NEUROVASCULAR MEDICINE - Pursuing Cellular Longevity for Healthy Aging Part 5 pdf

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Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson’s disease. Stem Cells. 24:781–792. Wernig M, Brustle O. 2002. Fifty ways to make a neuron: shifts in stem cell hierarchy and their implications for neuropathology and CNS repair. J Neuropathol Exp Neurol. 61:101–110. Wislet-Gendebien S, Hans G, Leprince P, Rigo JM, Moonen G, Rogister B. 2005. Plasticity of cultured mesenchymal stem cells: switch from nestin-positive to excitable neuron-like phenotype. Stem Cells. 23:392–402. 231 Chapter 9 MOTONEURONS FROM HUMAN EMBRYONIC STEM CELLS: PRESENT STATUS AND FUTURE STRATEGIES FOR THEIR USE IN REGENERATIVE MEDICINE K. S. Sidhu ABSTRACT Human embryonic stem (ES) cells are pluripotent and can produce the entire range of major somatic cell lineage of the central nervous system (CNS) and thus form an important source for cell-based therapy of various neurological diseases. Despite their potential use in regenerative medicine, the progress is ham- pered by diffi culty in their use because of safety issues and lack of proper protocols to obtain puri- fi ed populations of specifi ed neuronal cells. Most neurological conditions such as spinal cord injury and Parkinson’s disease involve damages to projection neurons. Similarly, certain cell populations may be depleted after repeated episodes of attacks such as the myelinating oligodendrocytes in multiple sclerosis. Motoneurons are the key effector cell type for control of motor function, and loss of motoneurons is associated with a number of debilitating diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy; hence, repair of such neurological conditions may require transplantation with exogenous cells. Transplantation of neural progenitor cells in animal models of neurological disorders and in patients from some clinical trial cases has shown survival of grafted cells and contribution to functional recovery. Recently a considerable progress has been made in understanding the biochemical, molecular, and developmental biology of stem cells. But translation of these in vitro studies to the clinic has been slow. Major hurdles are the lack of effective donor cells, their in vivo survival, and diffi culty in remodeling the non-neurogenic adult CNS environment. Several factors play a role in maintaining their functions as stem cells. It is becoming increasingly apparent that the role of developmental signaling molecules is not over when embryogenesis has been completed. In the adult, such molecules might function in the maintenance of stem cell proliferation, the regeneration of tissues and organs, and even in the maintenance of their differentiated state. A major challenge is to teach the naïve ES cells to choose a neural fate, especially the subclasses of neurons and STEM AND PROGENITOR CELLS IN DEGENERATIVE DISORDER 232 glial cells that are lost in neurological conditions. I review the progress that has been achieved with ES cells to obtain motoneurons and discuss how close we are to translating this research to the clinics. Keywords: central nervous system, neuroectoderm, motoneurons, cell replacement therapy, growth factors, neural induction. T he development of CNS involves spatial distribution and networking (circuitry) of neuronal and glial cells. These anatomical developments undergo modi cations during functional maturation. Insults, injury, or disease causes damage or loss of certain elements in the CNS circuitry that disrupts the neural network. Repair of these circuits would require sequential reactivation of the developmental signals in a particular spatial order, for which the adult mammalian brain and spinal cord have limited capacity (Steiner, Wolf, Kempermann 2006). Consequently, the adult brain often fails to repair the neural framework assembled by projection neurons despite the presence of stem cells or progenitors. These stem/progenitor cells in adult life appear to be designed for replenishing other parts of the CNS, because they differentiate primarily into interneurons and glial cells (Steigner, Wolf, Kempermann 2006). Most neurological conditions such as spinal cord injury and Parkinson’s disease involve damages to projection neurons. In other circumstances, certain cell populations may be depleted after repeated episodes of attacks such as the myelinating oligodendrocytes in multiple sclerosis. Motoneurons are the key effector cell type for control of motor function, and loss of motoneurons is associated with a number of debilitating diseases such as ALS and spinal muscular atrophy (Lefebvre, Burglen, Reboullet et al. 1995; Cleveland, Rothstein 2001). Hence, repair of such neurological conditions may require transplantation with exogenous cells. Transplantation of neural progenitor cells in animal models of neurological disorders and in patients from some clinical trial cases has shown survival of grafted cells and contribution to functional recovery. Laboratory investigation into understanding the biochemical, molecular, and developmental biology of stem cells has progressed rapidly in the last few years. However, until relatively recently, translation of these in vitro studies to the clinic has been slow. Neural replacement as a therapy still needs further laboratory investigations. Major hurdles are the lack of effective donor cells, their in vivo survival, and dif-  culty in remodeling the non-neurogenic adult CNS environment. Several factors play a role in maintaining their functions as stem cells. It is becoming increasingly apparent that the role of developmental signaling molecules is not over when embryogenesis has been completed. In the adult, such molecules might function in the maintenance of stem cell proliferation, the regeneration of tissues and organs, and even in the maintenance of their differentiated state (Maden 2007). Derivation of functional neurons from human embryonic stem cells (hESCs) as surrogate in regen- erating medicine for treating various neurodegenerative diseases is the subject of intensive investigation. Three basic features of hESCs, that is, self-renewal, proliferation, and pluripotency, make them immortal, capable of unlimited expansion and differentiation into all 230 different type of cells in the body, and thus hold great potential for regenerative medicine (Hardikar, Lees, Sidhu et al. 2006; Valenzuela, Sidhu, Dean et al. 2007). Most published protocols for guiding the differentiation of these cells result in heterogeneous cultures that comprise neurons, glia, and progenitor cells, which makes the assessment of neuronal function problematic. However, many recent studies including from our laboratory (Lim, Sidhu, Tuch 2006) have demonstrated that enough puri ed neurons could be generated from hESCs and used for carrying out gene expression and protein analyses and for examining whether they can form functional networks in culture (Benninger, Beck, Wernig et al. 2003; Zhang 2003; Keirstead, Nistor, Bernal et al. 2005; Muotri, Nakashima, Toni et al. 2005; Ben-Hur 2006; Soundararajan, Miles, Rubin et al. 2006; Lee, Shamy, Elkabetz et al. 2007; Soundararajan, Lindsey, Leopold et al. 2007; Wu, Xu, Pang et al. 2007; Zeng, Rao 2007). This review will discuss how recent advancement in stem cell technology offers hope for generating potential effective donor cells for replacement therapy with a special emphasis on developmental potentials of ES cells. POTENTIAL USE OF HUMAN EMBRYONIC STEM CELLS Adult stem cells are restricted during development to a particular fate of the tissue in which they are found. Brain-derived neural stem cells may generate neurons and glia. However, the subclasses of neurons and glia differentiated from neural stem cells depend on the regions and developmental stages in which the progenitor cells are isolated and expanded. Thus, the ideal stem cell population would be those that can generate most or all subtypes of neurons and glial cells. Presently, the best known cells that possess such traits are ES cells. ES cells are able to differentiate into all cell and tissue types of the body. Technology has been developed to selectively maintain and expand mouse and human ES cells in a synchronized, undif- ferentiated state. Compared to adult stem cells, ES Chapter 9: Motoneurons from Embryonic Stem Cells 233 recently some of the studies have been successful in purifying enough hESC-derived neurons to carry out gene expression and protein analyses and examine whether they can form functional networks in culture (Lim, Sidhu, Tuch 2006; Lee, Shamy, Elkabetz et al. 2007; Soundararajan, Lindsey, Leopold et al. 2007). However, different hESC lines behave very differ- ently in cultures and have variable potential to produce neurons (Lim, Sidhu, Tuch 2006; Wu, Xu, Pang et al. 2007). NEUROECTODERMAL INDUCTION Neuroectodermal Induction and Neuronal Specifi cation The production of neurons involves several sequential steps precisely orchestrated by signaling events (Wilson, Edlund 2001). The initial step is the speci - cation of neuroepithelia from ectoderm cells, the process known as neural induction, which is accomplished by inductive interaction with nascent mesoderm and de nitive endoderm. Despite being