**2. Different types of mesenchymal stem cells**

Mesenchymal stem cells (MSCs) are plastic-adherent, fibroblast-like cells, which are conven‐ tionally able to self-renew and differentiate into tissues of the mesodermic lineage, such as bone, adipose tissue and cartilage. Whereas those cells have traditionally been isolated from bone marrow stroma, many reports have now described the presence of MSCs in a variety of fetal, perinatal and adult tissues, including peripheral blood, umbilical cord Wharton's Jelly and blood, fetal liver and lungs, adipose tissue, skeletal muscles, amniotic fluid, syno‐ vium and circulatory system, where they work as supportive cells and maintain tissue ho‐ meostasis.

#### **2.1. Bone marrow mesenchymal stem cells**

The initial study of non-hematopoietic bone marrow (BM) cells was performed by Frieden‐ stein et al. in the late 80's [4, 5]. After establishing single cell suspensions of BM, they showed that those cells were able to generate colonies of adherent fibroblast-shaped cells when cultured *in vitro*. They demonstrated that these colony-forming unit - fibroblasts (CFU-F) presented the ability to undergo osteogenic differentiation [5, 6]. These bone mar‐ row mesenchymal stem cells (BM-MSCs) were then demonstrated as multipotent progeni‐ tors that were able to self-renew [7] and could differentiate into any cell of the mesodermic lineage, like osteoblasts, chondrocytes, or adipocytes [8, 9]. More interestingly, it has been showed that BM-MSCs were able to "trans-differentiate" into cells with endodermal or ecto‐ dermal characteristics [10], and particularly into neuron-like cells [11-15]. These stem cells are therefore raising huge interest, since they represent a promising source of material for cell therapy protocols, such as mesenchymal tissue engineering or neurological disorder treatments as well.

**Figure 1.** Mesenchymal and neural crest stem cells from different perinatal and adult tissues. The upper part describes the presence of mesenchymal and/or neural crest stem cells in various adult tissue. The lower part describes cell fate that have been demonstrated for each type of stem cells regarding the mesodermic, endodermic or ectodermic cell

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The main debate concerning BM-MSCs resides in the lack of exact phenotypic characteriza‐ tion, due to the absence of specific membrane markers and non-standardized culture meth‐ ods. Consequently, several groups described BM-MSCs with a wide variety of different phenotypes: Verfaillie's group described a rare population of cells in human BM stroma as

lineage.

regard, mesenchymal stem cells (MSCs) and neural crest-derived stem cells (NCSCs) that can be found in various locations of the adult organism (and even in perinatal tissues) represent an important source of easily-accessible multipotent cells to use in a cell therapy

In this chapter, we will describe the major features of MSCs and NCSCs isolated from five different tissues, which constitute the main exploited and accessible sources for cell isolation in an objective of cell therapy protocols for neurological disorders (Figure 1). Moreover, we will detail the multiple ways they can generate neuron-like cells *in vitro*. Indeed, numerous culture conditions and differentiation protocols do exist and are demonstrated as efficient, supporting the fact that neural differentiation can occur through different cellular signaling mechanisms. Therefore, we will review the various signaling pathways that could trigger the neural fate adoption of MSCs and NCSCs, and the related cell-based therapy experi‐

Mesenchymal stem cells (MSCs) are plastic-adherent, fibroblast-like cells, which are conven‐ tionally able to self-renew and differentiate into tissues of the mesodermic lineage, such as bone, adipose tissue and cartilage. Whereas those cells have traditionally been isolated from bone marrow stroma, many reports have now described the presence of MSCs in a variety of fetal, perinatal and adult tissues, including peripheral blood, umbilical cord Wharton's Jelly and blood, fetal liver and lungs, adipose tissue, skeletal muscles, amniotic fluid, syno‐ vium and circulatory system, where they work as supportive cells and maintain tissue ho‐

The initial study of non-hematopoietic bone marrow (BM) cells was performed by Frieden‐ stein et al. in the late 80's [4, 5]. After establishing single cell suspensions of BM, they showed that those cells were able to generate colonies of adherent fibroblast-shaped cells when cultured *in vitro*. They demonstrated that these colony-forming unit - fibroblasts (CFU-F) presented the ability to undergo osteogenic differentiation [5, 6]. These bone mar‐ row mesenchymal stem cells (BM-MSCs) were then demonstrated as multipotent progeni‐ tors that were able to self-renew [7] and could differentiate into any cell of the mesodermic lineage, like osteoblasts, chondrocytes, or adipocytes [8, 9]. More interestingly, it has been showed that BM-MSCs were able to "trans-differentiate" into cells with endodermal or ecto‐ dermal characteristics [10], and particularly into neuron-like cells [11-15]. These stem cells are therefore raising huge interest, since they represent a promising source of material for cell therapy protocols, such as mesenchymal tissue engineering or neurological disorder

purpose.

meostasis.

treatments as well.

ments that have been done downstream.

