Fig. 4. Population measurements can obscure the heterogeneity of the cells within the embryoid body. For instance, western blot or RT-PCR are not able to determine whether only a few cell are expressing the gene of interest on high level (A), every cell on middle level (B) or there is a polarity (C).

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## **8. Conclusions**

Embryonic cells are widely used model system to understand the early steps of embryonic development. Over the last years, many RA-regulated pathways have been discovered in EC and ES cells using a diverse set of techniques. In this chapter we have summarized the results of recent studies using high-throughput approaches (microarray, ChIP-chip, ChIP-Seq) to understand the mechanistic of retinoid signaling in early embryonic development. However, many questions remained to be unanswered. We should face the new technical challenges to be able to work out these issues.

## **9. References**

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**Part 2**

**Neural and Retinal Differentiation**

## **Part 2**

**Neural and Retinal Differentiation**

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**4**

*Brazil*

**Pluripotent Stem Cells as an** *In Vitro*

Irina Kerkis1, Mirian A. F. Hayashi2, Nelson F. Lizier1, Antonio C. Cassola3,

*1Laboratory of Genetics, Butantan Institute and Department of Morphology and Genetics,*

Embryonic stem (ES) cells are derived directly from inner cell mass (ICM) of mouse or human preimplantation embryos (Evans & Kaufman 1981, Martin 1981, Thomson et al., 1998). They are pluripotent, once they are able to differentiate *in vitro* and *in vivo* into derivatives of the three embryonic germ cell lines: mesoderm, endoderm and ectoderm (Fig. 1). The establishment of protocols for direct *in vitro* differentiation of pluripotent stem cell (PSC) into desirable cell type is extremely important for their use in therapies, for the studies of human diseases, and also for biochemical, toxicological and pharmacological studies (Pederson 1999, Sukoyan et al., 2002, Wobus & Löser 2011). Therefore, focusing on the PSCs use in these studies, many efforts have been devoted to the establishment of stem cells models with a

particular emphasis to their *in vitro* differentiation into mature and functional neurons.

The nervous system is the most complex system in the organism and its formation usually involves four stages: specification of the neural cells identity (neural or glia), neural migration and axon formation, synapse formation (with target neurons, muscle or gland cells) and synaptic connection refinement (elimination of axons branches and cells death) (Müller 2006). It is well-known that many genes are involved in the process of neuronal stem cells fate specification (Aiba et al., 2006). This process depends on the specific environment during organogenesis, after birth and during adult life. The temporal and spatial factors are essential for neuronal differentiation, due to the multilayer nature of cortex (Müller 2006). Although numerous publications have reported PSCs differentiation toward neurons, many important questions are not answered yet, especially in respect to the equivalency of the *in vitro* PSCs model and *in vivo* central nervous system (CNS) development. Accomplishments in these directions would represent a crucial starting point for the stem cell therapies and drug discovery. A number of important protocols have been set up for the differentiation of PSCs into neurons, which mainly lead to the coexistence in the culture of differentiated neurons and non-neural cells, together with neural precursors

**1. Introduction**

**Model of Neuronal Differentiation**

*2Departament of Pharmacology, Federal University of Sao Paulo, 3Department of Physiology and Biophysics, University of Sao Paulo,*

Lygia V. Pereira4 and Alexandre Kerkis5

*4Institute of Biosciences, University of Sao Paulo,*

*Federal University of Sao Paulo,*

*5Celltrovet (Genética Aplicada), Ltda.*

## **Pluripotent Stem Cells as an** *In Vitro* **Model of Neuronal Differentiation**

Irina Kerkis1, Mirian A. F. Hayashi2, Nelson F. Lizier1, Antonio C. Cassola3, Lygia V. Pereira4 and Alexandre Kerkis5

*1Laboratory of Genetics, Butantan Institute and Department of Morphology and Genetics, Federal University of Sao Paulo, 2Departament of Pharmacology, Federal University of Sao Paulo, 3Department of Physiology and Biophysics, University of Sao Paulo, 4Institute of Biosciences, University of Sao Paulo, 5Celltrovet (Genética Aplicada), Ltda. Brazil*

### **1. Introduction**

Embryonic stem (ES) cells are derived directly from inner cell mass (ICM) of mouse or human preimplantation embryos (Evans & Kaufman 1981, Martin 1981, Thomson et al., 1998). They are pluripotent, once they are able to differentiate *in vitro* and *in vivo* into derivatives of the three embryonic germ cell lines: mesoderm, endoderm and ectoderm (Fig. 1). The establishment of protocols for direct *in vitro* differentiation of pluripotent stem cell (PSC) into desirable cell type is extremely important for their use in therapies, for the studies of human diseases, and also for biochemical, toxicological and pharmacological studies (Pederson 1999, Sukoyan et al., 2002, Wobus & Löser 2011). Therefore, focusing on the PSCs use in these studies, many efforts have been devoted to the establishment of stem cells models with a particular emphasis to their *in vitro* differentiation into mature and functional neurons.

The nervous system is the most complex system in the organism and its formation usually involves four stages: specification of the neural cells identity (neural or glia), neural migration and axon formation, synapse formation (with target neurons, muscle or gland cells) and synaptic connection refinement (elimination of axons branches and cells death) (Müller 2006). It is well-known that many genes are involved in the process of neuronal stem cells fate specification (Aiba et al., 2006). This process depends on the specific environment during organogenesis, after birth and during adult life. The temporal and spatial factors are essential for neuronal differentiation, due to the multilayer nature of cortex (Müller 2006). Although numerous publications have reported PSCs differentiation toward neurons, many important questions are not answered yet, especially in respect to the equivalency of the *in vitro* PSCs model and *in vivo* central nervous system (CNS) development. Accomplishments in these directions would represent a crucial starting point for the stem cell therapies and drug discovery. A number of important protocols have been set up for the differentiation of PSCs into neurons, which mainly lead to the coexistence in the culture of differentiated neurons and non-neural cells, together with neural precursors

Pluripotent Stem Cells as an *In Vitro* Model of Neuronal Differentiation 83

within cell aggregates, when feeder layers and required factors to maintain pluripotency are removed. These aggregates, denominated embryoid bodies (EBs), resemble early post implantation embryos, although chaotically organized inside. It is assumed that EBs formation initiates spontaneous differentiation of ES cells to the three embryonic germ layers (Evans & Kaufman 1981). Innumerous studies have addressed the issue of cell specific differentiation of ES cells. In Figure 2 we summarize the main cell phenotypes, which can be induced to differentiate from ES cells *in vitro* under specific culture conditions. Those accomplishments are the result of a dynamic interaction between knowledge of embryonic development and empirical testing, targeted at reproducing *in vitro* cell specification

Fig. 2. ES cell differentiation *in vitro*. ES cells are isolated from the ICM of the blastocyst. These cells can be induced to form EBs, which are structures that contain representatives of the three embryonic germ layers. Under the appropriate culture condition, the EBs can be

Mesoderm is the germ layer responsible for the development of muscle, bone, cartilage, blood, and connective tissue. Blood and endothelial cells are the first cell types to form in the developing vertebrate embryo at around six days of gestation. This event leads to the formation of the yolk sac, an extraembryonic membrane composed of adjacent mesodermal

induced to differentiate into several types of cells *in vitro*.

**2.1 Mesoderm specification**

conditions found in the developing embryo.

and undifferentiated PSCs (Okabe et al., 1996, Li et al., 1998, Mujtaba et al., 1999, Baharvand et al., 2007). Most of these protocols are short-lasting, which therefore does not allow a careful analysis of the neurons maturation, aging, and death processes.

In this chapter, we describe principal methods of PSCs differentiation into neurons *in vitro*. Next, we present a method developed by our group, which established a long-term culture of committed neuronal precursors and functional neurons from mouse embryonic stem (mES) cells. In addition, using this long-term protocol we demonstrated the temporal and spatial localization of microtubule-associated proteins, such as, Lis1 (Lissencephaly-1) and Ndel1 (nuclear distribution element-like) in neuronal precursors and differentiated neurons. These both proteins have been shown to be essential for neuronal differentiation during the CNS development. Regardless of the relevance of these proteins for neuronal differentiation, their expression during PSCs differentiation was marginally explored.

Fig. 1. ES cell isolation and differentiation.

## **2. Pluripotent stem cells as a model of** *in vitro* **differentiation**

ES cells are powerful biological model, which can provide important information for our knowledge regarding the cell commitment and differentiation during development process (O'Shea 1999, Wobus & Boheler 2005). Multiple methods have been developed in order to induce *in vitro* PSCs differentiation and to obtain the desirable cell phenotype (Baharvand et al., 2004, Keller 2005). It has been found that ES cells are able to differentiate spontaneously

and undifferentiated PSCs (Okabe et al., 1996, Li et al., 1998, Mujtaba et al., 1999, Baharvand et al., 2007). Most of these protocols are short-lasting, which therefore does not allow a

In this chapter, we describe principal methods of PSCs differentiation into neurons *in vitro*. Next, we present a method developed by our group, which established a long-term culture of committed neuronal precursors and functional neurons from mouse embryonic stem (mES) cells. In addition, using this long-term protocol we demonstrated the temporal and spatial localization of microtubule-associated proteins, such as, Lis1 (Lissencephaly-1) and Ndel1 (nuclear distribution element-like) in neuronal precursors and differentiated neurons. These both proteins have been shown to be essential for neuronal differentiation during the CNS development. Regardless of the relevance of these proteins for neuronal differentiation,

careful analysis of the neurons maturation, aging, and death processes.

their expression during PSCs differentiation was marginally explored.

