true that the exact developmental fate of a stem cell will be defined by the microenvironment. For example, a hematopoietic stem cell would produce liver cells only in the liver microenvironment, and heart tissue in the heart microenvironment. Therefore, in order to explore the regulation of any plasticity phenomena, it will be crucial to focus on microenvironmental signals.
In order to understand the exact nature of the mechanisms responsible for the stem cell supporting properties we reasoned that molecular approaches would be fruitful. To begin these studies we performed a series of RT-PCR expression analyses on a panel of supporting and non-supporting stromal cell lines to evaluate the mRNA levels encoding a number of cytokines and other factors suggested to be stem cell regulators. No obvious differences were detected that could potentially be responsible for the dramatically different stem cell supporting abilities of the individual cell lines. These observations supported our underlying hypothesis that either novel stem cell regulatory factors are produced by AFT024, and by other supporting cell lines, or that stem cell support is mediated by a collection of regulatory molecules, none of which are individually responsible for the overall biological activity. It should be pointed-out that an implicit assumption in our studies is that at least some of the stem cell regulatory factors produced by AFT024 would act in a positive manner to support stem cells and the stem/progenitor cell hierarchy. Therefore, these factors would be expressed preferentially in stem cell supporting stromal cell lines, and not in non-supporting lines. In support of this notion we had already shown that the trans-membrane protein dlk/pref1 is expressed in AFT024 but not in the non-supporting 2018 or BFC012 cell lines. Expression of the dlk/pref1 gene-product in BFC012 cells that do not natively express dlk/pref-1 allowed these cells to maintain stem cells for a short time; though by no means for as long or as effectively as AFT024 [49]. It is also likely that AFT024 does not express overwhelming amounts or classes of negative stem cell regulators, such as potent differentiation promoting. High levels of such regulatory factors may be characteristic of at least some of the non-stem cell supporting stromal cell lines. For our present studies we have focused exclusively on potential positive regulatory molecules.
In order to expand our analyses we have attempted to identify most, if not all, gene-products preferentially expressed by stem cell supporting stromal cells. In essence, in the accompanying manuscript, we have provided a first, comprehensive, molecular phenotype of a stem cell niche. The complete results of our studies are presented in this on-line resource, called Stromal Cell Data Base (StroCDB). The information in StroCDB complements the molecular phenotype of the stem cell that is presented in the Stem Cell Database (SCDb). We anticipate that StroCDB will provide a foundation for the future functional identification of the entire spectrum of positively acting stem cell regulators provided by the microenvironment. In addition, we propose that the microenvironmental regulatory molecules responsible for mediating at least some of the suggested stem cell plasticity phenomena will also be identified.
