**4. Signaling pathways involved in neural differentiation of MSCs and NCSCs**

As already mentioned, a wide range of culture settings for MSCs/NCSCs neural differentia‐ tion were commonly experienced, with some of them giving rise to substantial results. Hence, it can be inferred that MSCs/NCSCs can give rise to neuronal cells through the acti‐ vation/deactivation of many intracellular signaling pathways. In this chapter, we will focus on the analysis of several differentiating protocols that highlighted specific signaling path‐ ways in neuronal fate decision. We will dissect those pathways from the ligand molecules to the different cellular and molecular effectors that are involved within, up to the gene expres‐ sion modulation. We will then describe the different downstream effects experimentally ob‐ served after activation of those pathways, in the context of neural fate adoption by different type of MSCs/NCSCs and summarized in Figure 2 and Table 1.

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

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

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‐

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

**4. Signaling pathways involved in neural differentiation of MSCs and**

As already mentioned, a wide range of culture settings for MSCs/NCSCs neural differentia‐ tion were commonly experienced, with some of them giving rise to substantial results. Hence, it can be inferred that MSCs/NCSCs can give rise to neuronal cells through the acti‐ vation/deactivation of many intracellular signaling pathways. In this chapter, we will focus on the analysis of several differentiating protocols that highlighted specific signaling path‐ ways in neuronal fate decision. We will dissect those pathways from the ligand molecules to the different cellular and molecular effectors that are involved within, up to the gene expres‐ sion modulation. We will then describe the different downstream effects experimentally ob‐ served after activation of those pathways, in the context of neural fate adoption by different

of significant interest in cell therapy.

332 Trends in Cell Signaling Pathways in Neuronal Fate Decision

**3.3. Dental neural crest stem cells**

**NCSCs**

uous teeth (SHED cells), identified as immature DP-SCs.

terms of tumorigenicity and genomic modifications [24, 50].

type of MSCs/NCSCs and summarized in Figure 2 and Table 1.

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

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

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 confirmed by several others studies [57,58].

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‐ rived cells showed significant voltage-dependent ionic currents (Na+ , K+ and Ca2+ currents).

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

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) over time, whereas transiently increased nestin expression. In parallel, a substantial increase of phosphorylated-CREB level was observed in the GM-CSF-treated BM-MSCs compared with control (CREB levels were not different between the two groups), and that the kinetics of this increase was consistent with the progress in neural differentiation.

*Cell type Passage Pathways*

**MSCs** ns cAMP-PKA-

P12-P24

P3-P6

**MSCs** ns cAMP

**MSCs** ns NT, SHH

P3-P8 cAMP, RA, SHH

P3-P5 cAMP, NT, RA, SHH

P3-P9 cAMP, NT, RA, SHH

RA, SHH

P5-P8 cAMP-PKA, NT, RA

P4 cAMP, PKC, RA

**MSCs** ns cAMP, NT,

CREB

cAMP-PKA-CREB, NT

cAMP-PKA-CREB

P0? cAMP-PKA Forskolin,

**1**

**2**

**3**

**4**

**5**

**6**

**7**

**8**

**9**

**10 AT-**

**11 UCB-MSCs**

**12 BM-MSCs**

**UCB-**

**BM-MSCs**

**BM-MSCs**

**BM-MSCs**

**BM-**

**BM-**

**BM-MSCs**

**BM-MSCs**

**MIAMI cells**

*Induction protocol*

1) EGF, bFGF. 2) cAMP, IBMX, BDNF

GM-CSF

IBMX

Forskolin / 8-bromocAMP

SHH, FGF8, bFGF (+ BDNF)

Forskolin, SHH and/or RA

1) bFGF, Forskolin. 2) Forskolin, IBMX, RA, SHH, BDNF

1) bFGF. 2) NT-3, SHH, RA, FGF8. 3) Forskolin, NT-3, BDNF, NGF, GDNF.

bFGF, IBMX. 2) SHH,RA. 3) BDNF, GNDF

RA, IBMX, NGF, bFGF

Forskolin, IBMX, TPA / RA

*Protocol length*

1) 1 week. 2) 12 hours

6 to 96 hours

1 hour to 2 days

6 and 24 hours / 1 and 4 days

2 days

7 days

1) 24h. 2- 2 days. 3) 3-7 days

6 hours. 2) 1 week. 3) ns

8 hours to 7 days

Up to 48 hours / 7 days

*Neural phenotype*

Forskolin 1 to 7 days NF, GFAP No H89,

NF-200, NFM, NeuroD, MAP2, NeuN, GABA

Nestin, NSE, GFAP

βIII-tubulin, GFAP, NSE

tubulin, DAT

Nestin, Sox2, NSE, GFAP, synapsin, ACh

GATA3, Sox10, GluR4, Irx2, calretinin, MAP2, NeuN, βIII-tubulin

NSE, GFAP, βIII-tubulin, NF-L, NF-M, Nurr1, TH

βIII-tubulin, ChAT, Nkx2.2, Pax6, Hb9, Olig2

GFAP, NF-L, NF-M, NF-H, NSE, Nurr-1, TH, Tau

βIII-tubulin, GFAP, NSE, NF-

M

12 days NeuN, TH, βIII-

*Electrophysiological profile*

Neural Fate of Mesenchymal Stem Cells and Neural Crest Stem Cells ...