328 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**2.1. Bone marrow mesenchymal stem cells**

**2. Different types of mesenchymal stem cells**

**Figure 1.** Mesenchymal and neural crest stem cells from different perinatal and adult tissues. The upper part describes the presence of mesenchymal and/or neural crest stem cells in various adult tissue. The lower part describes cell fate that have been demonstrated for each type of stem cells regarding the mesodermic, endodermic or ectodermic cell lineage.

The main debate concerning BM-MSCs resides in the lack of exact phenotypic characteriza‐ tion, due to the absence of specific membrane markers and non-standardized culture meth‐ ods. Consequently, several groups described BM-MSCs with a wide variety of different phenotypes: Verfaillie's group described a rare population of cells in human BM stroma as mesodermal adult progenitor cells (MAPCs)[12, 16]; D'Ippolito and collaborators cultured cells in low oxygen tension and characterized marrow isolated adult multilineage inducible (MIAMI) cells [17, 18]; whereas a lot of other groups kept the mesenchymal stem cell con‐ cept as defined by Pittenger et al. [9].

including melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and en‐

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In the past few years, multipotent and self renewing neural crest stem cells (NCSCs) have been described to persist in the adult organism. Those post-migratory NCSCs were found in the gut [35], the skin [36-38], the cornea [39], the heart [40], the teeth [41, 42], the dorsal root ganglion and the bone marrow [21, 22, 43]. As the skin, the teeth and the bone marrow con‐ stitute the most easily-accessible and available sources of NCSCs to use in therapy protocols,

Regarding the striking similarities of bone marrow stromal cells and embryonic NCSCs con‐ cerning their neural differentiation potential, the question of the presence of a neural crestderived cell subpopulation in bone marrow was raised. Indeed, the mesodermal origin of bone marrow stromal cells was definitely queried since a study of Takashima et al. demon‐ strated that a first wave of mesenchymal stem cells in the embryo derives from the Sox1-

Lately, convincing evidence for the subsistence of NCSCs in bone marrow emerged from a study by Nagoshi et al. They isolated neural crest-derived cells from the bone marrow using Wnt1-Cre/FloxedEGFP mice for *in vivo* fate mapping. Those cells could be propa‐ gated in sphere cultures for a couple of passages. A bit more than 3% of these isolated EGFP+ cells had the capacity to differentiate into neurons, glia, and smooth muscle cells (this proportion was sustainably increased by collagenase treatment, suggesting their tight contact with bone surface) [21, 22]. The same group used another transgenic mouse (P0-Cre/FloxedEGFP) to isolate NCSCs among the bone marrow stromal cells, and dem‐ onstrated their ability to differentiate into neural crest lineages but also into mesenchy‐ mal lineages such as adipocytes, chondrocytes and osteocytes [22]. Using a Wnt1-Cre/ FloxedLacZ transgenic mouse, the group of Wislet-Gendebien generated neural-crest de‐ rived clones of passage 5 bone marrow stromal cells, and compared them with mesen‐ chymal clones. They showed that the two types of populations were surprisingly similar at the transcriptomic level and in terms of differentiation abilities, and that both of them could give rise to neurons [43]. Altogether, the different results about bone marrow NCSCs make those cells as exciting as their MSCs neighbors in a context of therapy. Moreover, their neural crest origin may confer them particular additional properties in a

Using the same type of Wnt1-Cre reporter mice, the groups of Sieber-Blum and Toma identi‐ fied neural crest-derived stem cells in the facial skin of adult mice and humans [36, 37, 44, 45]. Those skin-derived precursors (SKPs) are located in the dermal papillae and in the hair follicle, and are able to differentiate *in vitro* into neurons, smooth muscle cells, Schwann cells

teric neuron, and glia.

we will describe those three tissues more precisely.

positive neuroepithelium through a neural crest stage [20].

**3.1. Bone marrow neural crest stem cells**

perspective of nervous system repair.

**3.2. Skin-derived neural crest stem cells**

and melanocytes.

