Uses Of Stem Cells In Therapeutic Applications

The Parkinson's-Reversing Breakthrough

Natural Remedies for Parkinsons

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The most obvious use of stem cells is in replacement therapies where compromised tissues are replaced with functional equivalents. This had been envisioned as the goal in several disease processes, most commonly in diseases where a specific known cell type is compromised, such as diabetes, spinal cord injury, myocardial infarctions, Parkinson's disease (PD), and some blood cancers reviewed in (Daley et al., 2003; Shizuru et al., 2005). While initial impetus for the field of therapeutic transplantation was given by bone marrow transplantations for blood-related malignancies (Shizuru et al., 2005), it is now being contemplated for several of the somatic stem cells—including NSCs and MSCs and, in the longer term, ESCs as well—when the processes of uncontrolled differentiation and tumorigenesis in these latter cells is understood better. Amelioration of disease phenotypes in animal models have been attempted with various stem cells including hESCs, NSCs, HSCs, and MSCs (Kim et al., 2002; Yang et al., 2002; Eglitis and Mezey, 1997; Zhao et al., 2002). We will discuss the use of NSCs in transplantation with specific reference to PD.

20.3.1 NSCs: Parkinson's Disease

NSCs have been used with varying degrees of success in transplantation therapies in the central nervous system (CNS) (Snyder et al., 2004; Picard-Riera et al., 2004). The impact of NSCs in directly rescuing endangered host neurons was first evinced in a series of experiments in aged rodents in which the nigrostriatal system was impaired (Fig. 20.2). PD is a degenerative disorder characterized by a loss of midbrain dopamine (DA) neurons with a subsequent reduction in striatal DA (Rosenthal, 1998). The disease, in addition to incapacitating many thousands of patients, has also long served as a model for testing neural cell replacement strategies. Transplantation therapy for this CNS disorder has a long history [for review see (Dunnett (1999)]. It was the neural disease that was first treated clinically by neural transplantation, using primary tissue from human fetal ventral mesencephalon to replace DA-expressing cells (Lindvall et al., 1990ab). Indeed, it was in this disease that the limitations of fetal tissue grafts in not only rodent and primate models of PD (Mehta et al., 1998), but also in clinical trials (Lindvall et al., 1990a,b), was first recognized. These limitations include (a) short graft survival and limited integration of the grafts and (b) the possibility of unregulated DA production in improper regions, leading adversely to dyskinesias. Given PD's storied role in the development of cellular therapies, it is appropriate that a model of this disease should have also played a pivotal role in revealing a little-suspected but powerful therapeutic action that NSCs may play in preserving degenerating host cells by some heretofore-unheralded mechanisms that are nevertheless inherent to stem cell biology.

