**5. Implications in cell therapy**

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

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

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

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‐

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‐

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.

p75NTR inhibitor Pep5 instead of Trk receptor inhibitor K252a.

342 Trends in Cell Signaling Pathways in Neuronal Fate Decision

MEK-ERK inhibitor [57].

**4.4. Wnt signaling pathway**

ferentiation, or proliferation..

tion regulation [97, 98].

an overexpression of neuron-related genes in differentiated SKPs [83].

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‐ marized in the Table 2.

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

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‐ ent locations of the brain [111, 112].

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‐ jective [115].

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 usefulness of stem cells in regenerative therapy).

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].


**Cell type Differentiation protocol Animal model Histology Behavioral aspects Ref.**

cells

cells

Survival rate of Reduced cavity size Myelination of endogenous axons Recruitment of endogenous Schwann

Survival rate of Reduced cavity size Myelination of endogenous axons Recruitment of endogenous Schwann

Bypass of the lesion Reduced gliosis Axonal growth

No migration, no proliferation

neuritis

No GFAP

("No" indicates that no behavioral testing is described in the study. Protocols describing *in vitro* differentiation in the meantime than *in vivo* studies are also included in the table, as well as some *in vivo* studies using non-differentiated

On the other hand, Khoo et al showed that neuronal-primed human BM-MSCs (with SHH, FGF8, GDNF and other growth factors) did survive transiently in the brain of 6-OHDAtreated rats, but no further differentiation in functional dopaminergic neurons was ob‐ served, even when a co-transplantation with olfactory ensheating cells (OECs) was

The same cocktail was used by Wang et al. to differentiate SHED cells (DP-SCs) into dopa‐ minergic neurons, this time supplemented with forskolin. Differentiated cells were βIII-tu‐ bulin, MAP2 and TH positive *in vitro*. They further characterized naïve SHED cells by transplanting them into the striatum of a hemiparkinsonian rat. While some TH-positive cell

Close contact with host

βIII-tubulin, GAD67, RIP and MBP positive cells.

Heightened sensory

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

Increased BBB score and Basso locomotor subscore

Decease in the number of hind limb errors No impact of sensitivity to sensory stimuli

No [109]

[108]

345

responses

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Rat with contused SC

Rat with contused SC

Rat with contused SC

**Table 2.** Different results obtained in cell-based therapy experiments using MSCs and NCSCs.

performed to enhance to graft efficiency [100].

SKPs

Nothing

Forskolin (+ neuregulin-1β)

EPI-NCSCs Nothing

cells).


**Cell type Differentiation protocol Animal model Histology Behavioral aspects Ref.**

6-OHDA rat Survival after 20 days

(in vitro) In vitro expression of βIII-

tubulin, MAP2, and TH

A2B5, NCAM and B3T

MAP2, NF and GFAP positive cells

Rescue of surviving

Integration in slice ChAT positive cells

In vitro expression of MAP2, NSE, nestin, βIII-

Reduced cystic cavity

Accumulation near the

Reduction of lesion size Enhanced axon regrowth

Reduction of cystic cavity

positive cells

size

neurons

tubulin

lesion

<4 weeks survival Few TH expression Many GABA positive cells

Nothing 6-OHDA rat TH-positive grafted cells

6-OHDA rat

Rat with contused SC

Rat with transected SC

Injured organotypic SC

slice

Rat with contused SC

Rat with hemisection SC

injury

TH positive grafted cells.

Transient survival No differentiation Decrease of

Decrease of

No

amphetamine-induced rotations over time.

apomorphine-induced

No [100]

rotations over time. [101]

No [102]

Recovery of hind limb stepping and hind limb weight support

Improvement of hindlim

No [105]

Improvement in motor

performances

Enhancement of functional locomotor

abilities

locomotion

[99]

[103]

[104]

[106]

[107]

Dopaminergic neurons - Parkinson's Disease

SHH, FGF8 (+ brainconditioned medium)

344 Trends in Cell Signaling Pathways in Neuronal Fate Decision

BM-MSCs SHH, FGF8, GDNF 6-OHDA rat

Forskolin, SHH, FGF8,

Forskolin, IBMX, dbcAMP and TPA

8-bromo-AMP and

GDNF

Motoneurons - Spinal Cord Injuries

Rolipram

BM-MSCs RA + NT-3 overexpression

Hb9 and Olig2 overexpression + RA, forskolin, SHH

IBMX

UCB-MSCs Nothing

RA, db-cAMP, forskolin,

WJ-MSCs

DP-SCs / SHED

BM-MSCs

BM-MSCs

BM-MSCs

BM-MSCs

("No" indicates that no behavioral testing is described in the study. Protocols describing *in vitro* differentiation in the meantime than *in vivo* studies are also included in the table, as well as some *in vivo* studies using non-differentiated cells).