a topic of intensive study, there is still no consensus on the mechanisms and signals involved in neural induction. Bone morphogenetic protein (BMP) antagonism has been viewed as the central and initiating event in neural induction. According to this concept, neuroepithelial speci cation occurs as a default pathway (Munoz- Sanjuan, Brivanlou 2002). However, recent  ndings challenge this neural default model and indicate some positive instructive factors, such as  broblast growth factors (FGFs) and Wnt. For example, interference cells can be expanded in vitro with current technology for a prolonged period, and yet they retain the genetic normality. Hence, ES cells can provide a large number of normal cells for deriving the desired cells for transplant therapy. A major challenge is to teach the naïve ES cells to choose a neural fate, especially the subclasses of neurons and glial cells that are lost in neurological conditions. hESCs are pluripotent cells derived from the inner cell mass of preimplantation embryos (Thomson 1998). Like mouse embryonic stem (ES) cells, theo- retically they can differentiate into various somatic cell types (Fig. 9.1) with a stable genetic background (Thomson 1998; Amit, Carpenter, Inokuma et al. 2000; Reubinoff, Pera, Fong et al. 2000; Thomson, Odorico 2000; Sidhu, Ryan, Tuch 2008). These unique features make hESCs a favorable tool for biomedical research as well as a potential source for therapeutic application in a wide range of diseases such as Parkinson’s disease, Alzheimer’s disease, and spinal cord injuries. Directing ES cells to differentiate to cells of interest, such as neural lineages, depends on strategies based on the understanding of mammalian neural development (Lee Lumelsky, Studer et al. 2000; Tropepe, Hitoshi, Sirard et al. 2001; Billon, Jolicoeur, Ying et al. 2002; Wichterle, Lieberam, Porter et al. 2002; Ying, Stavridis, Grif ths et al. 2003). Mass-scale production of functional neurons from hESCs for treating neurodegenerative diseases is the subject of intensive investigation. Most published protocols for guiding the differentiation of these cells result in heterogeneous cultures that comprise neurons, glia, and progenitor cells, which makes the assessment of neuronal function problematic. However, Skin cells of epidermis Neuron of brain Gastrula Pigment cell Sperm Egg Skeletal muscle cell Smooth muscle (in gut) Tubule cell of the kidney Skin Nerves Eyes Bones Blood Muscles Cardiac muscle Red blood cells Mesoderm (middle layer) Ectoderm Endoderm Mesoderm Endoderm (internal layer) Ectoderm (external layer) Germ cells Lungs Lining of gut Liver Lung cell (alveolar cell) Thyroid cell Pancreatic cell Zygote Inner-cell mass Some Embryonic Cell Types at Gastrulation Blastocyst Blastocyst Rostral neural Figure 9.1 Pluripotency in embryonic stem cells and the potential derivation of various lineage-specifi ed cells. STEM AND PROGENITOR CELLS IN DEGENERATIVE DISORDER 234 the neural plate acquires a rostral character and is subsequently caudalized by exposure to Wnt, FGF, BMP, and RA signals (Jessell 2000; Lee, Pfaff 2001; Munoz-Sanjuan, Brivanlou 2001; Panchision, Mckay 2002; Gunhaga, Marklund, Sjodal et al. 2003) to establish the main subdivisions of the CNS: the forebrain, midbrain, hindbrain, and spinal cord. Furthermore, along the dorsoventral axis, the neural tube is patterned into more subdivisions by three signals, SHH ventrally from the notochord and BMP and Wnt dorsally from the roof plate (Jessell 2000; Lee, Pfaff 2001; Panchision, Mckay 2002; Gunhaga, Marklund, Sjodal et al. 2003). Therefore, the precursor cells in each subdivision along the rostrocaudal axes are fated to subtypes of neurons and glia, dep- ending on its exposure to unique sets of morphogens at speci c concentrations. NEURAL DIFFERENTIATION FROM ES CELLS: METHODOLOGY Enrichment of neural progenitors from differentiating hESCs has been achieved by exploiting the observa- tion that cells of neural morphology form spontaneously within hESC colonies after prolonged culture. Reubinoff et al. (2001) demonstrated the mechanical isolation of these neural progenitors, and repeating the culture in chemically de ned medium supplemented with B27, human epidermal growth factor (hEGF), and basic  broblast growth factor 2 (bFGF-2) (Fig. 9.3) resulted in the formation of spherical structures called neurospheres. These neurospheres have highly enriched neural progenitor cells, with 99% of cells expressing neural cell adhesion molecule (N-CAM), 97% expressing nestin, and 90.5% expressing A2B5 (Reubinoff, Itsykson, Turetsky et al. 2001). According to Zhang et al. (2001), hESC-generated