Figure 20.2 NSCs possess an inherent mechanism for rescuing dysfunctional neurons. Shown here is evidence from the effects of NSCs in the restoration of mesencephalic dopaminergic function. [Modified from Ourednik et al. (2002)]. I. TH expression in mesencephalon and striatum of aged mice following MPTP lesioning and unilateral NSC engraftment into the substantia nigra/ventral tegmental area (SN/VTA). A model that emulates the slow dysfunction of aging dopaminergic neurons in substantia nigra (SN) was generated by giving aged mice repeated high doses of MPTP. Schematic on top indicates the levels of the analyzed transverse sections along the rostrocaudal axis of the mouse brain. Representative coronal sections through the striatum are presented in the left column (A, C, E, G) and through the SN/VTA area in the right column (B, D, F, H). A and B. Immunodetection of TH (black cells) shows the normal distribution of DA-producing TH+ neurons in coronal sections in the intact SN/VTA (B) and their projections to the striatum (A). C and D. Within 1 week, MPTP treatment caused extensive and permanent bilateral loss of TH immunoreactivity in both the mesostriatal nuclei (C) and the striatum (D), which lasted for a lifetime. Shown in this example, and matching the time point in g and h, is the situation in a mock-grafted animal 4 weeks after MPTP treatment. E and F. Unilateral (right side) stereotactic injection of NSCs into the nigra is associated, within 1 week after grafting, with substantial recovery of TH synthesis within the ipsilateral DA nuclei (F) and their ipsilateral striatal projections (E). By 3 weeks posttransplant, however (G, H), the asymmetric distribution of TH expression disappeared, giving rise to TH immunoreactivity in the midbrain (H) and striatum (G) of both hemispheres that approached the immunoreactivity of intact controls (A, B) and gave the appearance of mesostriatal restoration. Similar observations were made when NSCs were injected 4 weeks after MPTP treatment (not shown). Bars: 2 mm (left), 1 mm (right). Note the ectopically placed TH+ cells in H. These are analyzed in greater detail, along with the entire SN, in II. II. Immunohistochemical analyses of TH, DAT, and BrdU-positive cells in MPTP-treated and grafted mouse brains. The presumption was initially that the NSCs had replaced the dysfunctional TH neurons. However, examination of the reconstituted SN with dual bgal (green) and TH (red) ICC showed that 90% of the TH+ cells in the SN were host-derived cells that had been rescued [a c], and only 10% were donor-derived [d]. Most NSC-derived TH+ cells were actually just above the SN ectopically (blocked area in a, enlarged in b). These photomicrographs were taken from immunostained brain sections from aged mice exposed to MPTP, transplanted 1 week later with NSCs, and sacrificed after 3 weeks. The following combinations of markers were evaluated: TH (red) with bgal (green) [a-d]; NeuN (red) with bgal (green) [e]; GFAP (red) with bgal (green) [f]; CNPase (green) with bgal (red) [g]; TH (brown) and BrdU (black) [k]; GFAP (brown) with BrdU (black) [l]; CNPase (brown) with BrdU (black) [m]. Anti-DAT-stained areas are revealed in green in the SN of intact [h], mock-grafted [i], and NSC-grafted [j] brains. Three different fluorescence filters specific for Alexa Fluor 488 (green), Texas Red (red), and a double-filter for both types of fluorochromes (yellow) were used to visualize specific antibody binding: c, d, and h-j are single-filter exposures; a, b, and e-g are double-filter exposures. a shows a low-power overview of the SN/VTA of both hemispheres. The majority of TH+ cells (red cells in a) within the nigra are actually of host origin (~90%), with a much smaller proportion being donor-derived (green cells, ~10%) (representative close-up of such a donor-derived TH+ cell in d). Although a significant proportion of NSCs did differentiate into TH+ neurons, many of these actually resided ectopically, dorsal to the SN (boxed area in a, enlarged in b; high-power view of donor-derived (green) cell that was also TH+ (red) in c), where the ratio of donor-to-host cells was inverted: ~90% donor-derived compared with ~ 10% host-derived. Note the almost complete absence of a green b gal-specific signal in the SN+VTA, whereas ectopically, many of the TH+ cells were double-labeled and thus NSC-derived (appearing yellow-orange in higher power under ared/green double-filter in panel b). cg. NSC-derived non-TH neurons (NeuN+) [e, arrow], astrocytes (GFAP+) [f], and oligodendrocytes (CNPase+) [g, arrow] were also seen, both within the mesencephalic nuclei and dorsal to them. h-j. Any proliferative BrdU+ cells after MPTP insult and/or grafting were confined to glial cells, whereas the TH+ neurons [k] were BrdU-. This finding suggested that the reappearance of TH+ host cells was not the result of neurogenesis but rather the recovery of extant host TH+ neurons. Bars: ~10 mm [a]; 20 mm [c, d, e]; 30 mm [f]; 10 mm [g]; 20 mm [h-j]; 25 mm, [k]; 10 mm, [l]; and 20 mm [m]. k-m. The green DAT-specific signal in j suggests that the reconstituted mesencephalic nuclei in the NSC-grafted mice (as in H) were functional DA neurons comparable to those seen in intact nuclei [h] but not in MPTP-lesioned sham-engrafted controls [i]. This further suggests that the TH+ mesostriatal DA neurons affected by MPTP are, indeed, functionally impaired. (Note that sham-grafted animals [i] contain only punctate residual DAT staining within their dysfunctional fibers, whereas DAT staining in normal [h] and, similarly, in engrafted [j] animals was normally and robustly distributed both within processes and throughout their cell bodies.) See color plates.

In addition to "replacing" the injured tissue, neural stem cells also appear to engage the host in a dynamic series of ongoing reciprocal interactions, each instructing the other. Under instruction from exogenous NSCs, the injured host nervous system also contributes to its own repair. The impact of NSCs in directly rescuing endangered host neurons was first evinced in a series of experiments in aged rodents in which the nigrostriatal system was impaired (Fig. 20.2). In the hope that NSCs might spontaneously differentiate into DA neurons when implanted into a DA-depleted region of the CNS, unmanipulated murine NSCs were implanted unilaterally into the substantia nigra of aged mice that been exposed systemically to high-dose MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine), a neurotoxin that produces a persistent impairment of mesencephalic DA neurons and their striatal projections (Ourednik et al., 2002). The NSCs not only migrated from their point of implantation and integrated extensively within both hemispheres, but also were, indeed, associated with a dramatic reconstitution of DA function throughout the mesostriatal system. While there was spontaneous conversion of a subpopulation of donor NSCs into dopaminergic neurons in DA-depleted areas, contributing to nigral reconstitution, the majority (^80-90%) of dopaminergic neurons in the "reconstituted midbrain" were actually host cells that had been "rescued" by factors produced constitutively by the NSCs with which they were juxtaposed and that had not, themselves, become neurons. These "chaperone cells" constitutively produced substantial amounts of neurosupportive agents. One such prominent molecule among many in this case was glial-cell-line-derived neurotrophic factors (GDNF), a factor known to be neuroprotective of ventrally located neurons (including DA neurons and spinal ventral horn cells). A similar observation is beginning to emerge from the implantation of human NSCs into the MPTP-lesioned subhuman primate model of PD.