Fig. 1. ES cell isolation and differentiation.

**2. Pluripotent stem cells as a model of** *in vitro* **differentiation**

ES cells are powerful biological model, which can provide important information for our knowledge regarding the cell commitment and differentiation during development process (O'Shea 1999, Wobus & Boheler 2005). Multiple methods have been developed in order to induce *in vitro* PSCs differentiation and to obtain the desirable cell phenotype (Baharvand et al., 2004, Keller 2005). It has been found that ES cells are able to differentiate spontaneously within cell aggregates, when feeder layers and required factors to maintain pluripotency are removed. These aggregates, denominated embryoid bodies (EBs), resemble early post implantation embryos, although chaotically organized inside. It is assumed that EBs formation initiates spontaneous differentiation of ES cells to the three embryonic germ layers (Evans & Kaufman 1981). Innumerous studies have addressed the issue of cell specific differentiation of ES cells. In Figure 2 we summarize the main cell phenotypes, which can be induced to differentiate from ES cells *in vitro* under specific culture conditions. Those accomplishments are the result of a dynamic interaction between knowledge of embryonic development and empirical testing, targeted at reproducing *in vitro* cell specification conditions found in the developing embryo.

Fig. 2. ES cell differentiation *in vitro*. ES cells are isolated from the ICM of the blastocyst. These cells can be induced to form EBs, which are structures that contain representatives of the three embryonic germ layers. Under the appropriate culture condition, the EBs can be induced to differentiate into several types of cells *in vitro*.

### **2.1 Mesoderm specification**

Mesoderm is the germ layer responsible for the development of muscle, bone, cartilage, blood, and connective tissue. Blood and endothelial cells are the first cell types to form in the developing vertebrate embryo at around six days of gestation. This event leads to the formation of the yolk sac, an extraembryonic membrane composed of adjacent mesodermal

Pluripotent Stem Cells as an *In Vitro* Model of Neuronal Differentiation 85

The embryonic ectoderm is an embryonic germ layer, which can produce various cell lineages during development. Of particular interest, the differentiation of ES cells into neuronal cells was published independently by three groups in 1995 (Bain et al., 1995, Fraichard et al., 1995, Strubing et al., 1995). Gene expression and electrophysiological studies of cell derivatives from PSCs indicated the presence of the all three major cell types of the brain: astrocytes, oligodendrocytes, and neurons (dopaminergic, GABAergic (gamma-aminobutyric-acidreleasing), serotonergic, glutamatergic and cholinergic neurons) (Lee et al., 2000, Rolletschek at al., 2001, Aubert et al., 2002) (Fig. 3). Thus, these studies opened first perspectives regarding ES cell models for the study of neurodegenerative disorders. Human ES cells are also able to generate the neural epithelium (Thomson et al., 1998, Reubinoff et al., 2000, Zhang et al., 2001). However, although neural progenitors derived from ES cells could be enriched and directed to differentiate into mature neurons, astrocytes, and oligodendrocytes (Carpenter et al., 2001), experimental data obtained until recently could not demonstrate the formation of a given neuron subtype (Lee et al., 2000, Rolletschek at al., 2001, Aubert et al., 2002). The possibility of generating neurons *in vitro* signals for a first step towards exploring the therapeutic potential

Fig. 3. All three major cell types of CNS derivative from PSCs: oligodendrocytes, astrocytes,

Currently, common protocol comprises three steps: EBs (3-D model) formation, derivation of primitive neuroepithelial cells from EBs and generation of differentiated neural cell types. The most widely used method to induce neuronal differentiation is to enzymatically or mechanically lift the PSCs colonies and place them into low-adherence culture dishes or flasks

**2.3.1 Three dimensional (3-D) model of PSCs differentiation**

**2.3 Ectoderm specification**

and neurons.

of ES cells for Parkinson's disease (Svendsen, 2008).

and primitive (visceral) endodermal cell layers, which give rise to blood and endothelial cells (Baron 2001). *In vitro* differentiation of ES cells in EBs allows the generation of blood islands containing erythrocytes and macrophages (Doetschman et al., 1985), whereas differentiation in semisolid medium is efficient for the formation of neutrophilis, macrophages, and erythroid lineages (Wiles & Keller 1991). In an attempt to identify potential inducers of the hematopoietic lineage, researchers indicated Wnt3 (Proto-oncogene protein) as an important signaling molecule that plays a significant role in enhancing hematopoietic commitment during *in vitro* differentiation of ES cells (Lako et al., 2001). The hematopoietic cells derived from ES cells have been characterized by specific gene expression patterns and by cell surface antigens (Wiles & Keller 1991, Wang et al., 1992). However, the most important aspect was to characterize these cells in the functional capacity, by demonstrating long-term multilineage hematopoietic repopulating properties in an animal model (Palacios et al., 1995).

Cardiomyocytes readily differentiate from aggregates composed of mES cells in the presence of high concentration of serum (around 20%), and display properties comparable to those observed *in vivo*: they express similar cardiac gene expression patterns, present sarcomeric structures, and demonstrate contractility triggered by cardiac-specific ion currents, as well as the expression of membrane-bound ion channels. This type of differentiation can develop spontaneously or be induced by differentiation factors including dimethyl sulfoxide (DMSO) and retinoic acid (RA), or small molecules, such as Dynorphin B and cardiogenol derivatives (Fassler et al., 1996). Human ES cells also hold the ability to differentiate into cardiomyocytes, which show similar properties to those derived from mES cells (Kehat et al., 2001). Furthermore, it is well established that ES cells can efficiently differentiate into several other mesodermal cells types, including mesenchymal cell-derived adipogenic (Dani et al., 1997), chondrogenic (Kramer et al., 2000), osteoblast (Buttery et al., 2001), and myogenic cells (Rohwedel et al., 1994). In all of these experiments, the cell type derivation was induced by specific differentiation factors.

#### **2.2 Endoderm specification**

Endoderm is responsible for deriving the pancreas and liver. Regarding the therapeutic interest for the treatment of hepatic failure and diabetes mellitus, hepatic and pancreatic cells are of special interest. Thus, since these cells could be derived from ES cells new hope has emerged (Soria 2001). These *in vitro* derived cells showed hepatic-restricted transcripts and proteins, and were able to integrate and to function in a host liver following transplantation (Chinzei et al., 2002). Recently, researchers demonstrated that hepatocytelike endodermal markers were also detected in ES cell derivatives (Yamada et al., 2002).

The potential use of ES cells for treatment of diabetes was enhanced by the perspective of deriving insulin-producing pancreatic endocrine cells. Researchers at NovoCell, Inc., a biotechnology company in the USA, have developed an *in vitro* differentiation process that mimics pancreatic organogenesis. By directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor, they were able to convert human ES cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin (D'Amour et al., 2006). Moreover, in pre-clinical trials, the same group showed that those ES-derived pancreatic cells efficiently generated glucose-responsive endocrine cells after implantation into mice, and those insulin producing cells, in turn protected animals from streptozotocininduced hyperglycemia (Kroon et al., 2008).

#### **2.3 Ectoderm specification**

84 Embryonic Stem Cells – Differentiation and Pluripotent Alternatives

and primitive (visceral) endodermal cell layers, which give rise to blood and endothelial cells (Baron 2001). *In vitro* differentiation of ES cells in EBs allows the generation of blood islands containing erythrocytes and macrophages (Doetschman et al., 1985), whereas differentiation in semisolid medium is efficient for the formation of neutrophilis, macrophages, and erythroid lineages (Wiles & Keller 1991). In an attempt to identify potential inducers of the hematopoietic lineage, researchers indicated Wnt3 (Proto-oncogene protein) as an important signaling molecule that plays a significant role in enhancing hematopoietic commitment during *in vitro* differentiation of ES cells (Lako et al., 2001). The hematopoietic cells derived from ES cells have been characterized by specific gene expression patterns and by cell surface antigens (Wiles & Keller 1991, Wang et al., 1992). However, the most important aspect was to characterize these cells in the functional capacity, by demonstrating long-term multilineage hematopoietic repopulating properties

Cardiomyocytes readily differentiate from aggregates composed of mES cells in the presence of high concentration of serum (around 20%), and display properties comparable to those observed *in vivo*: they express similar cardiac gene expression patterns, present sarcomeric structures, and demonstrate contractility triggered by cardiac-specific ion currents, as well as the expression of membrane-bound ion channels. This type of differentiation can develop spontaneously or be induced by differentiation factors including dimethyl sulfoxide (DMSO) and retinoic acid (RA), or small molecules, such as Dynorphin B and cardiogenol derivatives (Fassler et al., 1996). Human ES cells also hold the ability to differentiate into cardiomyocytes, which show similar properties to those derived from mES cells (Kehat et al., 2001). Furthermore, it is well established that ES cells can efficiently differentiate into several other mesodermal cells types, including mesenchymal cell-derived adipogenic (Dani et al., 1997), chondrogenic (Kramer et al., 2000), osteoblast (Buttery et al., 2001), and myogenic cells (Rohwedel et al., 1994). In all of these experiments, the cell type

Endoderm is responsible for deriving the pancreas and liver. Regarding the therapeutic interest for the treatment of hepatic failure and diabetes mellitus, hepatic and pancreatic cells are of special interest. Thus, since these cells could be derived from ES cells new hope has emerged (Soria 2001). These *in vitro* derived cells showed hepatic-restricted transcripts and proteins, and were able to integrate and to function in a host liver following transplantation (Chinzei et al., 2002). Recently, researchers demonstrated that hepatocytelike endodermal markers were also detected in ES cell derivatives (Yamada et al., 2002). The potential use of ES cells for treatment of diabetes was enhanced by the perspective of deriving insulin-producing pancreatic endocrine cells. Researchers at NovoCell, Inc., a biotechnology company in the USA, have developed an *in vitro* differentiation process that mimics pancreatic organogenesis. By directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor, they were able to convert human ES cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin (D'Amour et al., 2006). Moreover, in pre-clinical trials, the same group showed that those ES-derived pancreatic cells efficiently generated glucose-responsive endocrine cells after implantation into mice, and those insulin producing cells, in turn protected animals from streptozotocin-

in an animal model (Palacios et al., 1995).