Inward Na+ currents and outward K+ currents

βIII-tubulin No No [63]

Inward Na+ currents and outward K+ currents

Neuronal resting potential

Inward Na+ currents and outward K+ currents

*Inhibitor Reference*

335

[59]

U0126 [56]

http://dx.doi.org/10.5772/53260

No [65, 66]

No [67]

No [69]

PKAi fragment 6–22 amide

No No [61]

No H89 [62]

No No [68]

No No [70]

No H89 [60]

No No [71]

**Figure 2.** Different signaling pathways are involved in neural differentiation of MSCs and NCSCs.Each pathway and modulating substances are described in the text.

While cAMP pathway is supposed to be involved in neural differentiation of MSCs, its pre‐ cise role in this differentiation process still needs to be defined. Zhang et al. showed evi‐ dence for the involvement of cAMP in two differentially-regulated processes, which are early transient neuron-like morphology changes, like cytoskeleton rearrangement, and later neural markers expression associated with neuronal function, but demonstrated that cAMPtreated BM-MSCs did not achieve complete differentiation [62]. Another hypothesis was made by Rooney et al., who examined the effect of intracellular cAMP elevation on BM-MSCs' fate [63]. They demonstrated that forskolin and 8-bromo-cAMP induced a transient increase in βIII-tubulin expression and changes in cell morphology, but no expression of growth associated protein 43 (GAP-43) [64] was seen in the neural-like BM-MSCs, excluding authentic neurite formation. They therefore concluded that this effect was mostly due to a modification of culture conditions rather than in a differentiation process.


over time, whereas transiently increased nestin expression. In parallel, a substantial increase of phosphorylated-CREB level was observed in the GM-CSF-treated BM-MSCs compared with control (CREB levels were not different between the two groups), and that the kinetics

**Figure 2.** Different signaling pathways are involved in neural differentiation of MSCs and NCSCs.Each pathway and

While cAMP pathway is supposed to be involved in neural differentiation of MSCs, its pre‐ cise role in this differentiation process still needs to be defined. Zhang et al. showed evi‐ dence for the involvement of cAMP in two differentially-regulated processes, which are early transient neuron-like morphology changes, like cytoskeleton rearrangement, and later neural markers expression associated with neuronal function, but demonstrated that cAMPtreated BM-MSCs did not achieve complete differentiation [62]. Another hypothesis was made by Rooney et al., who examined the effect of intracellular cAMP elevation on BM-MSCs' fate [63]. They demonstrated that forskolin and 8-bromo-cAMP induced a transient increase in βIII-tubulin expression and changes in cell morphology, but no expression of growth associated protein 43 (GAP-43) [64] was seen in the neural-like BM-MSCs, excluding authentic neurite formation. They therefore concluded that this effect was mostly due to a

modification of culture conditions rather than in a differentiation process.

modulating substances are described in the text.

of this increase was consistent with the progress in neural differentiation.

334 Trends in Cell Signaling Pathways in Neuronal Fate Decision


*Cell type Passage Pathways*

**MSCs** ns cAMP-NT

**<sup>25</sup> DP-SCs** P1-P4 cAMP, PKC,

**<sup>27</sup> SKPs** ns cAMP, NT,

P3-P4

RA

**<sup>26</sup> SKPs** P3 NT, RA 1) RA. 2)

RA

cAMP-MAPK-MEK-ERK-Raf

cAMP-PKA, PKC, MAPK-MEK-ERK

P0? cAMP-Wnt

**23 AT-**

**24**

**29 BM-MSCs**

**30 BM-MSCs** ns

**31 BM-MSCs**

**BM-MSCs & DP-SCs**

*Induction protocol*

BDNF, IBMX 3-5 days.

1) bFGF, 5 azacytidine. 2) bFGF, IBMX, TPA, db-cAMP, Forskolin, NT-3, NGF. 3) db-cAMP, NT-3.

NT-3

1) RA, NT-3, BDNF, NGF, db-cAMP

1) bFGF. 2) Forskolin.

bFGF, IBMX, Forskolin, Wnt1

1) bFGF, EGF, BDNF. 2) IBMX

P3 cAMP-NT bFGF, EGF,

*Protocol length*

1) 3 days. 2) 48 hours

1) 48 hours. 2) 3 days. 3) 3-7 days.

1) 7 days. 2) 7 days.

2 weeks to 1 month

Forskolin 48 hours βIII-tubulin,

3 to 7 days

**Table 1.** In vitro protocols for neural differentiation of different types of MSCs/NCSCs and detailed results.

("ns" indicates that the passage or the incubation length are not specified. "No" indicates that no electrophysiological results are described in the study or that no inhibitor has been tested to confirm the pathway). **Hedgehog signaling pathway**

1) Overnight. 2) Up to 7 days

*Neural phenotype*

βIII-tubulin, GFAP

GFAP, c-fos, NF, HNK1, enolase-2, βIIItubulin, MAP2, Sox2, Tenascin-C, Connexin-43 and nestin

βIII-tubulin, NF-M, GFAP, NeuN, NSE

βIII-tubulin, GFAP, MAP-2, NeuN

PGP9.5, NF, FMI-43, NMDAR

NF200, NSE

NF No

Ngn1, Brn3a, NeuroD, P2X3, GluR2, GluR4

*Electrophysiological profile*

Neural Fate of Mesenchymal Stem Cells and Neural Crest Stem Cells ...