A sense for the extent of the cross-talk also became evident when examining rodent models of hypoxia-ischemia (HI), a common cause of neurological disability in adults and children. HI causes much of its damage from extensive loss of cerebral parenchyma and of the cells and connections that reside there. When NSCs are implanted into these regions of extensive degeneration (particularly when transiently supported by biodegradable scaffolds), robust reciprocal interactions ensue spontaneously between the exogenous implant and the injured host brain that result in substantial reconstitution of parenchyma and anatomical connections as well as reduction of parenchymal loss, secondary cell loss, inflammation, and scarring. Similar results are observed in the hemi-resectioned adult rodent spinal cord in which evidence of an upregulated host neuronal regenerative response is noted, resulting in significant functional improvement. Indeed, the ability of engrafted NSCs to exert a protective and regenerative influence on degenerating host neural systems by virtue of their intrinsic expression of trophic factors is being observed in an increasing number of conditions. For example, the implantation of murine and human NSCs into the spinal cords of the SOD 1 transgenic mouse model of ALS, a disease characterized by virulent motor neuron degeneration, has been pivotal in protecting these ventral horn cells from death and in preserving motor and respiratory function, blunting disease progression, and extending life. NSCs can similarly protect other neuron pools, promote motor axonal outgrowth following traumatic spinal cord injury, preserve infarcted regions of cerebrum, induce vascularization of reconstituted regions of cortical parenchyma, and inhibit inflammation and scarring following traumatic or ischemic insult.

20.3.2 MSCs: Cardiac Illness

MSCs are relatively robust stem cells that are isolated from bone marrow, where they are present at a frequency of about 0.001% nucleated cells and of about a tenth as much as HSCs. While MSCs are usually cultured in FBS and resemble fibroblast cultures in appearance, unlike fibroblasts they have the capacity to differentiate into fates in the mesodermal lineage, including chondrocytes, adipocytes, and osteoblasts (Pittenger et al., 1999). Two properties of these cells make them attractive candidates for cellular therapies: their apparent lack of immunogenecity after transplantation and their potential to help in remodeling cardiac tissue after myocardial infarction (MI). While allogeneic MSCs have been used in animal models (rodent and porcine) with good functional and histological recovery, autologous bone marrow (presumably including several populations of cells in addition to MSCs) has also been used in the clinic with promising results (Silva et al., 2004). While MSCs have been injected into the site of cardiac ischemia resulting from MI, they have also been administered systemically and have been shown to home to the site of injury (Kraitchman et al., 2005). Subsequent histological examination of the site of transplantation indicates some incorporation and survival of the transplanted cells. However, the extent of recovery of the heart tissue is not concomitant with the numbers of surviving cells, suggesting that there is remodeling of the host cells and the extracellular matrix indicating possible paracrine effects originating from the transplanted cells. This is similar to the trend seen with the NSC transplantations. MSCs also appear to block T-cell responses in the host immune system (Aggarwal and Pittenger, 2005), thus leading to their possible use as adjunct therapy in transplantation of other tissues, including bone marrow. In addition, MSCs have been used with some success in therapies related to bone replacement (Caplan, 2005). Similar work is in progress with ADSCs, which also appear to ameliorate symptoms of MI and have the added advantage of ease of isolation of populations of interest from adipose tissue for autologous transplantation into the same patient.