**2.2 Endoderm specification**

induced hyperglycemia (Kroon et al., 2008).

derivation was induced by specific differentiation factors.

The embryonic ectoderm is an embryonic germ layer, which can produce various cell lineages during development. Of particular interest, the differentiation of ES cells into neuronal cells was published independently by three groups in 1995 (Bain et al., 1995, Fraichard et al., 1995, Strubing et al., 1995). Gene expression and electrophysiological studies of cell derivatives from PSCs indicated the presence of the all three major cell types of the brain: astrocytes, oligodendrocytes, and neurons (dopaminergic, GABAergic (gamma-aminobutyric-acidreleasing), serotonergic, glutamatergic and cholinergic neurons) (Lee et al., 2000, Rolletschek at al., 2001, Aubert et al., 2002) (Fig. 3). Thus, these studies opened first perspectives regarding ES cell models for the study of neurodegenerative disorders. Human ES cells are also able to generate the neural epithelium (Thomson et al., 1998, Reubinoff et al., 2000, Zhang et al., 2001). However, although neural progenitors derived from ES cells could be enriched and directed to differentiate into mature neurons, astrocytes, and oligodendrocytes (Carpenter et al., 2001), experimental data obtained until recently could not demonstrate the formation of a given neuron subtype (Lee et al., 2000, Rolletschek at al., 2001, Aubert et al., 2002). The possibility of generating neurons *in vitro* signals for a first step towards exploring the therapeutic potential of ES cells for Parkinson's disease (Svendsen, 2008).

Fig. 3. All three major cell types of CNS derivative from PSCs: oligodendrocytes, astrocytes, and neurons.

#### **2.3.1 Three dimensional (3-D) model of PSCs differentiation**

Currently, common protocol comprises three steps: EBs (3-D model) formation, derivation of primitive neuroepithelial cells from EBs and generation of differentiated neural cell types. The most widely used method to induce neuronal differentiation is to enzymatically or mechanically lift the PSCs colonies and place them into low-adherence culture dishes or flasks

Pluripotent Stem Cells as an *In Vitro* Model of Neuronal Differentiation 87

plated in culture flask, which allows rapid adherence of feeder cells. The EBs were obtained in low serum (5%) basal culture medium, following routine protocol of hanging drop method. Next, EBs were transferred into low-adherence culture dishes without substrate that allows adherence, and the neuronal differentiation was induced by the addition of RA (at final concentration of 0.1 µM). The EBs were maintained under non-adherent serum-free culture conditions (neurobasal (NB) medium supplemented with B27), for additional 4 days. Next, RA was removed and the EBs was transferred to poly-lysine treated plastic dishes in order to form ANs. The ANs were maintained in serum-free conditions for additional 7 days. At this moment of neuronal differentiation, outgrowth of neuron-like cells on the periphery of ANs was clearly observed (Fig. 5). These ANs were caught in small pieces and mechanically transferred into another Petri dish. After 3-4 days, these small ANs start to produce outgrowth of neurons. This process of ANs mechanical splitting and transfer can be

Fig. 4. Differentiation of ES cells towards neurons. 1-2: EBs formation; 3-6: 3-D model; 7-11:2-

D model. (1) Pluripotent ES cells in basal culture medium. (2) EBs formation using suspension cell culture or hanging drop protocol. (3-5) Adherence of EBs, in serum-free medium with or without RA, production of neuroepithelial cells, (4) rosette formation, (5) neurons (white-brilliant) and glial cells (black) production. (6-11) culture of floating EBs in serum-free medium in the presence of RA, (7, 9, 10) RA removal, EBs adherence and ANs formation, production of neuronal precursor and mature neurons, (8, 10) ANs mechanical splitting and transfer using glass pipette. White arrows showed in (2) EBs (phase contrast), in (3) rosette (Hematoxylin & Eosin staining) in (5) neurons (phase contrast), in (7, 9, 11) AN

with outgrowing neurons (phase contrast).

repeated several times continuously producing ANs and neurons.

without substrate, feeder cells, or mitogens, where they form EBs (Carpenter et al., 2001, Colombo et al., 2006, Baharvand et al., 2007). The culture media formulations for EBs vary significantly between different works (Ng et al., 2005, Yoon et al., 2006). Next, EBs are transferred to serum-free culture media and are plated onto laminin (poly-lysine)-coated dishes in order to generate an adherent culture and to differentiate into neuroepithelial cells. The EBs undergo spontaneous differentiation and the formation of clusters of small elongated cells surrounding a central zone, free of cells, so-called neural rosettes was showed (Pankratz et al., 2007, Pankratz & Zhang 2007). These rosettes resemble the morphology of the primitive neural tube and express early neural marker antigens such as Nestin (type VI intermediate filament (IF) protein) and Musashi-1 (RNA-binding proteins expressed in the CNS), but not markers of more mature neural cells. These rosettes were observed in majority of the studies of induction of neural differentiation of PSCs. PSCs neuroepithelial differentiation method are widely used to generate neural progenitors and mature neural cell types. The neuroepithelial cells obtained by this approach express the neuroepithelial transcription factors, such as PAX6 (Paired box gene 6), Sox1 (Sex determining region Y-box 1), and Sox2 (sex determining region Y-box 2), in about 90% of the total differentiated progenies (Li et al., 2007, Pankratz & Zhang 2007). Neuronal differentiation can also occur without EBs formation. In this case, specific growth factors or the co-culture of PSCs with cells of a particular origin that have been found to produce factors of neuronal cell specification are used to accelerate the differentiation towards one cell type or lineage of interest.

RA is an important regulator of the nervous system development, regeneration and maintenance (Zhang 2006, Maden 2007). Although, rosette formation occurrs during spontaneous *in vitro* differentiation of PSC-derived EBs, addition of RA enhances significantly the yielding of rosettes and mature neurons. Therefore, the predominant number of studies uses RA alone or in combination with other factors, e.g. bFGF (*basic fibroblast growth factor*). Additionally, neural differentiation can also be induced by the withdrawal of bFGF/EGF (*epithelial growth factor*) and exposure to BDNF (*brain-derived neurotrophic factor*), NGF (*neuronal growth factor*) or other factors into the culture medium.

The above described protocol and its modifications commonly produce mixed population of neuronal cells, which contain precursors, neurons and glial cells. This mixed population needs the application of further protocols for selection and enrichment, in order to obtain almost pure population of precursors or neurons, suitable for pharmacological screening or therapeutic applications.

#### **2.3.2 Bi-dimensional (2-D) model of PSCs differentiation**

Primary neural stem cells (NSC) can proliferate *in vitro,* forming multicellular floating spherical clusters, commonly referred as neurospheres, which are mainly composed by committed progenitor cells. When adhered on substrate, these neurospheres differentiate into functional neurons (Reynolds & Weiss, 1992; Chojnacki & Weiss, 2008). Our group aimed at developing a protocol for PSCs differentiation into neurons, which resemble the differentiation pattern of NSC-derived adherent neurosphere (AN). This protocol comprises five steps: EBs formation, culturing of floating EBs in the presence of RA, EBs adherence, formation of AN (2-D model), composed by commuted neuronal precursors and generation of neurons from AN. We further referred adherent EBs as ANs. It is worth mentioning that this protocol avoids the formation of rosettes.