neuroectodermal cells usually do not form typical neurospheres. Instead, they from aggregates of columnar cells in the form of neural tube–like rosettes, where only after the long- term expansion of the neural rosette clusters will they form the morphology of neurospheres. Therefore, neurospheres formed in the spontaneous differentiation cultures may represent neural precursors at a much later developmental stage. with FGF and Wnt signaling abolishes neural induction at an early stage in the chick (Wilson, Graziano, Harland et al. 2000; Wilson, Rydstrom, Trimborn et al. 2001). FGF might act by antagonizing the BMP signal pathway indirectly or by directly inducing speci c transcription factors, which determine neuroectoderm induction and inhibit mesoderm differentiation (Bertrand, Hudson, Caillol et al. 2003; Sheng, Dos, Stern et al. 2003). Hence, a balanced view of neural induction most likely needs to include both instructive and inhibitory factors. FGF may induce a neural state at an early stage, and BMP antagonists may subsequently stabilize the neural identity. Once a neuroectodermal fate is speci ed, the neural plate folds to form the neural tube, from which cells differentiate into various neurons and glia. However, this process does not occur homogenously and simultane- ously throughout the neural tube. Instead, the neural tube is patterned along its rostrocaudal and dorsoventral axes to establish a grid-like set of positional cues (Altmann, Brivanlou 2001). The neural plate initially acquires a rostral character, and it is then gradually caudalized by exposure to Wnt, FGF, BMP, and retinoic acid (RA) signals (Munoz-Sanjuan, Brivanlou 2001; Agathon, Thisse, Thisse et al. 2003) to establish the main subdivisions of the CNS: the forebrain, midbrain, hindbrain, and spinal cord. Along the dorsoventral axis, the neural tube is patterned into more subdivisions by the two opposing signals: sonic hedgehog (SHH) ventrally from the notochord and BMP dorsally from the roof plate (Jessell 2000; Lee, Pfaff 2001). Precursor cells in each subdivision along the rostrocaudal and dorsoventral axes, by exposure to a unique set of morphogens at speci c concentrations, are fated to subtypes of neurons and glial cells (Oster eld, Kirschner, Flanagan 2003). It is this unique positional code that endows a neuron with a speci c target. Thus, it will be crucial to imprint the positional information into the neurons that are generated in vitro to achieve their potential for cell replacement. Roles of Growth Factors in Neural Tube Formation The transition from neuroectoderm to neural plate and then to the neural tube sets up a platform from which cells differentiate into various neurons and glia (O’Rahilly, Muller 1994; O’Rahilly, Muller 2007). The neural tube is patterned along its rostrocaudal and dorsoventral axes to establish a grid-like set of positional cues (Altmann, Brivanlou 2001). Figure 9.2 depicts the central dogma of motor neuron development, where primitive ectodermal cells are converted to motor neurons through the caudalizing action of RA and the ventralizing action of SHH. Similarly, Neural induction Caudalization Ventralization Primitive ectoderm Rostral neural Caudal neural Motor neurons RA SHH Figure 9.2 Central dogma of motor neuron development. Neural inductive signals convert primitive ectodermal cells to a rostral neural fate. Signals including retinoic acid (RA) convert rostral neural cells to more caudal identities. Spinal progenitors are converted to motor neurons by sonic hedgehog (SHH) signaling. Adapted from Wichterle, Lieberam, Porter et al. 2002. Chapter 9: Motoneurons from Embryonic Stem Cells 235 is done either by enzymatic treatment or by mechanical dissection. Both groups utilize serum-free media (DMEM/F12) (1:1) supplemented with different types of nutrients for neural induction. The neurospheres are then plated on laminin- or ornithin-coated plates for further neural differentiation. Another commonly used technique for the neural differentiation from ES cells is the aggregation of ES cells into so-called embryoid bodies (EBs) in suspension cultures and treatment of these EBs with RA after withdrawing pluripotent growth factors such as bFGF. The EB structure recapitulates certain aspects of early embryogenesis with the appearance of lineage-speci c regions similar to that found in the embryo (Doetschman et al. 1985). After 2 to 4 days in suspension culture, primitive endoderm cells form on the surface of EBs and epiblast-like cells form inside. These EBs are termed simple EBs. With further culturing, differentiation of a columnar epithelium with a