In addition to the phenotypic differences of BM-MSCs which are inherent to culture settings, it has been demonstrated that BM stroma was a mixed population of cells arising from dif‐ ferent embryonic lineages. Although adult BM-MSCs were commonly considered to be of mesodermal origin (bone marrow mesenchymal stem cells) [19], several studies have shown that some adult BM-MSCs derive from the embryonic neural crest [20-24] (see paragraph be‐ low). Hence, the different studies that are detailed below often describe BM-MSCs without distinguishing mesenchymal and neural-crest derived cells.

#### **2.2. Adipose tissue stem cells**

Similarly to the main part of BM stroma, adipose tissue derives from mesodermic lineage and contains stem cells able to differentiate into bone, cartilage, fat and muscle. Likewise, adipose tissue mesenchymal stem cells (AT-MSCs) can adopt a neural-like phenotype [25, 26], which makes them another potential source of cells to use in replacement therapy.

#### **2.3. Umbilical cord blood and Wharton's jelly mesenchymal stem cells**

Wharton's jelly constitutes the gelatinous matrix of umbilical cord. Mainly composed of col‐ lagen fibers, proteoglycans and stromal cells, this tissue has also been described to enclose mesenchymal cells endowed with stem cells properties (WJ-MSCs); They are easily cultured and expanded *in vitro,* and are able to differentiate into a wide range of cell types, including neural cells [27-29].

Whereas adult peripheral blood only contains a tiny number of MSCs, umbilical cord blood is a richer source and allows to culture adherent MSCs more efficiently. These umbilical cord blood MSCs (UCB-MSCs) are considered as a more primitive population, but can be largely expanded and maintained in long term culture [30], and were described to be an os‐ teogenic, adipogenic, chondrogenic and even a neurogenic cell population [31-33]. Altogeth‐ er, these data confirmed umbilical cord as a new source of cells for cellular therapeutics for stromal, bone, and, potentially, neural repair [34].

## **3. Different types of neural crest stem cells**

During embryonic development of vertebrates, neural crest is specified at the border of the neural plate and the non-neural ectoderm after gastrulation. During neurulation, the neural folds both join at the dorsal midline to form the neural tube. Subsequently, neural crest cells (NCC) from the roof plate of the neural tube undergo an epithelial to mesenchymal transi‐ tion (EMT), delaminating from the neuroectoderm. Those multipotent NCC then migrate to‐ wards different locations in the body where they differentiate into various cell types, including melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and en‐ teric neuron, and glia.

In the past few years, multipotent and self renewing neural crest stem cells (NCSCs) have been described to persist in the adult organism. Those post-migratory NCSCs were found in the gut [35], the skin [36-38], the cornea [39], the heart [40], the teeth [41, 42], the dorsal root ganglion and the bone marrow [21, 22, 43]. As the skin, the teeth and the bone marrow con‐ stitute the most easily-accessible and available sources of NCSCs to use in therapy protocols, we will describe those three tissues more precisely.

#### **3.1. Bone marrow neural crest stem cells**

mesodermal adult progenitor cells (MAPCs)[12, 16]; D'Ippolito and collaborators cultured cells in low oxygen tension and characterized marrow isolated adult multilineage inducible (MIAMI) cells [17, 18]; whereas a lot of other groups kept the mesenchymal stem cell con‐

In addition to the phenotypic differences of BM-MSCs which are inherent to culture settings, it has been demonstrated that BM stroma was a mixed population of cells arising from dif‐ ferent embryonic lineages. Although adult BM-MSCs were commonly considered to be of mesodermal origin (bone marrow mesenchymal stem cells) [19], several studies have shown that some adult BM-MSCs derive from the embryonic neural crest [20-24] (see paragraph be‐ low). Hence, the different studies that are detailed below often describe BM-MSCs without

Similarly to the main part of BM stroma, adipose tissue derives from mesodermic lineage and contains stem cells able to differentiate into bone, cartilage, fat and muscle. Likewise, adipose tissue mesenchymal stem cells (AT-MSCs) can adopt a neural-like phenotype [25, 26], which makes them another potential source of cells to use in replacement therapy.