The details of 2-D protocol are presented in Figure 4. An enzymatic digestion with trypsin of mES cells were used in order to obtain a feeder-free cell suspension. The mES cells were

without substrate, feeder cells, or mitogens, where they form EBs (Carpenter et al., 2001, Colombo et al., 2006, Baharvand et al., 2007). The culture media formulations for EBs vary significantly between different works (Ng et al., 2005, Yoon et al., 2006). Next, EBs are transferred to serum-free culture media and are plated onto laminin (poly-lysine)-coated dishes in order to generate an adherent culture and to differentiate into neuroepithelial cells. The EBs undergo spontaneous differentiation and the formation of clusters of small elongated cells surrounding a central zone, free of cells, so-called neural rosettes was showed (Pankratz et al., 2007, Pankratz & Zhang 2007). These rosettes resemble the morphology of the primitive neural tube and express early neural marker antigens such as Nestin (type VI intermediate filament (IF) protein) and Musashi-1 (RNA-binding proteins expressed in the CNS), but not markers of more mature neural cells. These rosettes were observed in majority of the studies of induction of neural differentiation of PSCs. PSCs neuroepithelial differentiation method are widely used to generate neural progenitors and mature neural cell types. The neuroepithelial cells obtained by this approach express the neuroepithelial transcription factors, such as PAX6 (Paired box gene 6), Sox1 (Sex determining region Y-box 1), and Sox2 (sex determining region Y-box 2), in about 90% of the total differentiated progenies (Li et al., 2007, Pankratz & Zhang 2007). Neuronal differentiation can also occur without EBs formation. In this case, specific growth factors or the co-culture of PSCs with cells of a particular origin that have been found to produce factors of neuronal cell specification are used to accelerate the differentiation

RA is an important regulator of the nervous system development, regeneration and maintenance (Zhang 2006, Maden 2007). Although, rosette formation occurrs during spontaneous *in vitro* differentiation of PSC-derived EBs, addition of RA enhances significantly the yielding of rosettes and mature neurons. Therefore, the predominant number of studies uses RA alone or in combination with other factors, e.g. bFGF (*basic fibroblast growth factor*). Additionally, neural differentiation can also be induced by the withdrawal of bFGF/EGF (*epithelial growth factor*) and exposure to BDNF (*brain-derived neurotrophic factor*), NGF (*neuronal growth factor*) or other factors into the culture medium. The above described protocol and its modifications commonly produce mixed population of neuronal cells, which contain precursors, neurons and glial cells. This mixed population needs the application of further protocols for selection and enrichment, in order to obtain almost pure population of precursors or neurons, suitable for pharmacological screening or

Primary neural stem cells (NSC) can proliferate *in vitro,* forming multicellular floating spherical clusters, commonly referred as neurospheres, which are mainly composed by committed progenitor cells. When adhered on substrate, these neurospheres differentiate into functional neurons (Reynolds & Weiss, 1992; Chojnacki & Weiss, 2008). Our group aimed at developing a protocol for PSCs differentiation into neurons, which resemble the differentiation pattern of NSC-derived adherent neurosphere (AN). This protocol comprises five steps: EBs formation, culturing of floating EBs in the presence of RA, EBs adherence, formation of AN (2-D model), composed by commuted neuronal precursors and generation of neurons from AN. We further referred adherent EBs as ANs. It is worth mentioning that

The details of 2-D protocol are presented in Figure 4. An enzymatic digestion with trypsin of mES cells were used in order to obtain a feeder-free cell suspension. The mES cells were

towards one cell type or lineage of interest.

**2.3.2 Bi-dimensional (2-D) model of PSCs differentiation**

this protocol avoids the formation of rosettes.

therapeutic applications.

plated in culture flask, which allows rapid adherence of feeder cells. The EBs were obtained in low serum (5%) basal culture medium, following routine protocol of hanging drop method. Next, EBs were transferred into low-adherence culture dishes without substrate that allows adherence, and the neuronal differentiation was induced by the addition of RA (at final concentration of 0.1 µM). The EBs were maintained under non-adherent serum-free culture conditions (neurobasal (NB) medium supplemented with B27), for additional 4 days. Next, RA was removed and the EBs was transferred to poly-lysine treated plastic dishes in order to form ANs. The ANs were maintained in serum-free conditions for additional 7 days. At this moment of neuronal differentiation, outgrowth of neuron-like cells on the periphery of ANs was clearly observed (Fig. 5). These ANs were caught in small pieces and mechanically transferred into another Petri dish. After 3-4 days, these small ANs start to produce outgrowth of neurons. This process of ANs mechanical splitting and transfer can be repeated several times continuously producing ANs and neurons.

Fig. 4. Differentiation of ES cells towards neurons. 1-2: EBs formation; 3-6: 3-D model; 7-11:2- D model. (1) Pluripotent ES cells in basal culture medium. (2) EBs formation using suspension cell culture or hanging drop protocol. (3-5) Adherence of EBs, in serum-free medium with or without RA, production of neuroepithelial cells, (4) rosette formation, (5) neurons (white-brilliant) and glial cells (black) production. (6-11) culture of floating EBs in serum-free medium in the presence of RA, (7, 9, 10) RA removal, EBs adherence and ANs formation, production of neuronal precursor and mature neurons, (8, 10) ANs mechanical splitting and transfer using glass pipette. White arrows showed in (2) EBs (phase contrast), in (3) rosette (Hematoxylin & Eosin staining) in (5) neurons (phase contrast), in (7, 9, 11) AN with outgrowing neurons (phase contrast).

Pluripotent Stem Cells as an *In Vitro* Model of Neuronal Differentiation 89

The expression of other neuron-specific proteins, such as MAP2 (microtubule-associated protein 2), NF-M (neurofilament medium protein), Tau (a microtubule-associated protein), NeuN (neuronal nuclei marker), GABA and 5-HT (5-hydroxytryptamin), was observed in neurons derived from ANs each time after splitting and mechanical transfer, which was maintained during three months. Electrophysiological analysis, by using the patch-clamp technique, in long-lasting culture of AN-derived mature neurons, showed the presence of ionic channels and membrane electrical potentials typical of electrically excitable cells,

This method of mechanical splitting and transfer of ANs is advantageous because it avoids the trauma associated to the trypsin treatments and mechanical dissociation and, so forth, may improve the survival of committed precursors able to differentiate into neurons. It is well-known that CNS precursors are localized in stem cell niche of organisms, which guarantees their continuous growth and renewing, and also the production of differentiated cells. In our model, ANs provide a constant microenvironment (*in vitro* niche) for the neuronal progenitor cells, which can be maintained for at least twelve weeks in culture, following repetitive mechanical splitting and transfer. Since expression of GFAP (Glial fibrillary acidic protein) gene has not been detected, it seems that AN direct the fate of non-

**2.3.3 Importance of lineage selection for transplantation studies in regenerative**

A majority of available protocols for neural differentiation result in the generation of multiple cell types of committed neural precursor to a fully differentiated, post-mitotic neural cell. The selection and expansion of ES-derived neural precursors is a material for transplantation studies focusing on diseases as Parkinson's or Alzheimer's disease, or neural damage following stroke or injury. Such protocol is important due to the elimination of PSCs from the transplanted cell population, which can generate teratocarcinomas (Zhang et al., 1996, Deacon et al., 1998, Bjorklund et al., 2002). Commonly genetic engineering methods are used for lineage selection on differentiating ES cells to purify neural precursors. These techniques rely on the introduction of a reporter/selection cassette into a locus with restricted expression in the desired cell type by homologous recombination. Thus, to address lineage selection of neuronal precursor one copy of the pan-neural gene Sox1 has been replaced by the dual selection/reporter cassette egfpIRESpac in ES cell line, which confers cell-autonomous green fluorescence and puromycin resistance to cells that express Sox1. This gene is not expressed in ES cells. Its expression is limited to neuroectodermal cell, and undifferentiated ES cells are not fluorescent. Upon neural differentiation, Sox1 is activated and the cells produce green fluorescence enabling further purification of both neural and non-neural cells generated during differentiation. Fluorescence-activated cell sorting (FACS) is used for the isolation of both Sox1–GFP-positive and -negative cells allowing further analysis (Li et al., 1998, Pevny et

The comparison of AN and the above described techniques demonstrated that we succeeded to establish very simple and long-term protocol for generation of Sox1 positive cells. It is useful to note that 3 months of several mechanical splitting, Sox1-positive cells maintain the expression and continuously produce outgrowing beta III-tubulin-positive cells, while expression of GFAP gene has not been detected (Hayashi et al., 2010). Quantification of precursors and mature neurons demonstrated stable production of both Sox1 and beta IIItubulin proteins during the first 2 months. At the end of the third month, Sox1-nestin-

which is a characteristic feature of functional CNS neurons.

committed precursors toward the neurons generation.

al., 1998, Wood et al., 1999, Ying et al., 2003).

**medicine**

Fig. 5. Differentiation of PSC-derived adherent neurospheres. A) AN (black) producing a network of connected neurons. B) AN (white) after mechanical splitting and transfer. Migration of neurons also can be observed. Light microscopy (phase contrast).

These ANs present expression of the neural progenitor cell markers, such as Sox1 and Nestin just after plating. Following differentiation, the inner part of the AN, continuously expressed Sox 1 and Nestin proteins, while outgrowing neurons, which form an extensive neurite net around the AN expressed beta III-tubulin (neuron-specific marker) (Fig. 6).

Fig. 6. Expression of neuroepithelial and neuronal markers in AN. A) Expression of Sox 1 protein (red), which express in the nucleus of progenitor cells localized in the inner part of AN. B) Expression of beta-III tubulin protein (green) at the periphery of AN and in outgrowing neurons. C) Higher magnification of extensive neurite net in (B) showing interconnected neurons.

Fig. 5. Differentiation of PSC-derived adherent neurospheres. A) AN (black) producing a network of connected neurons. B) AN (white) after mechanical splitting and transfer. Migration of neurons also can be observed. Light microscopy (phase contrast).