20.3.3 Stem Cells as Anti-Cancer Agents

Tangential to the above discussion, NSCs and MSCs have been used to deliver therapeutics to tumors, particularly gliomas (Aboody et al., 2000; Lee et al., 2003; Nakamizo et al., 2005). Aboody et al. (2000) first reported that modified NSCs introduced into the parenchyma or cerebral ventricle as well as the systemic circulation could migrate over great distances to sites of intracranial pathology, as modeled by a glioma in rodent hosts and could position themselves in direct juxtaposition to glioma cells migrating away from the tumor bulk to invade normal tissue. This ability to track invading tumor cells signified a potentially powerful way to treat a phenomenon notorious to primary gliomas that has made their management so vexing. The same group observed reduction of tumor bulk and improved host survival with the use of genetically modified NSCs. Combinations of NSCs from different sources have been shown to exhibit the same gliomatropic effect in experimental rodent brain tumor models and to effectively reduce tumor bulk and prolong host survival (Ehtesham et al., 2002, 2004; Barresi et al., 2003). Factors released and expressed by the glioma cells themselves, by the tumor stroma (composed of adjacent reactive astrocytes, microglia, and oligodendrocytes) and by tumor-derived endothelium as well as by the damaged surrounding normal brain itself all contribute to NSC gliomatropism. Some of these agents, such as stem cell factor (SCF) and monocyte chemoattractant protein-1 (MCP1), have been identified (Erlandsson et al., 2004; Widera et al., 2004; Sun et al., 2004), yet others are still to be characterized and their role in NSC gliomatropism defined (Werbowetski et al., 2004). Expression of SDF-1 a by tumor-derived endothelium serves to attract the migration of NSCs (Allport et al., 2004; Fears et al., 2004). Blocking SDF-1a/CXCR4 interactions also prevents gliomatropic migration of NSCs (Ehtesham et al., 2004). SDF-1a/CXCR4 interactions appear to be pivotal as well to the gliomatropism of circulating adult hemato-poietic progenitor cells (Tabatabai et al., 2005).

Exploiting the unique tropism of NSCs for gliomas, several groups have now confirmed the therapeutic efficacy of using genetically armed NSCs to target neoplasms in vivo in a variety of murine brain tumor models through the delivery of a variety of growth-regulating and anti-glioma gene products. Aboody et al. (2000) demonstrated in vivo efficacy of murine NSCs transduced with the gene for the enzyme cytosine deaminase (CDA). The enzyme converts the nontoxic pro-drug 5-fluorocytosine (5-FC) into the nucleoside analogue 5-fluorouracil (5-FU), which is then incorporated into the DNA of the neoplastic cell, causing chain termination and cell death. Tumor-bearing mice inoculated with CDA-expressing NSCs and given 5-FC demonstrated dramatic reduction of the intracranial tumor burden. This finding was subsequently corroborated in a different murine tumor model using a different NSC line (Barresi et al., 2003). Another approach utilizes NSCs as engraftable, mobile, gliomatropic viral packaging lines (Lynch et al., 1999). One study reported effective killing of tumor and escaping micro-deposits in a murine host by using murine NSCs to release replication-conditional HSV TK (Herrlinger et al., 2000), hence overcoming the typical low transduction frequency encountered in HSV glioma gene therapy by directing delivery of the virus to the intended cellular targets. This targeted cytotoxic effect, mediated by conversion of the pro-drug ganciclovir by TK into ganciclovir phosphate, is greatly amplified by virtue of the "bystander effect'' (Li et al., 2005b). Gliomatropic NSCs engineered to be a viral packaging cell line for adenoviral-based vectors also show similar efficacy (Arnhold et al., 2003). NSCs have also been used to deliver gene products that show therapeutic effects against gliomas such as IL4, IL12, and TRAIL (Benedetti et al., 2000; Ehtesham et al., 2002; Walczak et al., 1999) and a potent anti-angiogenic compound called endostatin (Bjerkvig et al., 2003).

It should be noted that although the tropism of stem cells for cancer was first unveiled by observing the behavior of NSCs, this phenomenon has also been observed in MSCs. MSCs engineered to express interferon-p (IFN-p) when injected into the carotid artery of brain-tumor-bearing mice also appeared to migrate in a gliomatropic fashion and destroyed the tumor via direct cytotoxicity, resulting in prolonged survival of the hosts (Nakamizo et al., 2005). Others have shown that MSCs over-expressing the immunomodulatory cytokine IL-2 migrate to the contralateral tumor-bearing hemisphere via the corpus callosum, helping promote tumor destruction (Nakamura et al., 2004). Lee et al. (2003) also suggested that cells derived from MSCs, which have some characteristics of NSCs, have the capacity to migrate toward an injury or glioma in the brain. Furthermore, MSCs administered systemically appeared to localize to prostate and breast cancers metastatic within the periphery. The question of which type of stem cell is best suited for which type of tumor within which region will need to be determined empirically. At present, we favor the view that stem cells derived from the lineage-of-origin of the cancer are best suited for "hunting it down'' and eradicating it.