basal lamina and the formation of a central cavity occur, at which point the EBs are termed cystic EBs. Cystic EBs are similar to egg cylinder–stage embryos, consisting of a double-layered structure with an inner ectodermal layer and outer layer of endoderm enclos- ing a cavity. Continued culture of EBs results in the appearance of mesodermal and endodermal cell types. Hence, the differentiation of ES cells in the Selection of neural cells was also used by Zhang’s group as a method of enriching for neural progenitors (Zhang, Wernig, Duncan et al. 2001). hESCs were initially differentiated as EBs in chemically de ned medium supplemented with FGF-2 before culturing in adherent culture for a further 8 to 10 days (Fig. 9.3). Prominent outgrowths of neural progenitors, repre- senting 72% to 84% of the total cells, were seen in the cultures and could be isolated by limited enzymatic digestion. Culture in medium supplemented with FGF-2, but not epidermal growth factor or leukemia- inhibitory factor, was shown to promote proliferation of the isolated aggregates in suspension. Although the authors did not characterize the composition of these neurosphere-like aggregates, they demonstrated the presence of neural progenitors by differentiation potential, with the ability to form neurons, astrocytes, and oligodendrocytes on plating and withdrawal of FGF-2. The major difference between Zhang’s and Reubinoff’s method is that Zhang utilizes the embryoid body (EB) pathway whereas Reubinoff spontaneously differentiates hESC colonies for a prolonged time of 3 weeks (Fig. 9.3) (Reubinoff, Itsykson, Turetsky et al. 2001; Zhang, Wernig, Duncan et al. 2001). Both protocols require the isolation of neuroepithelial cells from other non-neural cells, and propagation of these neurospheres in culture. Isolation of these neural rosettes Zhang et al. (2001) EB formation in suspension Differentiating hESC colonies on feeder Plate on poly- D-lysine and laminin without growth factors Neurospheres Transfer to adherent tissue culture dishes with DMEM/F12 (1:1), B27, glutamine, penicillin, streptomycin, human EGF and bFGF 1–2 weeks 3 weeks 3 weeks 14–21 days 8–10 days 10–14 days 4 days Reubinoff et al. (2001) hESC grown on mouse feeder hESC grown on mouse feeder Mechanical dissection of neuroepithelial cells Enzymatic extraction of neuroepithelial cells EBs in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (1:1), supplemented with insulin, transferring, progesterone, petrescine, sodium selenite, heparin and FGF-2 Plate on ornithine and laminin substrate without FGF-2 Differentiation into neurons Differentiation into neurons Figure 9.3 Schematic procedures for neural differentiation. Comparative analysis of methodologies by Zhang et al. (2001) and Reubinoff et al. (2001) indicate some similarities and differences. Zhang et al. utilizes the EB pathway but not Reubinoff, et al. Both isolate neuroepithelial cells by mechanical dissection or enzymatic treatment. bFGF, basic fi broblast growth factor; EB, embryoid body; FGF-2, fi broblast growth factor 2; hEGF, human epidermal growth factor. STEM AND PROGENITOR CELLS IN DEGENERATIVE DISORDER 236 The absence of several rostral neural markers, such as BF-1 and Otx2 suggests that RA may selectively promote the differentiation of caudal neuronal types. RA is required for differentiation of spinal motoneurons (Billon, Jolicoeur, Ying et al. 2002). RA is a strong morphogen that appears to push ES cells toward postmitotic neurons and results in robust neuronal differentiation in a reproducible way. Hence, it is most widely used for neuronal differentiation from ES cells, including human ES cells. FGF-2 is a survival and proliferation factor used for early neural precursor cells. On the basis of this fact, McKay and colleagues developed a method to promote the proliferation of a neural precursor population selectively with FGF-2 (Okabe, Forsberg-Nilsson, Spiro et al. 1996). ES cell aggregates are cultured in suspension for 4 days and then plated on an adhesive substrate in the presence of FGF-2 in a serum-free ITSFn medium (DMEM/FIZ supplemented with insulin, transferrin, selenium, and  bronectin). Under this condition, the majority of cells die, but neural precursors survive and proliferate in the presence of FGF-2. After 6 to 8 days of selection and expansion, the nestin-positive neural precursor cells are enriched to approximately 80%. Withdrawal of FGF-2 induces spontaneous differentiation into various neurons and glia (Okabe, Forsberg-Nilsson, Spiro et al. 1996; Brustle, Jones, Learish et al. 1999), and the neuronal cells generated in this way ful ll the criteria