Inward Na+ currents and outward K+ currents

No K252a,

No No [83]

No PD98059 [58]

No No [84]

K252a, KT5720, AG879, KN-62, LY294002, PD98059

[57]

No No [79]

No No [80]

No [81]

Pep5 [82]

*Inhibitor Reference*

337

http://dx.doi.org/10.5772/53260


*Cell type Passage Pathways*

**14 DP-SCs** P0? RA

P3-P4 cAMP, RA (RARβ)

336 Trends in Cell Signaling Pathways in Neuronal Fate Decision

P5-P9 cAMP, NT

P5 clones MAPKs

**<sup>19</sup> SKPs** ns NT 1) bFGF,

P3 NT

NT-Raf1- MAPK/ERK, RA

NT (TrkB)- Raf1-MAPK-ERK, Bcatenin

NT (TrkC)- Rac1- Mek1/2- Erk1/2

**13 BM-MSCs**

**15**

**16**

**18 BM-NCSCs**

**20 UCB-MSCs** ns

**21 UCB-MSCs** ns

**22 WJ-MSCs**

**MIAMI cells**

**MIAMI cells** ns

**17 SKPs** P3-P9 NT

*Induction protocol*

1) RA. 2) Forskolin

1) EGF, bFGF. 2) bFGF. 3) bFGF, RA

1) EGF, bFGF. 2) bFGF. 3) bFGF, NT-3. 4) 3) Forskolin, NT-3, BDNF, NGF.

1) bFGF. 2) NT-3.

1) bFGF, EGF. 2) NT-3, NGF, BDNF

Co-culture

bFGF, RA, BDNF 7 days

BDNF / HCNP / rDHE

BDNF gene transfection

*Protocol length*

1) 24 hours. 2) ns

1) 7 days. 2) 7 days. 3) 7 days.

1) 10 days. 2) 24 hours. 3) 2 days. 4) 3 days

1) 24 hours. 2) 48 hours

1) 2-3 weeks. 2) 2-3 weeks.

with CGN 5 days βIII-tubulin

EGF. 2) NGF ns NF-M, βIII-

*Neural phenotype*

Nestin, NSE, MAP2

Nestin, βIIItubulin, NF-M, NFH, PSA-NCAM

βIII-tubulin, NF-M, NF-H, NF-L, GalC

βIII-tubulin, NF-M, NF-H, NF-L, Nestin

NF-M, GAP43, βIII-tubulin, MAP2

tubulin, GFAP

βIII-tubulin, NeuN, GFAP, MBP

βIII-tubulin, NeuN, GFAP, MBP

14 days MAP2, AChT No No [78]

No

Inward Na+ currents and outward K+ currents (PA for BM-MSCs clones)

*Electrophysiological profile*

Neuronal resting potential

Inward Na+ currents

*Inhibitor Reference*

No [72]

No [41]

No No [73]

U0126, K252a, NSC23766

No No [37]

No No [75]

No K252A [77]

No LY294002,

PD98059 [43]

PD98059 [76]

[74]

("ns" indicates that the passage or the incubation length are not specified. "No" indicates that no electrophysiological results are described in the study or that no inhibitor has been tested to confirm the pathway). **Hedgehog signaling pathway**

**Table 1.** In vitro protocols for neural differentiation of different types of MSCs/NCSCs and detailed results.

The hedgehog-mediated signalization is involved in the embryonic development, and its main ligand, Sonic Hedgehog (SHH) plays a role of morphogenic factor, defining which fate has to be applied to cells at each place of the embryo: its role in the nervous system organi‐ zation is crucial and depends on precise concentration gradients which are essential for a correct patterning of the embryo. Indeed, SHH signaling is the chief actor in the definition of the dorsoventral axis of the nervous system. This signalization mostly takes place in cell cil‐ ia, and implies two different receptors that, once activated, generate a tricky reorganization of cytoplasmic protein complexes. SHH binds its receptor Patched1 (Ptc), relieving Ptcmediated inhibition of a second receptor Smoothened (Smo): Ptc leaves the cilium where Smo then accumulates and induces the activation of Gli family of transcription factors (Gli1, Gli2, and Gli3) [85, 86].

**4.2. Retinoic acid signaling pathway**

dopaminergic profile.

Retinoic acid (RA) is physiologically metabolized from retinol, thanks to the sequential ac‐ tion of cellular retinol-binding protein (CRBP), retinol dehydrogenase (RoDH) and retinal‐ dehyde dehydrogenases (RALDHs). Once in the cytoplasm, RA is bound by cellular RAbinding protein (CRABP) and enters the nucleus to bind its specific receptors (RARs) and the retinoid X receptors (RXRs), which themselves heterodimerize and bind to DNA sequen‐ ces known as the RAREs (RA-response elements). This activates transcription of target genes (Hox genes, Oct4,...) [87]. This RA signalization is involved in brain development and more particularly in the definition of the anterio-posterior axis of the nervous system, by regulat‐

Neural Fate of Mesenchymal Stem Cells and Neural Crest Stem Cells ...

http://dx.doi.org/10.5772/53260

339

Whereas SHH was often coupled with RA in cell culture differentiation medium, the impli‐ cation of RA signaling in neuronal differentiation was also studied without a combination with SHH. RA was demonstrated to act on the up-regulation of NF-L expression in UCB-MSCs, while applied with IBMX, db-cAMP, nerve growth factor (NGF) and bFGF [60]. This study also highlighted the role of RA and cAMP/PKA pathways in the differential phos‐ phorylation of TH during differentiation. Indeed, neurally differentiated cells express neu‐ ronal markers as Tau or NSE, whereas TH and Nurr-1 expression assessed their

In the study of Scintu et al., two different protocols were used to differentiate BM-MSCs into neuronal cells. The first one was carried out by activating the cAMP and PKC pathway (with forskolin, TPA and IBMX), whereas the second one consisted in RA treatment. Both

Similarly, pre-treatment with RA before incubation with forskolin leaded BM-MSCs to ex‐ press higher levels of nestin, NSE, and microtubule associated protein 2 (MAP2) and exhibit neural-like resting membrane potential and increased intracellular calcium concentration [72]. They also demonstrated that only RA specific receptors RARa and RARc were ex‐ pressed in native BM-MSCs. Conversely, the expression of RARb was significantly increased