**Table 2.** Different results obtained in cell-based therapy experiments using MSCs and NCSCs.

On the other hand, Khoo et al showed that neuronal-primed human BM-MSCs (with SHH, FGF8, GDNF and other growth factors) did survive transiently in the brain of 6-OHDAtreated rats, but no further differentiation in functional dopaminergic neurons was ob‐ served, even when a co-transplantation with olfactory ensheating cells (OECs) was performed to enhance to graft efficiency [100].

The same cocktail was used by Wang et al. to differentiate SHED cells (DP-SCs) into dopa‐ minergic neurons, this time supplemented with forskolin. Differentiated cells were βIII-tu‐ bulin, MAP2 and TH positive *in vitro*. They further characterized naïve SHED cells by transplanting them into the striatum of a hemiparkinsonian rat. While some TH-positive cell bodies were found in the graft zone, a significant decrease in apomorphine-induced rota‐ tions was observed, attesting of a beneficial effect of the cell transplantation [101].

cord of rats. Some transplanted cells were positive for MAP2, NF and GFAP labeling. Moreover, cell transplantation leaded to the reduction of cystic cavity, improvement of lo‐ cal environment, rescue of surviving neurons from retrograde atrophy, and improvement

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Liqing et al. induced AT-MSCs to neural fate through the action of SHH and RA, and showed that those cells expressed the transcription factors Nkx2.2, Pax6, Hb9, and Olig2, suggesting those cells as attractive nominees to become mature motoneurons [70]. While no *in vivo* testing has been performed in this study, another experiment focusing on ventral spi‐ nal-specific transcription factors was carried out by Park and al., who genetically modified human BM-MSCs to express Hb9 and Olig2, just before treating them with neural induction medium consisting in sequential incubation with RA, forskolin, SHH and FGF. *In vitro*, those cells were excitable and were able to connect muscle fibers; while after transplantation into an injured organotypic spinal cord slice culture, they survive and integrate the slice,

Pedram et al. performed comparative study of the potencies of BM-MSCs to take part in the repair of spinal cord damages, either when neurally-differentiated than when used in their native state [106]. BM-MSCs were cultured following a multi-step protocol, in presence of RA and bFGF, db-cAMP, forskolin and IBMX. After assessing the neural nature of differenti‐ ated cells (expression of MAP2, NSE, nestin, and βIII-tubulin), they transplanted cells into the lesion cavity of contused rat spinal cords. Either undifferentiated BM-MSCs or neurallyinduced BM-MSCs transplantation leaded to a reduced cavity, but a significant improve‐ ment in motor performances was observed in rats that received neurally-differentiated BM-

Obtaining myelinating glial cells is another way to manage spinal cord injuries. In that pur‐ pose, Biernaskie et al. studied the effect of SKPs on spinal cord lesions, when transplanted in their naïve state or when pre-differentiated into Schwann cells (using forskolin and neure‐ gulin-1β) [108]. They showed that a graft of both the naïve SKPs and SKPs-derived Schwann cells leaded to a reduced cystic cavity size, and that the cells myelinated host axons and re‐ cruited host Schwann cells. Still, the SKPs-derived Schwann cells were the only ones to gen‐ erate a bridge across the lesion and to induce a growth-permissive environment, while a substantial improvement was observed at the behavioral level (Increased BBB score and Bas‐

Significant enhancement of functional locomotor abilities was observed by Schira et al. af‐ ter transplantation of unrestricted UCB-MSCs into the surrounding area of a hemisection injury, accompanied by cell accumulation near the lesion, reduction in its size and en‐ hanced axon regrowth [107]. This study gives an example of what can be observed using naïve stem cells in cell therapy, without any pre-differentiation. In the same way, Sieber-Blum et al. transplanted EPI-NCSCs in the core of a spinal cord lesion, and observed βIIItubulin, GAD67, RIP and MBP positive cells among the grafted cells. Those cells were tightly close to endogenous neuritis, but did not show any sign of proliferation nor migra‐

while expressing motoneurons-specific markers, e.g. ChAT [105].