of functional postmitotic neurons with both excitatory and inhibitory synaptic connections. In contrast to the RA approach, neural precursor cells expanded with FGF-2 are generally developmentally synchronized. They appear to be further induced to neuronal types with representatives of mid- and hindbrain, such as dopaminergic neurons (Lee, Lumelsky, Studer et al. 2000). Because FGF-2 also possesses caudalizing effects, it is reasonable to believe that FGF-2–induced neural precursors may give rise to neuronal types of a more caudal neuraxis. In addition to methods involving formation of ES cell aggregates, direct differentiation of indi- vidual or monolayer ES cells has been developed by several groups with the use of feeder cells or media conditioned from mesoderm-derived cell lines. The rationale behind these protocols is that signals from mesodermal cells are required to induce neural speci cation from the ectoderm in vivo. Sasai and colleagues  rst established this method to derive dopaminergic neurons (Kawasaki, Mizuseki, Nishikawa et al. 2000). Mouse ES cells are dissociated into single cells and plated on PA6 stromal feeder cells at a low density. After co-culturing in a serum-free medium for 6 days, 92% of the ES cell colonies contain nestin- positive cells. The authors name the inductive factor stromal cell–derived inducing activity (SDIA). SDIA induces co-cultured ES cells to differentiate into form of EBs in vitro obeys general rules of development that prevail in an embryo. However, EBs exhibit stochastic differentiation into a variety of cell lineages. Treatment with morphogens/growth factors and/or use of particular culture systems is necessary to achieve a directed differentiation and/or selective expansion of a speci c lineage. For neural differentiation, which occurs during early embryonic development, ES cell aggregates are usually treated with morphogens at an early stage in which these aggregates do not display the structure of embryonic germ layers. Hence, the name EBs in neural differentiation paradigms is rather misleading. Spontaneous differentiation of EBs yields only a small fraction of neural lineage cells. To promote neural differentiation, ES cell aggregates, cultured in the regular ES cell medium for 4 days, are exposed to RA (0.51 mM) for another 4 days. Hence, this method is often regarded as a 42/41 protocol (Bain, Kitchens, Yao et al. 1995). This method was optimized by Gotlieb and colleagues based on neuronal differentiation from teratocar- cinoma cells (Jones-Villeneuve, McBurney, Rogers et al. 1982). Other RA-triggered neural differentiation protocols are variations of the 42/41 protocol (Wobus, Grosse, Schoneich 1988; Strubing, Ahnert- Hilger, Shan et al. 1995; Fraichard, Chassande, Bilbaut et al. 1995; Dinsmore, Ratliff, Deacon et al. 1996; Renoncourt, Carroll, Filippi et al. 1998). Mouse ES cells treated with this protocol yield a good pro- portion (38%) of neuronal cells upon differentiation. The predominant population of neuronal cells is glu- taminergic and γ-aminobutyric acid (GABAergic) neurons (Jones-Villeneuve, McBurney, Rogers et al. 1982). These neuronal cells express voltage-gated Ca 2+ , Na + , K + ion channels and form functional synapses with neighboring neurons. They generate action potentials and are functionally coupled by inhibitory (GABAergic) and excitatory (glutamatergic) synapses, as revealed by measurement of postsynaptic currents (Strubing, Ahnert-Hilger, Shan et al. 1995). Signaling through RA is important during development, particularly in rostral/caudal patterning of the neural tube (Maden 2002). However, there is little evidence to sug- gest that RA in these protocols acts to induce neural speci cations. DIRECTED DIFFERENTIATION: USE OF SIGNALING MOLECULES/GROWTH FACTORS EBs treated with RA differentiate into neuronal cell types characteristic of ventral CNS: somatic motoneurons (islet1/2, Lim3, HB9), cranial motoneurons (islet1/2 and phox2b), and interneurons (lim1/2 or En1) (Renoncourt, Ahnert-Hilger, Shan et al. 1998). Chapter 9: Motoneurons from Embryonic Stem Cells 237 differentiation. The high ef ciency of neural induction with noggin treatment is consistent with its role in the default model of neural induction. Selection by FGF-2/bFGF FGF-2, also known as basic  broblast growth factor (bFGF), is a survival and proliferation factor for early neural precursor cells from mouse and human. As described previously, McKay and colleagues developed a method to promote the proliferation of neural precursor populations selectively with bFGF (Okabe, Forsberg-Nilsson, Spiro et al. 1996). Withdrawal of bFGF after 6 