Arthur et al. performed RA treatment of human DP-SCs, which subsequently showed neu‐ ral morphology and expression of βIII-tubulin, NF-M and NF-H, and more interestingly ex‐ hibited electrophysiological activity characteristic of sodium voltage-gated channels,

Neurotrophins are secreted growth factors that are involved in the development of neurons in the nervous system, as well as in their survival and functionality. This family of proteins is constituted by the "prototypical" nerve growth factor (NGF), brain-derived growth factor (BDNF), and the neurotrophins (NT-) 3, 4/5 and 6. Those members promote neural cells to survive, grow, differentiate and function through the activation of high-affinity tyrosinekinase (tropomyosin-related) receptors (TrkA, TrkB and TrkC are respectively bound by NGF/NT-6, BDNF/NT-4, and NT-3), and through the activation of a common low-affinity re‐

protocols leaded to NSE, βIII-tubulin, GFAP and NF positive cells [71].

in differentiated neurons, suggesting its major role in neural differentiation.

assessing for their potential ability to give rise to functional neurons [41].

**4.3. Neurotrophic factors and downstream signaling pathways**

ing the expression of Hox genes in defined localized domains of the embryo [88].

SHH was used to induce dopaminergic differentiation of human BM-MSCs, as demonstrat‐ ed by Trzaska et al. After a 12-days incubation with SHH, coupled with fibroblast growth factor 8 (FGF8) and basic fibroblast growth factor (bFGF), a great number of cells turned into putative dopaminergic neurons, as showed by tyrosine hydroxylase (TH) expression and electrophysiological features. Those cells showed higher expression of neuronal markers, and downregulated genes which are involved in cell cycle regulation, like cyclin-dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA), indicating that they entered a post-mitotic fate [65, 66].

Qi and al. analyzed the abilities of rhesus monkey BM-MSCs to differentiate into cholinergic neural cells. While SHH alone in the culture medium did not trigger any modification of resting potential, they demonstrated that BM-MSCs exhibited neuronal resting membrane potential when retinoic acid (RA) was present in the culture medium, and under the combi‐ nation of both SHH and RA. Moreover, cells from the SHH+RA inducing group expressed higher levels of synapsin and acetylcholine (ACh), indicating that the combination of both signals was the best way to obtain cholinergic neurons [67].

Many other studies demonstrated a synergic role of SHH and RA in neural induction of BM-MSCs. Kondo et al. identified those two signals as sensory factors, showing that SHH+RA application leaded to the expression of glutamatergic sensory neuron markers (including GATA3, Sox10 or GluR4) by treated BM-MSCs [68]. On the other hand, the combination of SHH+RA added to FGF8 (before neurotrophin incubation) was showed to promote dopami‐ nergic fate in MIAMI cells [69], which expressed TH and other molecules involved in dopa‐ minergic differenciation, like Nurr1.

Human AT-MSCs were induced to neural differentiation through the action of SHH and RA. After induction, immunochemical labeling showed βIII-tubulin, choline acetyltransfer‐ ase (ChAT), and NSE expression. The differentiated cells were then characterized by RT-PCR and results showed that those cells were restricted to a ventral spinal fate (Nkx2.2, Pax6, Hb9, and Olig2), suggesting that those cells could be good candidates for motoneur‐ ons generation, in the context of spinal cord injuries therapy [70].

#### **4.2. Retinoic acid signaling pathway**

The hedgehog-mediated signalization is involved in the embryonic development, and its main ligand, Sonic Hedgehog (SHH) plays a role of morphogenic factor, defining which fate has to be applied to cells at each place of the embryo: its role in the nervous system organi‐ zation is crucial and depends on precise concentration gradients which are essential for a correct patterning of the embryo. Indeed, SHH signaling is the chief actor in the definition of the dorsoventral axis of the nervous system. This signalization mostly takes place in cell cil‐ ia, and implies two different receptors that, once activated, generate a tricky reorganization of cytoplasmic protein complexes. SHH binds its receptor Patched1 (Ptc), relieving Ptcmediated inhibition of a second receptor Smoothened (Smo): Ptc leaves the cilium where Smo then accumulates and induces the activation of Gli family of transcription factors (Gli1,

SHH was used to induce dopaminergic differentiation of human BM-MSCs, as demonstrat‐ ed by Trzaska et al. After a 12-days incubation with SHH, coupled with fibroblast growth factor 8 (FGF8) and basic fibroblast growth factor (bFGF), a great number of cells turned into putative dopaminergic neurons, as showed by tyrosine hydroxylase (TH) expression and electrophysiological features. Those cells showed higher expression of neuronal markers, and downregulated genes which are involved in cell cycle regulation, like cyclin-dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA), indicating that they entered

Qi and al. analyzed the abilities of rhesus monkey BM-MSCs to differentiate into cholinergic neural cells. While SHH alone in the culture medium did not trigger any modification of resting potential, they demonstrated that BM-MSCs exhibited neuronal resting membrane potential when retinoic acid (RA) was present in the culture medium, and under the combi‐ nation of both SHH and RA. Moreover, cells from the SHH+RA inducing group expressed higher levels of synapsin and acetylcholine (ACh), indicating that the combination of both

Many other studies demonstrated a synergic role of SHH and RA in neural induction of BM-MSCs. Kondo et al. identified those two signals as sensory factors, showing that SHH+RA application leaded to the expression of glutamatergic sensory neuron markers (including GATA3, Sox10 or GluR4) by treated BM-MSCs [68]. On the other hand, the combination of SHH+RA added to FGF8 (before neurotrophin incubation) was showed to promote dopami‐ nergic fate in MIAMI cells [69], which expressed TH and other molecules involved in dopa‐

Human AT-MSCs were induced to neural differentiation through the action of SHH and RA. After induction, immunochemical labeling showed βIII-tubulin, choline acetyltransfer‐ ase (ChAT), and NSE expression. The differentiated cells were then characterized by RT-PCR and results showed that those cells were restricted to a ventral spinal fate (Nkx2.2, Pax6, Hb9, and Olig2), suggesting that those cells could be good candidates for motoneur‐

signals was the best way to obtain cholinergic neurons [67].

ons generation, in the context of spinal cord injuries therapy [70].