MSCs (compared to control group and native BM-MSCs-transplanted group).

so locomotor subscore and decrease in the number of hind limb errors).

of hind limb locomotion.

tion in the tissue [109].

cAMP pathway was recruited to differentiate BM-MSCs (after EGF/bFGF-induced sphere formation) in dopaminergic neurons, using forskolin, IBMX and db-cAMP (coupled with the PKC activator TPA) [102]. Differentiated cells showed *in vitro* expression of βIII-tubulin, neurofilament, Nurr-1, TH, AADC and GIRK2. After transplantation into the striatum of 6- OHDA rats, few cells were βIII-tubulin and TH positive, whereas a higher number of graft‐ ed cells became GABA-positive (maybe due to the striatal environment mainly composed of GABA neurons). Unfortunately, no behavioral observation was described by Suon et al.

#### **5.2. Motoneurons and spinal cord injuries**

Whereas peripheral nerves are able to regenerate when a lesion occurs, the motoneurons and nervous fibers in the spinal cord can't be replaced in case of spinal cord injury (SCI). Indeed, traumatic spinal cord injury results in a wide panel of pathophysiological events counteracting any possibility of neural regeneration, and those events are generally grouped in two phases. The primary injury phase can be due to either contusion or compression, and is characterized by section of axons, necrosis, degeneration, oligodendrocytes apoptosis, gliosis and macrophage infiltration. Altogether, those events lead to secondary lesions like ischemia, inflammation, alteration of ion balance, insult of the blood-brain-barrier, lipid per‐ oxidation and glutamate-induced excitotoxicity. Despite a slight spontaneous recovery, all those events collectively constitute an environment that hampers axonal regeneration [116]. Since the clinical consequences of such lesions are dramatic and rarely reversible (para-, hemi-, tetraplegy, respiratory problems, loss of sphincters control, all leading to important socio-economic issues), it's crucial to find out efficient therapies to improve the recuperation of motor function.

Stem cell grafting has been suggested as a therapeutic strategy for spinal cord repair, hence the obtainment of mature motoneurons is critical.

Human BM-MSCs were induced to differentiate into neural cells through the activation of cAMP signaling pathway, via the addition of 8-bromo-cAMP and Rolipram (inhibitor of phosphodiesterases) in the culture medium. Those neurally-induced BM-MSCs were then transplanted into a segment of the spinal cord of rats, previously wounded by contusion. After confirming neural nature of differentiated cells by immunostaining of A2B5, NCAM, B3T, *in vitro* as well as *in vivo* after transplantation, behavioral testing of rats revealed that the motor recovery (assessed by hind limb stepping and weight support) was significantly different at 2 to 12 weeks post-recovery in the group that was transplanted with neurallyinduced BM-MSCs when compared with the control groups that received non differentiated BM-MSCs and saline solution [103].

Another protocol was tested by Zhang et al., who treated BM-MSCs with RA before ge‐ netically modifying them to overexpress the gene coding for the neurotrophin 3 (NT-3) [104]. Once they've showed that RA pretreatment enhanced NT-3 expression and secre‐ tion by MSCs after genetic engineering, they transplanted cells into the transected spinal cord of rats. Some transplanted cells were positive for MAP2, NF and GFAP labeling. Moreover, cell transplantation leaded to the reduction of cystic cavity, improvement of lo‐ cal environment, rescue of surviving neurons from retrograde atrophy, and improvement of hind limb locomotion.

bodies were found in the graft zone, a significant decrease in apomorphine-induced rota‐

cAMP pathway was recruited to differentiate BM-MSCs (after EGF/bFGF-induced sphere formation) in dopaminergic neurons, using forskolin, IBMX and db-cAMP (coupled with the PKC activator TPA) [102]. Differentiated cells showed *in vitro* expression of βIII-tubulin, neurofilament, Nurr-1, TH, AADC and GIRK2. After transplantation into the striatum of 6- OHDA rats, few cells were βIII-tubulin and TH positive, whereas a higher number of graft‐ ed cells became GABA-positive (maybe due to the striatal environment mainly composed of GABA neurons). Unfortunately, no behavioral observation was described by Suon et al.