These ANs present expression of the neural progenitor cell markers, such as Sox1 and Nestin just after plating. Following differentiation, the inner part of the AN, continuously expressed Sox 1 and Nestin proteins, while outgrowing neurons, which form an extensive neurite net around the AN expressed beta III-tubulin (neuron-specific marker) (Fig. 6).

Fig. 6. Expression of neuroepithelial and neuronal markers in AN. A) Expression of Sox 1 protein (red), which express in the nucleus of progenitor cells localized in the inner part of

AN. B) Expression of beta-III tubulin protein (green) at the periphery of AN and in outgrowing neurons. C) Higher magnification of extensive neurite net in (B) showing

interconnected neurons.

The expression of other neuron-specific proteins, such as MAP2 (microtubule-associated protein 2), NF-M (neurofilament medium protein), Tau (a microtubule-associated protein), NeuN (neuronal nuclei marker), GABA and 5-HT (5-hydroxytryptamin), was observed in neurons derived from ANs each time after splitting and mechanical transfer, which was maintained during three months. Electrophysiological analysis, by using the patch-clamp technique, in long-lasting culture of AN-derived mature neurons, showed the presence of ionic channels and membrane electrical potentials typical of electrically excitable cells, which is a characteristic feature of functional CNS neurons.

This method of mechanical splitting and transfer of ANs is advantageous because it avoids the trauma associated to the trypsin treatments and mechanical dissociation and, so forth, may improve the survival of committed precursors able to differentiate into neurons. It is well-known that CNS precursors are localized in stem cell niche of organisms, which guarantees their continuous growth and renewing, and also the production of differentiated cells. In our model, ANs provide a constant microenvironment (*in vitro* niche) for the neuronal progenitor cells, which can be maintained for at least twelve weeks in culture, following repetitive mechanical splitting and transfer. Since expression of GFAP (Glial fibrillary acidic protein) gene has not been detected, it seems that AN direct the fate of noncommitted precursors toward the neurons generation.

#### **2.3.3 Importance of lineage selection for transplantation studies in regenerative medicine**

A majority of available protocols for neural differentiation result in the generation of multiple cell types of committed neural precursor to a fully differentiated, post-mitotic neural cell. The selection and expansion of ES-derived neural precursors is a material for transplantation studies focusing on diseases as Parkinson's or Alzheimer's disease, or neural damage following stroke or injury. Such protocol is important due to the elimination of PSCs from the transplanted cell population, which can generate teratocarcinomas (Zhang et al., 1996, Deacon et al., 1998, Bjorklund et al., 2002). Commonly genetic engineering methods are used for lineage selection on differentiating ES cells to purify neural precursors. These techniques rely on the introduction of a reporter/selection cassette into a locus with restricted expression in the desired cell type by homologous recombination. Thus, to address lineage selection of neuronal precursor one copy of the pan-neural gene Sox1 has been replaced by the dual selection/reporter cassette egfpIRESpac in ES cell line, which confers cell-autonomous green fluorescence and puromycin resistance to cells that express Sox1. This gene is not expressed in ES cells. Its expression is limited to neuroectodermal cell, and undifferentiated ES cells are not fluorescent. Upon neural differentiation, Sox1 is activated and the cells produce green fluorescence enabling further purification of both neural and non-neural cells generated during differentiation. Fluorescence-activated cell sorting (FACS) is used for the isolation of both Sox1–GFP-positive and -negative cells allowing further analysis (Li et al., 1998, Pevny et al., 1998, Wood et al., 1999, Ying et al., 2003).

The comparison of AN and the above described techniques demonstrated that we succeeded to establish very simple and long-term protocol for generation of Sox1 positive cells. It is useful to note that 3 months of several mechanical splitting, Sox1-positive cells maintain the expression and continuously produce outgrowing beta III-tubulin-positive cells, while expression of GFAP gene has not been detected (Hayashi et al., 2010). Quantification of precursors and mature neurons demonstrated stable production of both Sox1 and beta IIItubulin proteins during the first 2 months. At the end of the third month, Sox1-nestin-

Pluripotent Stem Cells as an *In Vitro* Model of Neuronal Differentiation 91

gene result in neuronal migration defects (Reiner et al., 1993, Youn et al., 2009, Saillour et al., 2009), while knockdown or ablation of cortical Ndel1 function also results in impaired migration of neocortical projection neurons (Sasaki et al., 2005, Youn et al., 2009). Lis1 and Ndel1 co-localize predominantly in the centrosome in early neuroblasts, and later, redistributes to axons during neuronal development (Shu et al., 2004, Guo et al., 2006, Bradshaw et al., 2008, Hayashi et al., 2010). Thus, Lis1 and Ndel1 are essential for normal

Currently, Lis1 and Ndel1 were shown to have additional, important functions in the cytoplasmic dynein pathway. They participate in nuclear and centrosomal transport in migrating neurons (Shu et al., 2004, Tsai et al., 2005).  McKenney and co-authors (2010), using biochemical and biophysical approaches, investigated whether and how Lis1 and NudE (Ndel1) affect dynein motor activity. Results obtained in this work apparently explain the requirement for Lis1 and NudE in the transport of nuclei, centrosomes, chromosomes, and the microtubule cytoskeleton. Additionally, they provide new insight into the molecular basis for lissencephaly, and the mechanism of action of

Regardless of the relevance of these proteins for neuronal differentiation, their expression during PSCs differentiation is not well explored yet. Our group was the first to analyze intracellular localization of both proteins in mES cells, undifferentiated and during *in vitro* neural differentiation (Hayashi et al., 2010). The expression of both Lis1 and Ndel1 proteins was observed in undifferentiated cells, which presented co-localization within the perinuclear region (Fig. 7). At early stages of differentiation, just after formation of ANs, Lis1 expression was observed in the cytoplasm, while Ndel1 was in the perinuclear region of committed cells. Following differentiation, when ANs grow in size, the expression of both proteins was no more observed in the area of committed cells. Both Lis1 and Ndel1 proteins were visualized in outgrowing neuritis. Additionally, they co-localized with Tau, which is a marker of MAPs, involved in the microtubule assembly and stabilization. In the same way, Ndel1 and MAP2 were also co-localized. In non-rosette MAP2 positive neurons, Lis1 and

In attempt to mimic the development of cortical layers *in vitro* and to study the cell migration during the differentiation process, which can be assessed by the analysis of the expression pattern of these proteins, the ANs were allowed to grow for 15 days without splitting. Significant variation in spatial distribution of Lis1 and Ndel1 proteins were observed within 2-D ANs. The expression of Lis1 was observed in the inner part of ANs, in the cells presenting rosette morphology. Unexpectedly, Ndel1 was not expressed in rosette forming cells. Both proteins were co-localized in the cytoplasm of the cells showing neuroblast-like morphology, which were found close to the periphery of AN. Lis1 protein was expressing in the cells very closely localized to Ndel1 expressing cells, which, in turn, were close to the region of outgrowing neurons. Co-localization of Lis1 and Ndel1 expression was detected in cells from upper layer of ANs. Ndel1 was found to interact with

Ndel1 proteins co-localized in neuronal cell body and growing axons (Fig. 7).

centrosomes, suggesting that these cells are early neuroblasts (Fig. 8).

cortical neuronal migration and neurite outgrowth.

these proteins in a broad range of biological functions.

**<IP_ADDRESS> Expression of Lis1 and Ndel1 in ANs**

positive cells were maintained at a similar level as before (~83%), whereas the number of immature neurons (~45%) decreased 1.5-fold, suggesting delay of the maturation process (~32%). Moreover, we showed that under the described conditions, dopaminergic, GABAergic, and serotonergic neurons can be produced. Therefore, generation of 2-D model is of great importance because allows expansion of neural progenitor without genetic modification from primary ES cell culture. Our AN protocol is especially advantageous for the future of regenerative medicine and treatment of neurodegenerative diseases, which will provide more tools for a safety clinical protocol with the advantage of lacking the intermediate effects from non-neural cells.

## **2.3.4 Importance of microtubule associated proteins for neuronal differentiation**

Neuronal migration has been studied extensively in diverse mammalian species and the sequence of events that occurs during cortical development is shared by all mammals. (Gleeson & Walsh 2000, Walsh & Goffinet 2000). During neurogenesis, neural precursors are generated, which proliferate and differentiate into immature postmitotic neurons. These immature cells migrate from the ventricular zone (VZ) to preplate, a layer at the surface of the developing cerebral cortex, splitting the preplate and forming the cortical plate, which further develops into the cortex. Following immature neurons migration from the VZ, cortical lamination is established in an inside-out fashion. In the deep of the cortex, the earliest-born neurons end up, while later-born neurons localize at more superficial layers of the cortex residing near the pial surface. Synaptogenesis and apoptosis of neurons occur at the final stages of cortical development. Indeed, the migration of neurons requires the same steps, which is necessary for migration of any cell type. The signals of environment for attraction and repulsion; the nucleus dislocation from central position to the periphery, a process called nucleokinesis; and a mechanism for migration end up. Microtubule associated proteins (MAPs), for instance, Lis1 and Ndel1, have been shown to be essential for neuronal differentiation and cell migration during the CNS development and also in the adult nervous system.