Gli2, and Gli3) [85, 86].

338 Trends in Cell Signaling Pathways in Neuronal Fate Decision

a post-mitotic fate [65, 66].

minergic differenciation, like Nurr1.

Retinoic acid (RA) is physiologically metabolized from retinol, thanks to the sequential ac‐ tion of cellular retinol-binding protein (CRBP), retinol dehydrogenase (RoDH) and retinal‐ dehyde dehydrogenases (RALDHs). Once in the cytoplasm, RA is bound by cellular RAbinding protein (CRABP) and enters the nucleus to bind its specific receptors (RARs) and the retinoid X receptors (RXRs), which themselves heterodimerize and bind to DNA sequen‐ ces known as the RAREs (RA-response elements). This activates transcription of target genes (Hox genes, Oct4,...) [87]. This RA signalization is involved in brain development and more particularly in the definition of the anterio-posterior axis of the nervous system, by regulat‐ ing the expression of Hox genes in defined localized domains of the embryo [88].

Whereas SHH was often coupled with RA in cell culture differentiation medium, the impli‐ cation of RA signaling in neuronal differentiation was also studied without a combination with SHH. RA was demonstrated to act on the up-regulation of NF-L expression in UCB-MSCs, while applied with IBMX, db-cAMP, nerve growth factor (NGF) and bFGF [60]. This study also highlighted the role of RA and cAMP/PKA pathways in the differential phos‐ phorylation of TH during differentiation. Indeed, neurally differentiated cells express neu‐ ronal markers as Tau or NSE, whereas TH and Nurr-1 expression assessed their dopaminergic profile.

In the study of Scintu et al., two different protocols were used to differentiate BM-MSCs into neuronal cells. The first one was carried out by activating the cAMP and PKC pathway (with forskolin, TPA and IBMX), whereas the second one consisted in RA treatment. Both protocols leaded to NSE, βIII-tubulin, GFAP and NF positive cells [71].

Similarly, pre-treatment with RA before incubation with forskolin leaded BM-MSCs to ex‐ press higher levels of nestin, NSE, and microtubule associated protein 2 (MAP2) and exhibit neural-like resting membrane potential and increased intracellular calcium concentration [72]. They also demonstrated that only RA specific receptors RARa and RARc were ex‐ pressed in native BM-MSCs. Conversely, the expression of RARb was significantly increased in differentiated neurons, suggesting its major role in neural differentiation.

Arthur et al. performed RA treatment of human DP-SCs, which subsequently showed neu‐ ral morphology and expression of βIII-tubulin, NF-M and NF-H, and more interestingly ex‐ hibited electrophysiological activity characteristic of sodium voltage-gated channels, assessing for their potential ability to give rise to functional neurons [41].

#### **4.3. Neurotrophic factors and downstream signaling pathways**

Neurotrophins are secreted growth factors that are involved in the development of neurons in the nervous system, as well as in their survival and functionality. This family of proteins is constituted by the "prototypical" nerve growth factor (NGF), brain-derived growth factor (BDNF), and the neurotrophins (NT-) 3, 4/5 and 6. Those members promote neural cells to survive, grow, differentiate and function through the activation of high-affinity tyrosinekinase (tropomyosin-related) receptors (TrkA, TrkB and TrkC are respectively bound by NGF/NT-6, BDNF/NT-4, and NT-3), and through the activation of a common low-affinity re‐ ceptor, the p75LNR, which has no intrinsic kinase property. While p75LNR activation is suffi‐ cient to induce events like neurite formation, its role seems to facilitate the binding of neurotrophins to Trk receptors. After trans-phosphorylation, Trk receptors function as acti‐ vators of three main signaling pathways, respectively mitogen-activated protein kinases (MAPKs), phospholipase C (PLC) and phosphatidyl-inositol-3-kinase (PI3K) [89,90].

The first assessment of the ability of SKPs to generate neural cells was achieved by the group of Toma, who first described that about 9,4% of SKPs were NF-M, GAP43, βIII-tubu‐

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When co-cultivated with immature cerebellar granules, nestin-positive BM-MSCs are able to differentiate into functional neuronal cells [93], as showed by βIII-tubulin expression and ac‐ tion potentials firings. After characterizing two subpopulations of BM-MSCs which were re‐ spectively derived and non-derived from the embryonic neural crest (after generating clonal cultures), Wislet-Gendebien and collaborators demonstrated that the neural differentiation of BM-NCSCs in co-culture conditions was abolished in the presence of a MAPKs inhibitor. Those results confirmed the importance of MAPKs pathway in neural differentiation of

After adding NGF in the culture medium of human SKPs, Bakthiari et al. obtained NF-M, βIII-tubulin, S100 and GFAP positive cells, and that after different conditions of cryopreser‐