Wharton's jelly constitutes the gelatinous matrix of umbilical cord. Mainly composed of col‐ lagen fibers, proteoglycans and stromal cells, this tissue has also been described to enclose mesenchymal cells endowed with stem cells properties (WJ-MSCs); They are easily cultured and expanded *in vitro,* and are able to differentiate into a wide range of cell types, including

Whereas adult peripheral blood only contains a tiny number of MSCs, umbilical cord blood is a richer source and allows to culture adherent MSCs more efficiently. These umbilical cord blood MSCs (UCB-MSCs) are considered as a more primitive population, but can be largely expanded and maintained in long term culture [30], and were described to be an os‐ teogenic, adipogenic, chondrogenic and even a neurogenic cell population [31-33]. Altogeth‐ er, these data confirmed umbilical cord as a new source of cells for cellular therapeutics for

During embryonic development of vertebrates, neural crest is specified at the border of the neural plate and the non-neural ectoderm after gastrulation. During neurulation, the neural folds both join at the dorsal midline to form the neural tube. Subsequently, neural crest cells (NCC) from the roof plate of the neural tube undergo an epithelial to mesenchymal transi‐ tion (EMT), delaminating from the neuroectoderm. Those multipotent NCC then migrate to‐ wards different locations in the body where they differentiate into various cell types,

cept as defined by Pittenger et al. [9].

330 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**2.2. Adipose tissue stem cells**

neural cells [27-29].

distinguishing mesenchymal and neural-crest derived cells.

stromal, bone, and, potentially, neural repair [34].

**3. Different types of neural crest stem cells**

**2.3. Umbilical cord blood and Wharton's jelly mesenchymal stem cells**

Regarding the striking similarities of bone marrow stromal cells and embryonic NCSCs con‐ cerning their neural differentiation potential, the question of the presence of a neural crestderived cell subpopulation in bone marrow was raised. Indeed, the mesodermal origin of bone marrow stromal cells was definitely queried since a study of Takashima et al. demon‐ strated that a first wave of mesenchymal stem cells in the embryo derives from the Sox1 positive neuroepithelium through a neural crest stage [20].

Lately, convincing evidence for the subsistence of NCSCs in bone marrow emerged from a study by Nagoshi et al. They isolated neural crest-derived cells from the bone marrow using Wnt1-Cre/FloxedEGFP mice for *in vivo* fate mapping. Those cells could be propa‐ gated in sphere cultures for a couple of passages. A bit more than 3% of these isolated EGFP+ cells had the capacity to differentiate into neurons, glia, and smooth muscle cells (this proportion was sustainably increased by collagenase treatment, suggesting their tight contact with bone surface) [21, 22]. The same group used another transgenic mouse (P0-Cre/FloxedEGFP) to isolate NCSCs among the bone marrow stromal cells, and dem‐ onstrated their ability to differentiate into neural crest lineages but also into mesenchy‐ mal lineages such as adipocytes, chondrocytes and osteocytes [22]. Using a Wnt1-Cre/ FloxedLacZ transgenic mouse, the group of Wislet-Gendebien generated neural-crest de‐ rived clones of passage 5 bone marrow stromal cells, and compared them with mesen‐ chymal clones. They showed that the two types of populations were surprisingly similar at the transcriptomic level and in terms of differentiation abilities, and that both of them could give rise to neurons [43]. Altogether, the different results about bone marrow NCSCs make those cells as exciting as their MSCs neighbors in a context of therapy. Moreover, their neural crest origin may confer them particular additional properties in a perspective of nervous system repair.

#### **3.2. Skin-derived neural crest stem cells**

Using the same type of Wnt1-Cre reporter mice, the groups of Sieber-Blum and Toma identi‐ fied neural crest-derived stem cells in the facial skin of adult mice and humans [36, 37, 44, 45]. Those skin-derived precursors (SKPs) are located in the dermal papillae and in the hair follicle, and are able to differentiate *in vitro* into neurons, smooth muscle cells, Schwann cells and melanocytes.

Other skin-derived NCSCs, termed epidermal NCSCs (EPI-NCSCs) were isolated from the bulge region of whisker follicles. Similarly to SKPs, EPI-NCSCs can give rise to neurons, smooth muscle cells, Schwann cells and melanocytes [37, 44, 46]. As an easy-accessible au‐ tologous source of highly multipotent stem cells, the skin and its SKPs and EPI-NCSCs are of significant interest in cell therapy.

**4.1. Cyclic-adenosine-monophosphate and PKA signaling pathway**

and other processes like long term memory and neuronal plasticity [53-55].

is long-acting because of its resistance to degradation by phosphodiesterases.

rived cells showed significant voltage-dependent ionic currents (Na+

pathway control on differential phosphorylation of TH isoforms [60].

confirmed by several others studies [57,58].