to 8 days of selection and expansion induces spontaneous differentiation into various neurons and glia (Okabe, Forsberg-Nilsson, Spiro et al. 1996; Brustle, Jones, Learish et al. 1999). Another role of bFGF is its ability to direct differentiation of ES cells to neural cell types, particularly motor neurons. A study by Shin et al. (2005) demonstrated that by using bFGF alone, there was a 2.64-fold increase of motor neurons differentiated from hESCs when compared to the control treatment, suggesting that bFGF may be an effective growth factor for in vitro differentiation to human motor neurons. FGF-2 is routinely used to expand central nervous system stem cells (CNS-SCs) in serum-free media (Ray et al. 1993; Kilpatrick, Bartlett 1995; Palmer et al. 1995; Gritti et al. 1996; Johe et al. 1996). This growth factor is considered to act simply as a neutral mitogen. Gabay et al. (2003), however, have demonstrated that contrary to this assumption, the spinal cord progenitor cells change their dorsoventral identity in FGF, even at concentrations two orders of magnitude lower than those used to grow the cells (0.2 ng/mL). In the case of dorsally derived cells, FGF causes an extinction of dorsal progenitor domain markers such as Pax3 and Pax7 and an induction of ventral markers such as Olig2 and Nkx2.2. FGF probably induces SHH signaling for ventralization in these cells. The evidence that FGF induces ventralization through SHH is based on induction of SHH mRNA and SHH antagonist (Frank-Kamenetsky et al. 2002; Williams, Guicherit, Zaharian et al. 2003), which attenuate the effect of FGF (Fig. 9.4). However, an SHH-independent mecha- nism does exist in telencephalon (Kuschel, Rüther, Theil 2003). Grb2-associated binder 1 (Gab1) has been iden- ti ed as an adaptor molecule downstream of many growth factors, including epidermal growth factor (EGF),  broblast growth factor, and platelet-derived growth factor, which have been shown to play crucial roles as mitotic signals for a variety of neural progenitor cells, including stem cells, both in vitro and in vivo (Hayakawa-Yano, Nishida, Fukami et al. 2007). rostral CNS precursor cells with both a ventral and dorsal character. Early exposure of SDIA-treated ES cells to bone morphogenetic protein 4 (BMP4) sup- presses neural differentiation and promotes epidermal differentiation, whereas late BMP4 exposure (after day 4 of co-culture) causes differentiation of neural crest cells and the dorsal-most CNS cells. In contrast, SHH promotes differentiation of ventral CNS cells such as motor neurons, and SHH at a high concentration ef ciently promotes differentiation of the ventral-most  oor plate cells. Thus, SDIA-treated ES cells generate precursors that have the compe- tence to differentiate into the full dorsal–ventral range of neuroectodermal derivatives in response to patterning signals (Mizuseki, Sakamoto, Watanabe et al. 2003). The neural inducing factor(s) does not appear to be restricted to PA6 cells. Studer and colleagues demonstrate that several mesoderm-derived cell lines promote the differentiation of mouse ES cells to different neuronal subtypes, astrocytes, and oligodendrocytes, in combination with morphogens at different concentrations and at different times (Barberi, Klivenyi, Calingasan, et al. 2003). Thus, neural precursor cells induced by stromal signals appear to be naive and are responsive to versa- tile signals for further differentiation into neurons and glia with speci c regional identities, although the phenotypes of these neural precursors are not characterized. Alternatively, the stromal signals can induce a wide range of neural precursors that can be selectively promoted by different morphogens. The identity of the SDIA remains unknown, which intro- duces an unknown component into the experimental paradigm. This co-culture system can be combined with ES cell aggregation to yield a more homoge- neous neuroectodermal differentiation (Rathjen, Haines, Hudson et al. 2002). The aforementioned neural differentiation protocols are designed on the basis of our understanding of neural development. However, introduction of unknown factors, empirically devised steps, and selective culture systems make them irrelevant to normal neural development. In recent years, more sophisti- cated and chemically de ned culture systems have been developed. Anti-BMP signaling is thought to play a crucial role in neural induction. Gratsch and O’Shea (2002) examined the role of BMP antagonists, noggin and chordin, in neural differentiation from mouse ES cells. Exposure of mouse ES cells to noggin in de ned medium or transfection with a noggin expression plasmid promotes widespread neural differentiation. After 72 hours of noggin treatment, about 90% cells become nestin positive neural precursor cells, which are strongly inhibited by BMP4. Interestingly, exposure to chordin produces a more complex pattern of neural cell differentiation as well as mesenchymal cell [...]