A brief spate of papers introduced the possibility of somatic cells differentiating into lineages removed from that of their origin—for instance, NSCs differentiating into skeletal muscle and hematopoietic lineages (Bjornson et al., 1999; Galli et al., 2000). Similarly, MSCs differentiate into mesodermal fates such as chondrocytes, adipocytes, and osteocytes as expected (Pittenger et al., 1999), but can also be induced to differentiate into ectodermal fates such as neurons and smooth muscle and endodermal fates such as liver. Whole bone marrow and MSCs have been shown to remain viable when transplanted into the CNS in models of spinal cord and ischemic injury and are thought to provide beneficial effects (Kopen et al., 1999; Chopp and Li, 2002; Akiyama et al., 2002; Hofstetter et al., 2002). While this "transdifferentiation" is an intriguing biological question in its own right, it may also provide alternative therapeutic options, especially in the case of somatic stem cells that are challenging to propagate, such as HSCs, or where the tissue in which they reside is relatively inaccessible, such as NSCs in the brain. However, it is also possible that the starting population of stem cells may not be as homogeneous as originally thought—that is, that the MSCs had some neural progenitors that were carried over from the bone marrow, or that some of the cells scored positive for a particular mature phenotype were scored aberrantly due to experimental artifacts (Daley et al., 2003). In addition, the occurrence of cell fusion of stem cells with other mature cells in the vicinity in vitro (Ying et al., 2002; Terada et al., 2002) and in vivo (Weimann et al., 2003a,b) introduces the possibility that some of these transdifferentiation events could be a result of cell fusion. In spite of all these caveats, the possibility of transdifferentiation of somatic cells merits further scrutiny because it has not been conclusively disproved.

In summary, the science of transplantation has made significant progress in the past decade, but several of the details remain to be investigated and conditions standardized. While transplantation of adult stem cells appears to be a more mature discipline than that of ESCs at the present time, mechanistic details of related phenomena will greatly enhance the efficacy of the treatment. These phenomena include incorporation and survival of transplanted cells in the compromised tissue, homing of transplanted cells to injured tissue, differentiation of stem cells to pertinent host tissue if any, paracrine/trophic effects of transplanted cells on host tissue (with or without differentiation), fraction of transplanted cells that are retained in the tissue of interest and location, and survival of remaining cells. The method of delivery of the cells may be determined somewhat by the disease being treated, but introduction of cells may be at the site of the injury or systemic. In either scenario, the cells could be delivered with several refinements that enhance survival and perhaps differentiation/ incorporation after transplantation, such as addition of growth factors that promote survival, genetic manipulation of the transplanted cells that may allow prolonged expression of genes of interest and may permit control of the transplanted cells in the patient, and, particularly in the case of ESCs, transplantation of cells that have been partially differentiated into appropriate lineages. Perhaps the site of transplantation may be pretreated to be favorable to receive and enhance the efficiency of the graft. Finally, knowledge of the precise mechanism(s) by which symptoms of the illness are reversed—that is, differentiation of transplanted cells and integration of host and transplanted tissues, trophic and paracrine functions of the transplanted cells, and cell fusion—will aid in the design of treatment protocols and in the possible use of combination therapies that include transplantation.

20.4 USES OF STEM CELLS IN RESEARCH APPLICATIONS 20.4.1 Experimental Systems to Study Basic Biology

Since the functional definition of a stem cell lies in its capacity to differentiate into several fates, the obvious follow-up to that question is the determination of the mechanism of these differentiation events. Stem cells provide a tractable system for a study of dynamic signaling processes that occur during fate choice and other events important in mammalian development, such as cell proliferation, differentiation, cell motility, cell survival, senescence, cell death, and formation of functional three-dimensional tissues. A wealth of literature exists describing (a) some of the molecular mechanisms of the complex differentiation phenotypes presented by stem cells and (b) the use of these cells to model three-dimensional cultures that resemble tissues and to model disease states so that the mechanism of diseased phenotypes may be studied. Three-dimensional tissues reminiscent of liver, capillaries, and blood have been described where stem or progenitor cells were cultured in three-dimensional matrices, including those composed of collagen (Imamura et al., 2004), matrigel or scaffolds composed of lactic acid/glycolic acid, and self-assembling peptide matrices (Semino et al., 2003). Ishikawa et al. (1996) studied hESCs, which were deficient for the NFkB precursor, and showed that NFkB is important in inflammatory responses involving p50 and RelA complexes. It is also conceivable that diseases caused by mutations in single genes, such as Lesch-Nyhan's disease, could be modeled in hESCs. With the advent of recent successes in cloning of hESCs, it is conceivable that the generation of mutant lines could expedite mechanistic studies of certain disorders, especially developmental disorders. The regulation of proliferation and differentiation of stem cells may also borrow a few lessons from research progressing in the field of cancer, such as the observation that dicer miRNA circumvents the G1-S boundary, thus allowing stem cells to proliferate in an environment that generally does not promote proliferation, a fact that may be pertinent to cancer (Hatfield et al., 2005). Alternatively, miRNA have been shown to be involved in lymphoma, gastric, and lung cancers (Croce and

Calin, 2005) and in the differentiation of cardiac and fat tissue (Esau et al., 2004; Zhao et al., 2005). The use of stem cells as in vitro models to study the mechanism of action of such observations is feasible, especially when differentiation-/devel-opment-related events are involved.