Cyclic-adenosine-monophosphate (cAMP) is a well-known intracellular messenger, which is physiologically synthesized from adenosine-triphosphate by a membrane-anchored adenyl‐ yl cyclase, when this last is induced by an active G protein-coupled receptor. In the cyto‐ plasm, cAMP essentially activates the protein kinase A (PKA), which then reach the nucleus where it supports the phosphorylation of a transcription factor (cAMP responsive element binding protein, or CREB protein). Once phosphorylated, CREB protein binds CREB-bind‐ ing protein (CREBB protein or CBP), and with the support of different co-factors, join specif‐ ic DNA sequences and regulate the expression of different genes (coding for c-fos, brain derived neurotrophic factor (BDNF)[51], or tyrosine hydroxylase (TH)[52]). The destruction of intracellular cAMP is mediated by phosphodiesterases (PDE), which convert cAMP into AMP, then regulating cAMP cytoplasmic concentrations. This cAMP-dependent pathway has been demonstrated to be fundamental in embryonic development, neural cells survival

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cAMP is frequently used in culture media to induce MSCs/NCSCs into neural lineage, as well as other molecules which raise the intracellular cAMP levels. For example, forskolin ac‐ tivates adenylyl-cyclase, dibutyryl-cAMP (db-cAMP) and 3-isobutyl-1-methylxanthine (IBMX) both act as inhibitors of phosphodiesterases, and 8-bromo-cAMP activates PKA and

The main cytoplasmic target of cAMP, which is the PKA, has effectively been demonstrated to mediate neural differentiation. Wang et al. studied the impact of PKA activation on neu‐ rite outgrowth and on neural markers glial acidic fibrillary protein (GFAP) and neurofila‐ ment (NF) expression. They observed that the complete inactivation of this kinase led to a total absence of neural differentiation in UCB-MSCs, while the level of phosphorylated CREB was upregulated in forskolin-treated cells (this effect was inhibited in presence of PKA inhibitor) [56]. The involvement of PKA in the neural differentiation of MSCs was also

According to Lepski et al., neuronal differentiation of human BM-MSCs resulted from a spe‐ cific mechanism dependent upon the PKA pathway. Indeed, they demonstrated that the presence of a PKA inhibitor in the induction medium impaired the differentiation process (induced by IBMX, coupled with brain-derived neurotrophic factor (BDNF), see paragraph 3.4.), and that CREB was phosphorylated in differentiated MSCs [59]. Moreover, MSC-de‐

Besides, UCB-MSCs were induced to neural outcome with db-cAMP and IBMX treatment, which was demonstrated to be necessary and sufficient for neurite-like outgrowth and for nuclear receptor related 1 protein (Nurr1) expression. Nurr1 is known to play a key role in dopaminergic system maintenance. In addition, those data showed evidence for cAMP-

Lin et al. studied the ability of granulocyte-macrophage colony-stimulating-factor (GM-CSF) to promote neural differentiation in BM-MSCs through the phosphorylation of CREB [61]. Indeed, GM-CSF-treated BM-MSCs expressed higher levels of neuron specific enolase (NSE)

, K+

and Ca2+ currents).

#### **3.3. Dental neural crest stem cells**

The dental pulp is the connective tissue that forms the inner part of the teeth, and contains ondotoblasts which are responsible of dentin formation. Few years ago, a population of stem cells has been identified in dental pulp, and is thought to arise from the embryonic cra‐ nial neural crest [47, 48]. These dental pulp stem cells (DP-SCs) are endowed with high pro‐ liferative potential, self-renewal ability and multi-lineage differentiation [42, 49], making them an attractive tool for stem cell therapeutic strategies. Whereas the DP-SCs are isolated from the adult teeth, the same type of stem cells can be found in the human exfoliated decid‐ uous teeth (SHED cells), identified as immature DP-SCs.

The properties of self-renewal and multi-lineage differentiation ability of all described stem cells make them truly attractive candidates for cell therapy. Furthermore, they offer the nonnegligible advantage of being easily obtained without invasive method. Indeed, whereas umbilical cord is usually intended to trash after birth and can rather be preserved in order to collect cells, bone marrow aspiration, lipo-aspiration, or teeth extraction are non-heavy pro‐ cedures that are commonly performed in clinical context. Those procedures could even be performed in patients when needed, allowing autologous grafts and avoiding immunologi‐ cal rejects. Additionally, the use of MSCs/NCSCs, from either adult origin or isolated from umbilical cord, get round the ethical problems related to fetal cells use. Moreover, those cells are supposed to be safer than embryonic stem cells or induced pluripotent stem cells in terms of tumorigenicity and genomic modifications [24, 50].