Lim et al. demonstrated the important role of BDNF in neural differentiation of human UCB-MSCs, and provided a deeper study of the different involved pathways. Addition of BDNF in the induction medium leaded to the phosphorylation of Raf-1 and ERK, then to the downstream up-regulation of p35 expression, which was not observed when ERK was blocked with a specific inhibitor. p35 is known as an anti-apoptotic factor able to block pro‐

They also analyzed the contribution of BDNF to cell viability, and demonstrated an up-regu‐ lation of the anti-apoptotic gene Bcl2, which was mediated by the activity of PI3K and Akt phosphorylation [76]. Few years later, after genetically modifying UCB-MSCs through trans‐ fection with a BDNF-expressing plasmid, they showed the expression of βIII-tubulin, NeuN, GFAP and myelin basic protein (MBP) in differentiated cells, associated with an up-regulat‐

BDNF was also used to induce bi-multipolar morphology and MAP2 expression in WJ-MSCs. When used in combination with hippocampal cholinergic neurostimulating peptide (HCNP) and/or denervated hippocampal extract (rDHE), WJ-MSCs turned into Choline-ace‐

Coupled with IBMX, BDNF was able to induce GFAP and βIII-tubulin expression in AT-MSCs, as demonstrated by Ying et al. [79]. The same combination of IBMX and BDNF was used to differentiate DP-SCs into GFAP, c-fos, NF, HNK1, enolase-2, βIII-tubulin, MAP2,

Neural induction of DP-SCs was achieved by Kiraly et al. through the activation of both cAMP and PKC signaling pathways. After reprogramming by 5-azacytidine treatment, cells where treated with IBMX, db-cAMP, forskolin, TPA, NGF and NT-3, and showed an in‐ crease in neurogenin-2, βIII-tubulin, NSE, NF-M and GFAP expression, while electrophysio‐

logical recordings revealed voltage dependent sodium channels activity [81].

lin and MAP2 positive after being treated with NGF, BDNF and NT-3 [37].

caspase maturation and to protect neurons from cell death [94, 95].

ed phosphorylation level of TrkB, Raf-1 and ERKs [77].

Sox2, Tenascin-C, Connexin-43 and nestin positive cells [80].

tyltransferase (ChAT) positive cells [96].

adult BM-NCSCs [43].

vation [75].

Briefly, the MAPKs pathway consists in a set of sequentially-activated kinase proteins grouped in three main connected cascades, involving regulators of alpha-foetoproteins (Raf), extracellular-regulated kinases (ERK), p38 or jun-kinase 1/2/3, resulting in the phos‐ phorylation of transcription factors and then regulating gene expression. MAPKs are abundantly expressed in the central nervous system (CNS), and ERKs are known to be involved in different processes, including neuronal maturation, survival, and synaptic functions.

The PLC signalization pathway mostly induces intracellular calcium mobilization, but fur‐ thermore stimulates protein kinase C (PKC) via the production of diacylglycerol (DAG). The PKC can also be directly activated by 12-*O*-tetradecanoylphorbol-13-acetate (TPA), and is al‐ so able to activate MAPKs pathway.

Finally, PI3K controls another downstream kinase called Akt (also named protein kinase B or PKB) that is a crucial player in cell survival through the regulation of apoptosis among other roles.

Several additional neurotrophic factors are regrouped in a second family, the GDNF family of ligands (GFL). Briefly, the glial neurotrophic factor (GDNF) is a protein that has been shown to promote the survival and differentiation of dopaminergic neurons and motoneur‐ ons, and constitutes then an important potential actor in the management of neurological diseases such as Parkinson's disease [91, 92], amyotrophic lateral sclerosis or spinal cord in‐ juries. Neurturin, artemin and persephin are similar ligands that also play a role in cell sur‐ vival, neurite outgrowth, and cell differentiation migration. Those four members act by the activation of a tyrosine kinase receptor RET, which in association with a co-receptor (GFRα), triggers auto-transphosphorylation and downstream signaling processes.

A cocktail of the three main neurotrophins (BDNF, NGF and NT-3) was used to direct neu‐ ral differentiation of MIAMI cells, which began to develop a complex neuritic arborization and to express neuronal markers, e.g. NF-L or NeuN [69]. Moreover, those differentiated cells showed inward Na+ and outward K+ currents. This study also highlighted the impor‐ tance of NT-3 in the neural commitment and differentiation step, through the demonstration of a dramatic decrease in viability, βIII-tubulin expression, neuron-like morphology and branching of differentiated cells without NT-3 treatment. Those results were then further de‐ tailed and showed the enhanced neural specification from MIAMI cells thanks to epidermal growth factor (EGF) and bFGF pre-treatment [73]; the implication of Rho-GTPase Rac1, which was thought to regulate Mek1/2-Erk1/2 phosphorylation, mediating transcription of genes involved in neural differentiation versus proliferation during NT-3-induced neuronal commitment [74].