We have studied glial differentiation of NSCs in relative detail and will discuss some of these findings below. NSCs differentiate into cells characteristic of the CNS—namely, neurons, astrocytes, and oligodendrocytes—upon withdrawal of the mitogen bFGF (Johe et al., 1996). The addition of specific factors also causes the directed differentiation of NSCs into CNS and non-CNS fates. While the addition of platelet-derived growth factor (PDGF) enhances neuronal differentiation, triiodothyronine (T3) causes oligodendrocyte differentiation, and ciliary neurotrophic factor (CNTF) along with the other gp130 family of cytokines is a robust inducer of the astrocytic fate (Johe et al., 1996; Bonni et al., 1997; Rajan and McKay, 1998). While the neuropoietic cytokines are robust inducers of glial differentiation of neural stem cells, other factors such as addition of bone morphogenetic proteins (BMPs), serum, and (to a much lesser extent) epidermal growth factor (EGF) also cause glial differentiation. Physical parameters are also important: NSC cultures that are maintained and passaged at high density are biased toward a glial fate. BMPs are a large family of soluble factors that have several important functions during development and are involved in the differentiation of several cell fates, including neural crest, cortical neurons, generation of germ cells, lung, tooth, kidney, cartilage, and bone, as well as in the formation of dorsalizing gradients during early differentiation (Liem et al., 1995, 1997; Nguyen et al., 2000; Sela-Donenfeld and Kalcheim, 1999; Weaver et al., 1999; Pizette and Niswander, 2000). There is only one major cytoplasmic signaling pathway described for BMPs, which seems paradoxical in light of the various BMP-mediated effects described. One mechanism by which a single factor may have such pleiotropic effects is by acting in conjunction with other factors present in the local environment. Specific cellular fates may be caused by the interaction of the signaling intermediates of two or more cytoplasmic pathways, leading to different transcriptional profiles and thus different phenotypes. Although this idea is conceptually simple, the details of the molecular interactions and their specific outcomes are not clearly defined in most cases. The complex differentiation phenotype that BMP exerts on NSCs was studied as a model system of this phenomenon.

BMPs cause differentiation into three fates in NSCs in our cultures: neurons, smooth muscle, and glia. The culture conditions used in this study do not support the survival of neurons, and thus the mechanism of differentiation of that lineage was not pursued. However, the differentiation of NSCs into smooth muscle and glia is mutually exclusive and can be regulated in culture by density. Dense cultures treated with BMP yield glia as a result of Stat3 activation, and sparse cultures differentiate into smooth muscle as a result of SMAD activation. BMP-mediated Stat3 activation occurs in the presence of a prior signal initiated by cell-cell contact in dense cultures and is blocked by the drug rapamycin. In dense cultures, the glial fate is dominant and occurs at the expense of smooth muscle (Rajan et al., 2003). In a developmental context, the increase to threshold levels of a differentiation signal by the concerted action of two or more pathways provides a model for the generation of various phenotypes by intersecting gradients of factors. This example shows the involvement of a clinically relevant molecule in regulation of fate choice. It is thus not surprising that rapamycin analogues have been used on occasion in the treatment of glial tumors (Galanis et al., 2005). Also, even though FRAP and Stat3 are relatively ubiquitous proteins, the complex of these two proteins could be considered a marker of glial differentiation in stem cells (Fig. 20.3).

Recent data suggest that acetylation is possible on lysine 685 present on Stat3. In overexpression studies, it was suggested that acetylation is required for optimal activation of Stat3 in addition to phosphorylation (Yuan et al., 2005). It has also been shown that lysine 685 is a substrate for the CBP/p300 protein that forms a complex with Stat3 (Wang et al., 2005). Although the physiological significance of this observation has already been called into question (O'Shea et al., 2005), it is an interesting additional level of control that may be relevant to the glial differentiation of stem cells. The gp130 family of cytokines including CNTF, LIF, oncostatin M, and IL6 cause the activation of STAT proteins in several cell types including liver, kidney, neurons, astrocytes, and neural stem cells. However, the outcome of STAT activation in these various cell types is distinctive. Also, neural stem cells isolated from rats of different ages exhibit varying capacities for astrocytic differentiation. NSCs isolated