#### **<IP_ADDRESS> Lis1 and Ndel1**

Haploinsufficiency of Lis1 results in lissencephaly, a human neuronal migration disorder (Reiner et al., 1993, Saillour et al., 2009).  Lis1 binds with high affinity to a protein called Ndel1. Both proteins can complex with cytoplasmic dynein, the retrograde microtubule motor. Lis1 and Ndel1 are proposed to be important for the regulation of dynein-related events in mitosis and migration (Shu et al., 2004, Yamada et al., 2008, Youn et al., 2009, Hippenmeyer et al., 2010, Zyłkiewicz et al., 2011). Thus, PSCs can provide an important model to study migration defects related to MAPs.

Lis1 is a central component of a protein complex, evolutionarily conserved from fungus to human that regulates nuclear migration (Morris, 2000). Lis1 is able to regulate neuronal migration efficiency in a dose-dependent manner (Gambello et al., 2003). Reduced Lis1 activity results in severe defects in the radial migration of multiple types of neurons, including neocortical projection neurons (Tsai et al., 2005).

Ndel1 is important for normal cortical development and it is involved in microtubule organization, nuclear translocation, and neuronal positioning, in concert with various other proteins, including Lis1 (Shu et al., 2004, Youn et al., 2009). Mutations in the mammals Lis1

positive cells were maintained at a similar level as before (~83%), whereas the number of immature neurons (~45%) decreased 1.5-fold, suggesting delay of the maturation process (~32%). Moreover, we showed that under the described conditions, dopaminergic, GABAergic, and serotonergic neurons can be produced. Therefore, generation of 2-D model is of great importance because allows expansion of neural progenitor without genetic modification from primary ES cell culture. Our AN protocol is especially advantageous for the future of regenerative medicine and treatment of neurodegenerative diseases, which will provide more tools for a safety clinical protocol with the advantage of lacking the

**2.3.4 Importance of microtubule associated proteins for neuronal differentiation**  Neuronal migration has been studied extensively in diverse mammalian species and the sequence of events that occurs during cortical development is shared by all mammals. (Gleeson & Walsh 2000, Walsh & Goffinet 2000). During neurogenesis, neural precursors are generated, which proliferate and differentiate into immature postmitotic neurons. These immature cells migrate from the ventricular zone (VZ) to preplate, a layer at the surface of the developing cerebral cortex, splitting the preplate and forming the cortical plate, which further develops into the cortex. Following immature neurons migration from the VZ, cortical lamination is established in an inside-out fashion. In the deep of the cortex, the earliest-born neurons end up, while later-born neurons localize at more superficial layers of the cortex residing near the pial surface. Synaptogenesis and apoptosis of neurons occur at the final stages of cortical development. Indeed, the migration of neurons requires the same steps, which is necessary for migration of any cell type. The signals of environment for attraction and repulsion; the nucleus dislocation from central position to the periphery, a process called nucleokinesis; and a mechanism for migration end up. Microtubule associated proteins (MAPs), for instance, Lis1 and Ndel1, have been shown to be essential for neuronal differentiation and cell migration during the CNS development and also in the

Haploinsufficiency of Lis1 results in lissencephaly, a human neuronal migration disorder (Reiner et al., 1993, Saillour et al., 2009).  Lis1 binds with high affinity to a protein called Ndel1. Both proteins can complex with cytoplasmic dynein, the retrograde microtubule motor. Lis1 and Ndel1 are proposed to be important for the regulation of dynein-related events in mitosis and migration (Shu et al., 2004, Yamada et al., 2008, Youn et al., 2009, Hippenmeyer et al., 2010, Zyłkiewicz et al., 2011). Thus, PSCs can provide an important model to study migration

Lis1 is a central component of a protein complex, evolutionarily conserved from fungus to human that regulates nuclear migration (Morris, 2000). Lis1 is able to regulate neuronal migration efficiency in a dose-dependent manner (Gambello et al., 2003). Reduced Lis1 activity results in severe defects in the radial migration of multiple types of neurons,

Ndel1 is important for normal cortical development and it is involved in microtubule organization, nuclear translocation, and neuronal positioning, in concert with various other proteins, including Lis1 (Shu et al., 2004, Youn et al., 2009). Mutations in the mammals Lis1

including neocortical projection neurons (Tsai et al., 2005).

intermediate effects from non-neural cells.

adult nervous system. **<IP_ADDRESS> Lis1 and Ndel1**

defects related to MAPs.

gene result in neuronal migration defects (Reiner et al., 1993, Youn et al., 2009, Saillour et al., 2009), while knockdown or ablation of cortical Ndel1 function also results in impaired migration of neocortical projection neurons (Sasaki et al., 2005, Youn et al., 2009). Lis1 and Ndel1 co-localize predominantly in the centrosome in early neuroblasts, and later, redistributes to axons during neuronal development (Shu et al., 2004, Guo et al., 2006, Bradshaw et al., 2008, Hayashi et al., 2010). Thus, Lis1 and Ndel1 are essential for normal cortical neuronal migration and neurite outgrowth.

Currently, Lis1 and Ndel1 were shown to have additional, important functions in the cytoplasmic dynein pathway. They participate in nuclear and centrosomal transport in migrating neurons (Shu et al., 2004, Tsai et al., 2005).  McKenney and co-authors (2010), using biochemical and biophysical approaches, investigated whether and how Lis1 and NudE (Ndel1) affect dynein motor activity. Results obtained in this work apparently explain the requirement for Lis1 and NudE in the transport of nuclei, centrosomes, chromosomes, and the microtubule cytoskeleton. Additionally, they provide new insight into the molecular basis for lissencephaly, and the mechanism of action of these proteins in a broad range of biological functions.

#### **<IP_ADDRESS> Expression of Lis1 and Ndel1 in ANs**

Regardless of the relevance of these proteins for neuronal differentiation, their expression during PSCs differentiation is not well explored yet. Our group was the first to analyze intracellular localization of both proteins in mES cells, undifferentiated and during *in vitro* neural differentiation (Hayashi et al., 2010). The expression of both Lis1 and Ndel1 proteins was observed in undifferentiated cells, which presented co-localization within the perinuclear region (Fig. 7). At early stages of differentiation, just after formation of ANs, Lis1 expression was observed in the cytoplasm, while Ndel1 was in the perinuclear region of committed cells. Following differentiation, when ANs grow in size, the expression of both proteins was no more observed in the area of committed cells. Both Lis1 and Ndel1 proteins were visualized in outgrowing neuritis. Additionally, they co-localized with Tau, which is a marker of MAPs, involved in the microtubule assembly and stabilization. In the same way, Ndel1 and MAP2 were also co-localized. In non-rosette MAP2 positive neurons, Lis1 and Ndel1 proteins co-localized in neuronal cell body and growing axons (Fig. 7).

In attempt to mimic the development of cortical layers *in vitro* and to study the cell migration during the differentiation process, which can be assessed by the analysis of the expression pattern of these proteins, the ANs were allowed to grow for 15 days without splitting. Significant variation in spatial distribution of Lis1 and Ndel1 proteins were observed within 2-D ANs. The expression of Lis1 was observed in the inner part of ANs, in the cells presenting rosette morphology. Unexpectedly, Ndel1 was not expressed in rosette forming cells. Both proteins were co-localized in the cytoplasm of the cells showing neuroblast-like morphology, which were found close to the periphery of AN. Lis1 protein was expressing in the cells very closely localized to Ndel1 expressing cells, which, in turn, were close to the region of outgrowing neurons. Co-localization of Lis1 and Ndel1 expression was detected in cells from upper layer of ANs. Ndel1 was found to interact with centrosomes, suggesting that these cells are early neuroblasts (Fig. 8).

Pluripotent Stem Cells as an *In Vitro* Model of Neuronal Differentiation 93

Interaction of Lis1 and Ndel1 with other cytosolic proteins had been well studied using cultured non-neuronal and/or neuronal cells and the expression of both Ndel1 and Lis1 genes in early neuroblasts derived from embryonic and adult tissues was observed (Sasaki et al., 2000, Shu et al., 2004). Our data demonstrated an expression of only Ndel1 without Lis1 expression in the centrosome region in neuroblast-like cells within differentiated AN. Variation of spatial distribution of Lis1 and Ndel proteins expression was also observed. The ES cells isolated from, for instance, Lis1 or Ndel1 knockout mice, followed by their differentiation into neuronal cells using the present protocol, will permit the elucidation of the real role of each protein during the neuronal differentiation process. Our data suggest that further analysis involving other important MAPs are necessary to allow a better comprehension of the migration mechanism(s) and of the specification fate of neuronal cells

PSCs have the capacity to differentiate *in vitro* into neuronal cells spontaneously through EBs formation or in monolayer culture. EBs 3-D model is shown to be more efficient model, which can be improved using serum-free culture conditions and inductors of differentiation (e.g. RA). Following this protocol, neuroepithelial cells could be obtained, which formed rosettes. Further selection and enrichment protocols are needed to isolate culture of committed neuronal precursor, neurons and/or glial cells. This 3-D model provides shortterm culture of neuronal cells, which did not allow analysis of neurons migration and survival. It is of note that these AN can be maintained even in the absence of growth factors,