The first assessment of the ability of SKPs to generate neural cells was achieved by the group of Toma, who first described that about 9,4% of SKPs were NF-M, GAP43, βIII-tubu‐ lin and MAP2 positive after being treated with NGF, BDNF and NT-3 [37].

ceptor, the p75LNR, which has no intrinsic kinase property. While p75LNR activation is suffi‐ cient to induce events like neurite formation, its role seems to facilitate the binding of neurotrophins to Trk receptors. After trans-phosphorylation, Trk receptors function as acti‐ vators of three main signaling pathways, respectively mitogen-activated protein kinases

Briefly, the MAPKs pathway consists in a set of sequentially-activated kinase proteins grouped in three main connected cascades, involving regulators of alpha-foetoproteins (Raf), extracellular-regulated kinases (ERK), p38 or jun-kinase 1/2/3, resulting in the phos‐ phorylation of transcription factors and then regulating gene expression. MAPKs are abundantly expressed in the central nervous system (CNS), and ERKs are known to be involved in different processes, including neuronal maturation, survival, and synaptic

The PLC signalization pathway mostly induces intracellular calcium mobilization, but fur‐ thermore stimulates protein kinase C (PKC) via the production of diacylglycerol (DAG). The PKC can also be directly activated by 12-*O*-tetradecanoylphorbol-13-acetate (TPA), and is al‐

Finally, PI3K controls another downstream kinase called Akt (also named protein kinase B or PKB) that is a crucial player in cell survival through the regulation of apoptosis among

Several additional neurotrophic factors are regrouped in a second family, the GDNF family of ligands (GFL). Briefly, the glial neurotrophic factor (GDNF) is a protein that has been shown to promote the survival and differentiation of dopaminergic neurons and motoneur‐ ons, and constitutes then an important potential actor in the management of neurological diseases such as Parkinson's disease [91, 92], amyotrophic lateral sclerosis or spinal cord in‐ juries. Neurturin, artemin and persephin are similar ligands that also play a role in cell sur‐ vival, neurite outgrowth, and cell differentiation migration. Those four members act by the activation of a tyrosine kinase receptor RET, which in association with a co-receptor (GFRα),

A cocktail of the three main neurotrophins (BDNF, NGF and NT-3) was used to direct neu‐ ral differentiation of MIAMI cells, which began to develop a complex neuritic arborization and to express neuronal markers, e.g. NF-L or NeuN [69]. Moreover, those differentiated

tance of NT-3 in the neural commitment and differentiation step, through the demonstration of a dramatic decrease in viability, βIII-tubulin expression, neuron-like morphology and branching of differentiated cells without NT-3 treatment. Those results were then further de‐ tailed and showed the enhanced neural specification from MIAMI cells thanks to epidermal growth factor (EGF) and bFGF pre-treatment [73]; the implication of Rho-GTPase Rac1, which was thought to regulate Mek1/2-Erk1/2 phosphorylation, mediating transcription of genes involved in neural differentiation versus proliferation during NT-3-induced neuronal

currents. This study also highlighted the impor‐

triggers auto-transphosphorylation and downstream signaling processes.

and outward K+

(MAPKs), phospholipase C (PLC) and phosphatidyl-inositol-3-kinase (PI3K) [89,90].

functions.

other roles.

cells showed inward Na+

commitment [74].

so able to activate MAPKs pathway.

340 Trends in Cell Signaling Pathways in Neuronal Fate Decision

When co-cultivated with immature cerebellar granules, nestin-positive BM-MSCs are able to differentiate into functional neuronal cells [93], as showed by βIII-tubulin expression and ac‐ tion potentials firings. After characterizing two subpopulations of BM-MSCs which were re‐ spectively derived and non-derived from the embryonic neural crest (after generating clonal cultures), Wislet-Gendebien and collaborators demonstrated that the neural differentiation of BM-NCSCs in co-culture conditions was abolished in the presence of a MAPKs inhibitor. Those results confirmed the importance of MAPKs pathway in neural differentiation of adult BM-NCSCs [43].

After adding NGF in the culture medium of human SKPs, Bakthiari et al. obtained NF-M, βIII-tubulin, S100 and GFAP positive cells, and that after different conditions of cryopreser‐ vation [75].

Lim et al. demonstrated the important role of BDNF in neural differentiation of human UCB-MSCs, and provided a deeper study of the different involved pathways. Addition of BDNF in the induction medium leaded to the phosphorylation of Raf-1 and ERK, then to the downstream up-regulation of p35 expression, which was not observed when ERK was blocked with a specific inhibitor. p35 is known as an anti-apoptotic factor able to block pro‐ caspase maturation and to protect neurons from cell death [94, 95].

They also analyzed the contribution of BDNF to cell viability, and demonstrated an up-regu‐ lation of the anti-apoptotic gene Bcl2, which was mediated by the activity of PI3K and Akt phosphorylation [76]. Few years later, after genetically modifying UCB-MSCs through trans‐ fection with a BDNF-expressing plasmid, they showed the expression of βIII-tubulin, NeuN, GFAP and myelin basic protein (MBP) in differentiated cells, associated with an up-regulat‐ ed phosphorylation level of TrkB, Raf-1 and ERKs [77].

BDNF was also used to induce bi-multipolar morphology and MAP2 expression in WJ-MSCs. When used in combination with hippocampal cholinergic neurostimulating peptide (HCNP) and/or denervated hippocampal extract (rDHE), WJ-MSCs turned into Choline-ace‐ tyltransferase (ChAT) positive cells [96].

Coupled with IBMX, BDNF was able to induce GFAP and βIII-tubulin expression in AT-MSCs, as demonstrated by Ying et al. [79]. The same combination of IBMX and BDNF was used to differentiate DP-SCs into GFAP, c-fos, NF, HNK1, enolase-2, βIII-tubulin, MAP2, Sox2, Tenascin-C, Connexin-43 and nestin positive cells [80].

Neural induction of DP-SCs was achieved by Kiraly et al. through the activation of both cAMP and PKC signaling pathways. After reprogramming by 5-azacytidine treatment, cells where treated with IBMX, db-cAMP, forskolin, TPA, NGF and NT-3, and showed an in‐ crease in neurogenin-2, βIII-tubulin, NSE, NF-M and GFAP expression, while electrophysio‐ logical recordings revealed voltage dependent sodium channels activity [81].