Figure 20.3 Schematic of intracellular signals activated by BMP4 in neural stem cells This model is based on the data presented in Rajan et al. (2003). BMP4 causes the activation of at least two signaling pathways in neural stem cells. Upon ligand-receptor binding, the BMPR receptor complex releases FKBP12 and activates SMAD proteins, resulting in smooth muscle differentiation. The released FKBP12 may bind with rapamycin to inhibit FRAP or may act in some modified form to activate FRAP. FRAP then catalyzes serine phosphorylation (S*) of STAT to augment its prior activation by tyrosine phosphorylation (Y*) by another density-mediated signal. High cell density acts to promote basal STAT activation and DNA binding by an unknown signaling mechanism. This enhanced activation of STAT (Y*S*) causes efficient glial differentiation. Levels of activated STAT proteins in the cell receiving the BMP signal dictate whether the STAT or SMAD signal acquires precedence in the fate choice between smooth muscle and glia. See color plates.

Figure 20.3 Schematic of intracellular signals activated by BMP4 in neural stem cells This model is based on the data presented in Rajan et al. (2003). BMP4 causes the activation of at least two signaling pathways in neural stem cells. Upon ligand-receptor binding, the BMPR receptor complex releases FKBP12 and activates SMAD proteins, resulting in smooth muscle differentiation. The released FKBP12 may bind with rapamycin to inhibit FRAP or may act in some modified form to activate FRAP. FRAP then catalyzes serine phosphorylation (S*) of STAT to augment its prior activation by tyrosine phosphorylation (Y*) by another density-mediated signal. High cell density acts to promote basal STAT activation and DNA binding by an unknown signaling mechanism. This enhanced activation of STAT (Y*S*) causes efficient glial differentiation. Levels of activated STAT proteins in the cell receiving the BMP signal dictate whether the STAT or SMAD signal acquires precedence in the fate choice between smooth muscle and glia. See color plates.

from E12 rat embryos do not respond to LIF by differentiating into glia, while those isolated from E15 embryos do (Molne et al., 2000). The differences in outcomes could possibly be due to the epigenetic state of the cell receiving the signal, as has been suggested by Song and Ghosh (2004). There is also increasing evidence that genes such as bmil (which is one of the Polycomb Group of genes, an "oncogene" that regulates cell-cycle-related genes such as INK4a and p16ARF) are involved in the maintenance, proliferation, and differentiation states of hematopoietic and neural stem cells, while bmil forms part of a complex called the polycomb repressive complex 1 (PRC1), thought to maintain stable maintenance of gene expression by regulation of epigenetic chromatin modifications (Valk-Lingbeek et al., 2004). Mice deficient in bmi1 display deficits in neural stem cells and cerebellar neurons (Leung et al., 2004). Interestingly, they also show an increase in astrocytes (Zencak et al., 2005). These observations, though fragmented, strongly suggest that epigenetic modifications play an important role in fate choice of glia and, in a broader sense, in the decision of a cell to remain stem-like or to differentiate.

Epigenetic modification includes processes such as DNA methylation, histone modification, and ATP-driven chromatin remodeling. These processes function cooperatively to establish and maintain active or inactive chromatin states in cellular development. In general, acetylation of core histones and methylation of K4 of histone H3 correlate with transcriptional active ("open") chromatin state, whereas deacetylation of core histones and methylation of K9 of histone H3 correlate with transcriptional repressed ("closed") chromatin state. The proteins that mediate these interactions include DNA cytosine methyltransferases, histone acetyl transferases, histone deactylases, histone methyltransferases, and other specialized proteins that mediate the interaction of proteins with nucleosomal DNA. The precise function of these proteins in plasticity of stem cells is not clear. It appears that the expression of Oct-4 is required for the maintenance of pluripotency, self-renewal, and survival (Kehler et al., 2004). A global study of hESC promoters that bind the three markers Oct-4, Nanog, and Sox2 yielded clues as to genes that remain inactive when bound to these transcription factors and others that appear to be activated (Boyer et al., 2005). Follow-up studies may delineate the epigenetic reasons for this observation, including a possible mechanism for the role of these three factors in global regulation of "sternness" (genes of promoters that are activated) and phenotypic commitment (genes of promoters that are repressed) in ESCs. While it is generally agreed that global transcriptional, translational, and epigenetic characteristics of ESCs are unique, the factors that orchestrate these changes are still elusive. In a preliminary experiment to identify these factors, it was determined that characteristics of ESCs could be conferred on somatic cells by fusion (Cowan et al., 2005). These authors showed that the nucleus from the mature fusion partner could be appreciably "reprogrammed" such that it acquired some of the epigenetic features resembling an undifferentiated hESC. It appears that cytoplasmic factors play an important role in the initiation and maintenance of the epigenetic state of the nucleus.