Our study demonstrated that ANs is a long-term protocol, which can be used to analyze the process of neuronal differentiation in dynamics. Plating of intact ANs also provides a window of time to the precursor cells for establishing their fate in a 2-D environment. ANs model avoid a stage of rosettes formation directly producing committed progenitors and non-rosette neurons, mimicking process of differentiation of neurospheres form CNS. Mature neurons, obtained from ANs, display ionic channels and membrane electrical potential, which are typical of electrically excitable cells and are also a characteristic feature

Following mechanical splitting and transfer, these ANs grow continuously, confirming their auto-renewing properties similarly to progenitors of CNS. When maintained untouched during prolonged period (at least 15 days), progenitors inside growing ANs undergo further cell specification. As we demonstrated by the analyses of expression of Lis1 and Ndel1 proteins, both presented differential spatial distribution within the ANs. The discrepancy between patterns of expression of these proteins in neuroblasts isolated from embryonic or adult mouse neuronal tissues, and in those AN-derived cells was observed. AN-derived neuroblasts demonstrated only Ndel1 location in centrosome region, instead of showing the location of both proteins in this region. This indicates that miss expression of proteins, which are responsible for neuronal cells division and migration, can occur during *in vitro*

Thus, our protocol provides an efficient experimental model for studying neuronal *in vitro* differentiation mimicking early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of drugs on functional

without lacking the capacity to produce functional neurons.

during differentiation.

of functional CNS neurons.

differentiation.

neuronal cells.

**3. Conclusions**

Fig. 7. Schematic presentation of expression pattern of Lis1 and Ndel1 during neuronal differentiation of PSCs. In undifferentiated ES cells, both Lis1 and Ndel1 show a perinuclear co-localization. In neuronal precursors, Lis1 presents a cytoplasmatic and Ndel1 a perinuclear localization. In neurons, at the periphery of ANs, both Lis1 and Ndel1 colocalize in the outgrowing neurites. In non-rosette neurons, these proteins co-localize in neuronal body and neurites.

Fig. 8. Schematic presentation of Lis1 and Ndel1 expression in different layers of AN. Lis1 (green) expression was observed in the inner part of AN in the cells organized in rosettes. Intermediate layer (yellow) is composed by the neuroblast-like cells, which express both proteins, Lis1 (green) in the cytoplasm and Ndel1 in centriolos (red) and less in cytoplasm. Upper layer is composed mainly by Lis1 and at the periphery of AN by Ndel1 expressing cells. Proteins expression is also demonstrated by immunofluorescence within AN located on both sides of schematic presentation (Confocal microscopy + Fluorescence).

Interaction of Lis1 and Ndel1 with other cytosolic proteins had been well studied using cultured non-neuronal and/or neuronal cells and the expression of both Ndel1 and Lis1 genes in early neuroblasts derived from embryonic and adult tissues was observed (Sasaki et al., 2000, Shu et al., 2004). Our data demonstrated an expression of only Ndel1 without Lis1 expression in the centrosome region in neuroblast-like cells within differentiated AN. Variation of spatial distribution of Lis1 and Ndel proteins expression was also observed. The ES cells isolated from, for instance, Lis1 or Ndel1 knockout mice, followed by their differentiation into neuronal cells using the present protocol, will permit the elucidation of the real role of each protein during the neuronal differentiation process. Our data suggest that further analysis involving other important MAPs are necessary to allow a better comprehension of the migration mechanism(s) and of the specification fate of neuronal cells during differentiation.

## **3. Conclusions**

92 Embryonic Stem Cells – Differentiation and Pluripotent Alternatives

Fig. 7. Schematic presentation of expression pattern of Lis1 and Ndel1 during neuronal differentiation of PSCs. In undifferentiated ES cells, both Lis1 and Ndel1 show a perinuclear

Fig. 8. Schematic presentation of Lis1 and Ndel1 expression in different layers of AN. Lis1 (green) expression was observed in the inner part of AN in the cells organized in rosettes. Intermediate layer (yellow) is composed by the neuroblast-like cells, which express both proteins, Lis1 (green) in the cytoplasm and Ndel1 in centriolos (red) and less in cytoplasm. Upper layer is composed mainly by Lis1 and at the periphery of AN by Ndel1 expressing cells. Proteins expression is also demonstrated by immunofluorescence within AN located

on both sides of schematic presentation (Confocal microscopy + Fluorescence).

co-localization. In neuronal precursors, Lis1 presents a cytoplasmatic and Ndel1 a perinuclear localization. In neurons, at the periphery of ANs, both Lis1 and Ndel1 colocalize in the outgrowing neurites. In non-rosette neurons, these proteins co-localize in

neuronal body and neurites.

PSCs have the capacity to differentiate *in vitro* into neuronal cells spontaneously through EBs formation or in monolayer culture. EBs 3-D model is shown to be more efficient model, which can be improved using serum-free culture conditions and inductors of differentiation (e.g. RA). Following this protocol, neuroepithelial cells could be obtained, which formed rosettes. Further selection and enrichment protocols are needed to isolate culture of committed neuronal precursor, neurons and/or glial cells. This 3-D model provides shortterm culture of neuronal cells, which did not allow analysis of neurons migration and survival. It is of note that these AN can be maintained even in the absence of growth factors, without lacking the capacity to produce functional neurons.

Our study demonstrated that ANs is a long-term protocol, which can be used to analyze the process of neuronal differentiation in dynamics. Plating of intact ANs also provides a window of time to the precursor cells for establishing their fate in a 2-D environment. ANs model avoid a stage of rosettes formation directly producing committed progenitors and non-rosette neurons, mimicking process of differentiation of neurospheres form CNS. Mature neurons, obtained from ANs, display ionic channels and membrane electrical potential, which are typical of electrically excitable cells and are also a characteristic feature of functional CNS neurons.

Following mechanical splitting and transfer, these ANs grow continuously, confirming their auto-renewing properties similarly to progenitors of CNS. When maintained untouched during prolonged period (at least 15 days), progenitors inside growing ANs undergo further cell specification. As we demonstrated by the analyses of expression of Lis1 and Ndel1 proteins, both presented differential spatial distribution within the ANs. The discrepancy between patterns of expression of these proteins in neuroblasts isolated from embryonic or adult mouse neuronal tissues, and in those AN-derived cells was observed. AN-derived neuroblasts demonstrated only Ndel1 location in centrosome region, instead of showing the location of both proteins in this region. This indicates that miss expression of proteins, which are responsible for neuronal cells division and migration, can occur during *in vitro* differentiation.

Thus, our protocol provides an efficient experimental model for studying neuronal *in vitro* differentiation mimicking early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of drugs on functional neuronal cells.

Pluripotent Stem Cells as an *In Vitro* Model of Neuronal Differentiation 95

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to microtubule anchoring at the mother centriole and is involved in both dyneindependent and -independent centrosomal protein assembly. *Mol. Biol. Cell 17(2),*

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Luo, L. (2010). Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration.

differentiate into myocytes with structural and functional properties of

Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by

## **4. Acknowledgments**

The authors thank Dr. Antonio C.M. Camargo and Dr. Juliano R. Guerreiro from Butantan Institute for their contribution in our research, as well as Alexsander Seixas de Souza for his technical assistance with confocal microscopy; and Dr. Toshie Kawano (in memoriam), Laboratory of Parasitology of Butantan Institute, for allowing the free access to the microscope whenever necessary. This work was financially supported by FAPESP.

## **5. References**

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**5**

*Italy*

**Characterization of Embryonic Stem (ES)**

The nervous system development is of tremendous fundamental importance, but it is immensely challenging because of the complexity of both its architecture and function (Staii et al., 2011). Understanding the formation of this ordered complexity is one of the greatest challenges of modern science. Mechanisms by which progenitor cells differentiate and acquire their cell identity are just beginning to be fully understood (Gilbert, 2000; Lai &

A potent tool for studying differentiation and development *in vitro* has been offered to researchers by the isolation of mouse embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos more than 20 years ago (Evans & Kaufman, 1981; Martin, 1981). Two unique characteristics distinguish them from all other organ-specific stem cells identified to date. First, they can be maintained and expanded as pure populations of undifferentiated cells for extended periods of time retaining normal karyotypes. Second, they are pluripotent, capable to generate every cell type in the body. The pluripotent nature of mouse ES cells was formally demonstrated by their ability to contribute to all tissues of adult mice, including the germline, following their injection into host blastocysts (Bradley et al., 1984). In addition to their developmental potential *in vivo*, ES cells display a remarkable

*In vitro* differentiation of embryonic stem (ES) cells recapitulates early events in the development of the mammalian nervous system: ES cells can both generate and respond *in vitro* to signals that normally regulate embryonic development (Fraichard et al., 1995; Keller, 2005). This knowledge has been transported *in vitro* designing several different protocols that have evolved to promote neuroectoderm differentiation (Cai & Grabel, 2007; Keller, 2005). Each of the three major neural cell types of the central nervous system—neurons, astrocytes, and oligodendrocytes—can be generated, and relatively pure populations of each can be isolated when cultured under appropriate conditions (Okabe et al., 1996; Barberi et al., 2003). In addition to the generation of these different neural populations, conditions have been established for the development of different subtypes of neurons. Thus neurons

capacity to form all differentiated cell types in culture (Keller, 2005).