... identified as causes of the early-onset form of AD (St George-Hyslop, Petit 20 05) The late-onset form is not inherited It appears generally at an older age (above age 65) The origin of the late-onset form of AD remains unknown; risk factors include expression of different forms of the gene apolipoprotein (Raber, Huang, Ashford et al 2004) and reduced expression of neuronal sortilin-related receptor gene (Rogaeva,... Nat Rev Neurosci 8: 755 –7 65 Marom K, Shapira E, Fainsod A 1997 The chicken caudal genes establish an anterior-posterior gradient by partially overlapping temporal and spatial patterns of expression Mech Dev 64:41 52 Marti E, Bumcrot DA, Takada R, McMahon AP 19 95 Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants Nature 3 75: 322–3 25 Matsunaga E, Araki... neurofibrillary tangles in the brain (Caselli, Beach, Yaari et al 2006) There are two forms of the disease: the early-onset, or familial, form, and the late-onset, or sporadic, form of AD The early-onset form of AD is a rare form of the disease Approximately 10% of patients with AD have the familial form It is the genetic form of the disease and is inherited It appears at a young age Mutations in three... (retinoic acid, BMP-2, BMP-4) Expansion (FGF-2, NT-3) Embryonic gonad Embryonic germ cells Embryoid bodies Cholinergic neurons Expansion Transplantation B Expansion (FGF-2, CEE) Spinal cord Isolation (immunopanning) Differentiation (plating on liminin) Cholinergic neurons Expansion (FGF-2, NT-3) Differentiation (retinoic acid, BMP-2, BMP-4) Cholinergic neurons Differentiation (FGF-2, CNTF, NGF, BDNF... without initial culture in RA, approximately 56 % and 65% of the cells expressed neuroectodermal markers, PSA-N-CAM and A2B5, respectively Initial EB culture in a medium supplemented with RA resulted in 87% of cells expressing PSA-N-CAM or A2B5, a 30% increase in marker expression Although it is difficult to determine from this report if this represents enrichment for neural progenitors, other reports have... retinoid-activated pathway of neurogenesis in the ventral spinal cord Cell 97:903–9 15 Plachta N, Bibel M, Tucker KL, Barde YA 2004 Developmental potential of defined neural progenitors derived from mouse embryonic stem cells Development 131 :54 49 54 56 Placzek M 19 95 The role of the notochord and floor plate in inductive interactions Curr Opin Genet Dev 5: 499 50 6 Placzek M, Dodd J, Jessell TM 2000 The case for. .. 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Biotechnol 18 :53 57 Tosney KW, Landmesser LT 1985a Growth cone morphology and trajectory in the lumbosacral region of the chick embryo J Neurosci 5: 23 45 2 358 Tosney KW, Landmesser LT 1985b Specificity of early motoneuron growth cone outgrowth in the chick embryo J Neurosci 5: 2336–2344 Trainor PA, Krumlauf R 2001 Hox genes, neural crest cells, and branchial arch patterning Curr Opin Cell Biol 13:698–7 05 Tropepe... in Parkinson’s disease of substantia nigra dopamine neurons containing calbindin-D28K Brain Res 52 6:303–307 Yamamoto S, Yoshino I, Shimazaki T et al 20 05 Essential role of Shp2-binding sites on FRS2alpha for corticogenesis and for FGF2-dependent proliferation of neural progenitor cells Proc Natl Acad Sci U S A 102: 159 83– 159 88 Ying QL, Stavridis M, Griffiths D, Li M, Smith A 2003 Conversion of embryonic... hypomyelination in PDGF-A knockout mice Development 126: 457 –467 Gabay L, Lowell S, Rubin LL, Andeson DJ 2003 Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro Neuron 40:4 85 499 Gard AL, Pfeiffer SE 1990 Two proliferative stages of the oligodendrocyte lineage (a2b5_0 4- and 04_galc-) under different mitogenic control Neuron 5: 6 15 6 25 Gibb WR 1992 . Pommey S, Galipeau J. 20 05. Allogeneic marrow stromal cells are immune rejected by MHC class I- and class II-mismatched recip- ient mice. Blood. 106:4 057 –40 65. Ellis-Behnke RG, Liang Y, You. increased 5- to 10-fold (Bain, Kitchens, Yao et al. 19 95) , and 50 % to 70% of surviv- ing cells exhibited properties of neural and glial cell populations, including expression of neuron-speci. approximately 56 % and 65% of the cells expressed neuroectodermal markers, PSA-N-CAM and A2B5, respectively. Initial EB culture in a medium supplemented with RA resulted in 87% of cells expressing

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