Thus, as expected, there appears to be an intimate relationship between cyto-plasmic and nuclear signals. Another facet of regulation acquiring importance is stable, nontranslated RNA molecules called micro RNAs (miRNAs), which lead to reduced translation of specific proteins by either mRNA degradation or inhibition of translation (Alvarez-Garcia and Miska, 2005). Although small interfering RNAs (siRNAs) were first discovered in C. elegans (Lee et al., 1993) and have since been used extensively as research reagents that inhibit eukaryotic—including mammalian—gene expression, functionally similar miRNAs have been isolated from mammalian cells, and two have been shown to be involved in cardiac muscle and adipocyte differentiation. Zhao et al. (2005) have shown that miR-1 targets a transcription factor hand-1 that controls the proliferation of ventricular cardiomyo-cytes during development. Similarly, miR-147 has been shown to regulate adipocyte differentiation, possibly by inhibiting translation of ERK5 (Esau et al., 2004). On a more global level, mutation of the enzyme dicer, which is part of the cellular machinery responsible for the generation of miRNAs, causes a G1-S block in stem cells in Drosophila, implying that miRNAs are essential for the proliferation of stem cells in their native environment (Hatfield et al., 2005).

The initial use of mouse ESCs was in the creation of "knock-out" and "knock-in" mice, which have contributed immensely to the study and understanding of mammalian developmental biology. The past decade has seen a revolution in this field with the isolation of murine, rodent, and human stem cells from a variety of sources, including blastocysts, the brain, and bone marrow. In spite of the obvious relevance of the human stem cells to the medical sciences, important discoveries about the basic biology of the stem cells themselves and some of the associated developmental phenomena in mammals are continuing to be made in the rodent and murine systems, and to a lesser extent in invertebrates and lower vertebrates such as Drosophila and Zebrafish. All these studies—molecular, cellular, systems biology, and clinical—will contribute to the gestalt of knowledge about stem cells and will aid immensely in the successful exploitation and manipulation of this powerful system to our advantage.

20.4.2 Creation of Applied Systems for Drug Discovery

Stem cells are potentially useful in several stages of the drug discovery process, including target discovery and validation, secondary screens of candidate molecules, and toxicological studies of potential drugs. Alternatively, stem cells could be used as in vitro experimental systems to determine the precise mechanism of action of a candidate drug. Using genetic manipulation technology, ESC lines may be created that express specifically mutated proteins or lines that lack specific genes, either of which may be used to study the mechanism of action of particular targets or as a platform for screening drugs that reverse a particular phenotype. Alternatively, "humanized" mice may be produced that express the human version of the gene of interest, which may then be used for some in vivo testing of drug action and ADME-TOX (administration, distribution, metabolism, excretion, and toxicity of candidate drug molecules). Mouse ESCs are already being used by companies in drug screening applications (McNeish, 2004). The creation of person-specific and disease-specific hESCs would also aid drug discovery efforts. The former has potential for autologous cellular therapies as well as for pharma-cogenomic studies where genomic profiles are correlated with the most effective therapeutic regimen. Disease-specific hESCs could lead to the design of experiment systems for target validation and subsequently to screening platforms that may be used for secondary screens of compounds of interest.

In addition to the advantages that stem cells present in the areas of drug discovery, they also represent a potentially limitless source of "normal," untrans-formed, diploid cells. This is especially relevant to human systems, because most primary human cells do not propagate beyond a few generations in culture. This property of stem cells could potentially be exploited for the generation of large quantities of cells needed to generate screening platforms for high-throughput screening of chemical libraries. Secondly, drug discovery platforms that would otherwise be challenging to generate on a large scale, such as platforms that employ neuronal cells, could be derived from stem cells or precursors that have been differentiated in vitro. Some groups have been successful in the generation of neurons from hESCs (Reubinoff et al., 2001; Schuldiner et al., 2001), which suggests that this may be possible in the future. Even specific types of neurons, such as dopaminergic or serotonergic, could possibly be derived by in vitro stem cell differentiation: The former has particular relevance to Parkinson's disease, and the latter has relevance to psychiatric illness. Finally, stem cells and their derivatives could be used for toxicity studies of candidate drugs. They would provide a preferable alternative to the transformed cell lines on which these studies are currently being performed. In addition, they could potentially be differentiated into liver cells, which would provide an ideal platform for toxicity studies to be performed upon in vitro, and for which a viable alternative does not exist.

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