The study of Zhang et al. demonstrated that RA induced SKPs to neural differentiation through the up-regulation of the transcription factor NeuroD and the cell-cycle regulatory protein p21 [82]. In the meantime, RA also induced p75NTR up-regulation that leaded to apoptotic cell death. They showed that when treated with NT-3 after RA induction, the sur‐ vival and neural differentiation of SKPs were improved significantly, and cell apoptosis in‐ duced by RA was decreased. These effects were reversible as confirmed by the way of a p75NTR inhibitor Pep5 instead of Trk receptor inhibitor K252a.

**5. Implications in cell therapy**

**5.1. Dopaminergic neurons and Parkinson's disease**

marized in the Table 2.

jective [115].

ent locations of the brain [111, 112].

usefulness of stem cells in regenerative therapy).

With regards to their accessibility and their multipotentiality, adult and perinatal MSCs and NCSCs constitute ideal stem cells to use in cell therapy. As it has been shown that those cells could give rise to neuron-like cells via multiple ways of induction, we can infer that they could be of valuable interest in the treatment of neurological lesions. In this paragraph, we will collect the results of some studies that focused on cell therapy of Parkinson's disease and spinal cord injuries, using different types of MSCs/NCSCs and different ways to differ‐ entiate them into neurons before being transferred in animal models. Those results are sum‐

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Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alz‐ heimer's disease, with a prevalence of 0,3% of the population in industrialized countries, reaching 1% after 60 years of age [110]. This pathology is characterized by typical clinical symptoms, like bradykinesia, rigidity, gait troubles and resting tremor, while the main pathological feature is the loss of dopaminergic neurons in the Substantia Nigra pars com‐ pacta (SNpc), associated with ubiquitinated protein aggregates called Lewy bodies in differ‐

In the last 80's, clinical trials have been started, using fetal mesencephalic dopaminergic neurons to transplant in PD patients [113, 114]. Despite the demonstration of several benefits in terms of clinical symptoms and pathology, few problems remain. Fetal tissue heterogenei‐ ty, influence of harvesting methods on the graft efficiency, need of too much fetuses for only one patient, altogether coupled with ethical concerns, left no option but finding other ways to proceed. One of the main goals in this field relies in the replacement of lost dopaminergic neurons in the nigrostriatal system, which could be achieved through the use of different types of stem cells. As explained earlier, MSCs/NCSCs are interesting candidates in this ob‐

In this paragraph, we will review the results of some studies aiming to differentiate diverse types of MSCs and NCSCs in dopaminergic neurons before grafting those cells *in vivo*, using animal models mimicking the symptoms of PD (which are required to study the putative

After *in vitro* differentiation of WJ-MSCs in dopaminergic neural cells using a SHH and FGF8 treatment (in combination with brain-conditioned medium), Fu et al transplanted those differentiated cells inside the striatum of hemiparkinsonian rats, previously treated with 6-hydroxydopamine (6-OHDA). 20 days after transplantation, TH positive cells were found around the implantation site, and those cells were shown to be grafted WJ-MSCs. Moreover, the number of amphetamine-induced rotations (giving idea of motor performan‐ ces of hemiparkinsonian rats) was decreased, and this decrease was gradual over time,

showing an important improvement in the nigrostriatal pathway function [99].

The three pathways of RA, cAMP and NT were recruited together to differentiate SKPs into neuronal cells. After adding RA, db-cAMP, NGF, BDNF and NT-3 to the culture medium, Lebonvallet et al. identified NF and PGP9.5 positive cells, which were also able to incorpo‐ rate FMI-43 staining, indicating the presence of synaptic vesicles. Furthermore, they showed an overexpression of neuron-related genes in differentiated SKPs [83].

Kim et al. studied the involvement of non-neurotrophin-activated MAPKs pathway. They showed that cAMP and PKA (resulting of forskolin treatment) promoted the phosphory‐ lation/activation of B-Raf, MEK and ERK [58]. Confirmation was specified with the use of an inhibitor of MAPKs pathway that induced a significant decrease in neural features of forskolin-treated BM-MSCs. The same observation was carried out by Jori et al., con‐ firming that neural-like BM-MSCs reverted to uncommitted cells when cultured with a MEK-ERK inhibitor [57].

#### **4.4. Wnt signaling pathway**

The Wnt signaling pathway is constituted by a network of proteins that are involved in the regulation of multiple developmental events during embryogenesis, but also in adulthood, in several physiological processes and tissue homeostasis through cell fate specification, dif‐ ferentiation, or proliferation..

Wnt proteins act on cells by binding Frizzled (Fzd)/low density lipoprotein (LDL) recep‐ tor-related protein (LRP) receptor complex. When Wnt signal is inactive, the levels of cy‐ toplasmic transcription factor β-catenin are kept low through continuous proteasomemediated degradation, which is regulated by a complex including glycogen synthase kinase-3β (GSK-3), Axin, and Adenomatous Polyposis Coli (APC). Once Wnt ligands acti‐ vate Fzd/LRP, the degradation pathway is inhibited (through the activity of Dishevelled (Dsh)) and β-catenin accumulates in the cytoplasm. After nuclear translocation, it inter‐ acts with T-cell specific transcription factors (TCF) among others, which allows transcrip‐ tion regulation [97, 98].

Kondo et al. exposed that BM-MSCs induced to neural differentiation (with forskolin and IBMX) showed significant dose-dependent upregulation of sensory neurons markers Ngn1, NeuroD, Brn3a and P2X3 when the induction medium was supplemented with recombinant Wnt1 (whereas Wnt3a exhibited comparable but slighter effects)[84]. Glutamate receptors GluR2 and GluR4 were also up-regulated in those conditions.