**1. Introduction**

Johnson, 2008).

**Neuronal Differentiation Combining**

**Atomic Force, Confocal and DIC**

Maria Elisabetta Ruaro, Jelena Ban and Vincent Torre

**Microscopy Imaging**

*International School for Advanced Studies (SISSA-ISAS), via Bonomea 265, Trieste*

## **Characterization of Embryonic Stem (ES) Neuronal Differentiation Combining Atomic Force, Confocal and DIC Microscopy Imaging**

Maria Elisabetta Ruaro, Jelena Ban and Vincent Torre *International School for Advanced Studies (SISSA-ISAS), via Bonomea 265, Trieste Italy*

## **1. Introduction**

98 Embryonic Stem Cells – Differentiation and Pluripotent Alternatives

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The nervous system development is of tremendous fundamental importance, but it is immensely challenging because of the complexity of both its architecture and function (Staii et al., 2011). Understanding the formation of this ordered complexity is one of the greatest challenges of modern science. Mechanisms by which progenitor cells differentiate and acquire their cell identity are just beginning to be fully understood (Gilbert, 2000; Lai & Johnson, 2008).

A potent tool for studying differentiation and development *in vitro* has been offered to researchers by the isolation of mouse embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos more than 20 years ago (Evans & Kaufman, 1981; Martin, 1981). Two unique characteristics distinguish them from all other organ-specific stem cells identified to date. First, they can be maintained and expanded as pure populations of undifferentiated cells for extended periods of time retaining normal karyotypes. Second, they are pluripotent, capable to generate every cell type in the body. The pluripotent nature of mouse ES cells was formally demonstrated by their ability to contribute to all tissues of adult mice, including the germline, following their injection into host blastocysts (Bradley et al., 1984). In addition to their developmental potential *in vivo*, ES cells display a remarkable capacity to form all differentiated cell types in culture (Keller, 2005).

*In vitro* differentiation of embryonic stem (ES) cells recapitulates early events in the development of the mammalian nervous system: ES cells can both generate and respond *in vitro* to signals that normally regulate embryonic development (Fraichard et al., 1995; Keller, 2005). This knowledge has been transported *in vitro* designing several different protocols that have evolved to promote neuroectoderm differentiation (Cai & Grabel, 2007; Keller, 2005). Each of the three major neural cell types of the central nervous system—neurons, astrocytes, and oligodendrocytes—can be generated, and relatively pure populations of each can be isolated when cultured under appropriate conditions (Okabe et al., 1996; Barberi et al., 2003). In addition to the generation of these different neural populations, conditions have been established for the development of different subtypes of neurons. Thus neurons

Characterization of Embryonic Stem (ES)

Park et al., 2010).

Neuronal Differentiation Combining Atomic Force, Confocal and DIC Microscopy Imaging 101

Confocal laser scanning microscopy (CLSM) utilizes the optical pathway of a regular optical microscope and in combination with immunofluorescence histochemistry has been widely used to simultaneously map the distribution and localization of different cellular components (extracellular and intracellular macro-molecules, including proteins, nucleic acids and lipids, as well as intracellular ions such as calcium) helping to understand intracellular mechanisms (Dailey et al., 1999; Rajwa et al., 2004). Moreover, it is possible to monitor the process and dynamics of living cells with high temporal resolution (Hell, 2007;

The 200 nm resolution limit of the CLSM is restricted by the diffraction limits of the microscope objective and does not permit accurate evaluation of the sample height. The zresolution of CLSM for any given wavelength is always at least 2 times less than the corresponding xy-resolution thus restricting a detailed structural image that can be achieved

Great advances in optical microscopy such as near field scanning optical microscopy (NSOM), stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) have been achieved to overcome the optical diffraction limit (Hell, 2007 and 2009; Schermelleh et al., 2010), but there are still constraints for realizing time-resolved dynamics or three dimensional imaging (Park et al., 2010). These techniques provide an excellent spatial resolution in the xy plane also in live specimens but relying on the presence of fluorophores

State-of-the-art transmission and scanning electron microscopy techniques are technically demanding, relatively costly, and time-consuming. Moreover, the possibility to specifically label and visualize multiple cellular structures or components in one specimen is still limited. Furthermore, chemical fixation, contrasting procedures and/or physical sectioning can cause artifacts and exclude the option to observe living cells (Schermelleh et al., 2010). With the discovery of **Atomic force microscopy (AFM)**, amazing progress has been made in the imaging of biomolecules with sub-nanometer resolution (Binnig et al., 1986; Engel et al. 1997; Fotiadis et al., 2002; Kasas et al, 1993; Lal & John, 1994; Müller et al., 1999), comparable to scanning electron microscopy. To date, AFM has been used increasingly and is progressively becoming a usual benchtop technique. The volume of scientific publications citing AFM increases continuously and papers with a biological emphasis reached more

AFM is a scanning probe device, which generates an image by systematically scanning a sharp tip mounted on the end of a flexible cantilever over the samples' surface. The tip interacts with the surface causing the cantilever to bend. A laser beam acting as an optical lever is deflected from the end of the cantilever to a position sensitive photo-diode that measures the cantilever

AFM can be used in ambient air or under liquid and provides an unprecedented way to image the morphological structure of the surfaces of cells and other biological samples, i.e. it images the surfaces where most of the regulatory biochemical and other signals are directed (Lal & John, 1994). By functionalizing the cantilever tip with appropriate molecules, (Reddy et al., 2004; Li et al., 2006), it is possible to localize specific molecules on the surface of the cell and to obtain a high resolution map of their localization. Furthermore, AFM, by measuring forces within and between biological molecules, can provide additional

deflection. In this way a topographic map of the surface is generated (Doak et al., 2008).

when exploring biological samples (Doak et al., 2008; Rajwa et al., 2004).

cannot provide information on the overall morphology of cells.

than 21% of total publications (as in 2006) (Parot et al., 2007).

of different parts of the neural tube were successfully generated including spinal motoneurons, midbrain dopaminergic neurons, spinal cord interneurons, Purkinje and granule cells of cerebellum, hypothalamus and finally cortical pyramidal cells (Gaspard & Vanderhaeghen, 2010).

These neurons do not only have the correct morphology and express specific markers but they are also functional: electrophysiological data from ES-derived neurons with different protocols validate their functional differentiation *in vitro* and there are evidence of the formation of synapse between ES-derived neurons or between an ES-derived neuron and a mature neuron in organotypic slices as well as integration of ES-derived neurons *in vivo*. Moreover, networks formed by ES-derived neurons display functional properties remarkably similar to those of hippocampal neurons (Ban et al., 2007 and references herein). The isolation of human ES cells in 1998 (Thomson et al., 1998) dramatically elevated the interest in the therapeutic promises of ES cells differentiation, in particular for brain repair. Therefore ES have emerged as a powerful tool in neurobiology, helping address longstanding questions in an entirely novel way (Gaspard & Vanderhaeghen, 2011).

If the cell is to become a neuron, the next decision is what type of neuron it will be. After fate is determined, still another decision gives the neuron a specific target (Gilbert, 2000). Neurons find their targets by protruding axons driven by the motile apparatus of growth cones. The surface of growth cones contains receptors for extracellular guidance cues that integrate this information into directional movement towards the target cell (Grzywa et al., 2006). Filamentous (F)-actin is the primary cytoskeletal element that maintains the growth cone shape and is essential for proper axon guidance, whereas microtubules are essential for giving the axon structure and serve an important function in axon elongation (Dent & Gertler, 2003). Thus growth cones act as sensors, signal transducers and motility devices and understanding their fate is a key question.

While the role of guidance molecules (Dickson, 2002) for growth cone movements, underlying signalling of both the F-actin and microtubules is well established (Dent & Gertler, 2003; Schaefer et al., 2002), relatively little is known about the three-dimensional structure of growth cones, which is difficult to determine *in vivo* in the mouse embryo and impossible in the human embryo (Keller, 2005).

Cellular three-dimensional structures can be analyzed with different imaging techniques. Despite the enormous advancements brought about by electron and scanning probe microscopy, about 80% of all microscopy investigations in the life sciences are still carried out with conventional lenses and visible light. Taking advantage of the optical transparency of cells, light microscopy uniquely provides noninvasive imaging of the interior of cells (Hell, 2007).

Discovered and first patented in 1957 by Marvin Minsky, confocal microscope, a predecessor to today's widely used **confocal laser scanning microscopy (CLSM)** has allowed a major advance in biological imaging of cell structure and physiology in thick specimens, in three dimensions and in time. CLSM offers high-quality three-dimensional fluorescent images over conventional wide-field optical microscopy, due to its ability to control the depth of a focal plane to eliminate out-of-focus light or glares in specimens and its capability to collect serial (i.e., z-stack) optical images for thick specimens without the need for physical sectioning of the tissue. In this way, thin, optical sections with greater resolution and contrast and with greater sensitivity than conventional, wide-field microscopes are produced (Dailey et al., 1999; Park et al., 2010; Pawley, 2006).

of different parts of the neural tube were successfully generated including spinal motoneurons, midbrain dopaminergic neurons, spinal cord interneurons, Purkinje and granule cells of cerebellum, hypothalamus and finally cortical pyramidal cells (Gaspard &