Whereas peripheral nerves are able to regenerate when a lesion occurs, the motoneurons and nervous fibers in the spinal cord can't be replaced in case of spinal cord injury (SCI). Indeed, traumatic spinal cord injury results in a wide panel of pathophysiological events counteracting any possibility of neural regeneration, and those events are generally grouped in two phases. The primary injury phase can be due to either contusion or compression, and is characterized by section of axons, necrosis, degeneration, oligodendrocytes apoptosis, gliosis and macrophage infiltration. Altogether, those events lead to secondary lesions like ischemia, inflammation, alteration of ion balance, insult of the blood-brain-barrier, lipid per‐ oxidation and glutamate-induced excitotoxicity. Despite a slight spontaneous recovery, all those events collectively constitute an environment that hampers axonal regeneration [116]. Since the clinical consequences of such lesions are dramatic and rarely reversible (para-, hemi-, tetraplegy, respiratory problems, loss of sphincters control, all leading to important socio-economic issues), it's crucial to find out efficient therapies to improve the recuperation

Stem cell grafting has been suggested as a therapeutic strategy for spinal cord repair, hence

Human BM-MSCs were induced to differentiate into neural cells through the activation of cAMP signaling pathway, via the addition of 8-bromo-cAMP and Rolipram (inhibitor of phosphodiesterases) in the culture medium. Those neurally-induced BM-MSCs were then transplanted into a segment of the spinal cord of rats, previously wounded by contusion. After confirming neural nature of differentiated cells by immunostaining of A2B5, NCAM, B3T, *in vitro* as well as *in vivo* after transplantation, behavioral testing of rats revealed that the motor recovery (assessed by hind limb stepping and weight support) was significantly different at 2 to 12 weeks post-recovery in the group that was transplanted with neurallyinduced BM-MSCs when compared with the control groups that received non differentiated

Another protocol was tested by Zhang et al., who treated BM-MSCs with RA before ge‐ netically modifying them to overexpress the gene coding for the neurotrophin 3 (NT-3) [104]. Once they've showed that RA pretreatment enhanced NT-3 expression and secre‐ tion by MSCs after genetic engineering, they transplanted cells into the transected spinal

tions was observed, attesting of a beneficial effect of the cell transplantation [101].

**5.2. Motoneurons and spinal cord injuries**

346 Trends in Cell Signaling Pathways in Neuronal Fate Decision

the obtainment of mature motoneurons is critical.

BM-MSCs and saline solution [103].

of motor function.

Liqing et al. induced AT-MSCs to neural fate through the action of SHH and RA, and showed that those cells expressed the transcription factors Nkx2.2, Pax6, Hb9, and Olig2, suggesting those cells as attractive nominees to become mature motoneurons [70]. While no *in vivo* testing has been performed in this study, another experiment focusing on ventral spi‐ nal-specific transcription factors was carried out by Park and al., who genetically modified human BM-MSCs to express Hb9 and Olig2, just before treating them with neural induction medium consisting in sequential incubation with RA, forskolin, SHH and FGF. *In vitro*, those cells were excitable and were able to connect muscle fibers; while after transplantation into an injured organotypic spinal cord slice culture, they survive and integrate the slice, while expressing motoneurons-specific markers, e.g. ChAT [105].

Pedram et al. performed comparative study of the potencies of BM-MSCs to take part in the repair of spinal cord damages, either when neurally-differentiated than when used in their native state [106]. BM-MSCs were cultured following a multi-step protocol, in presence of RA and bFGF, db-cAMP, forskolin and IBMX. After assessing the neural nature of differenti‐ ated cells (expression of MAP2, NSE, nestin, and βIII-tubulin), they transplanted cells into the lesion cavity of contused rat spinal cords. Either undifferentiated BM-MSCs or neurallyinduced BM-MSCs transplantation leaded to a reduced cavity, but a significant improve‐ ment in motor performances was observed in rats that received neurally-differentiated BM-MSCs (compared to control group and native BM-MSCs-transplanted group).

Obtaining myelinating glial cells is another way to manage spinal cord injuries. In that pur‐ pose, Biernaskie et al. studied the effect of SKPs on spinal cord lesions, when transplanted in their naïve state or when pre-differentiated into Schwann cells (using forskolin and neure‐ gulin-1β) [108]. They showed that a graft of both the naïve SKPs and SKPs-derived Schwann cells leaded to a reduced cystic cavity size, and that the cells myelinated host axons and re‐ cruited host Schwann cells. Still, the SKPs-derived Schwann cells were the only ones to gen‐ erate a bridge across the lesion and to induce a growth-permissive environment, while a substantial improvement was observed at the behavioral level (Increased BBB score and Bas‐ so locomotor subscore and decrease in the number of hind limb errors).

Significant enhancement of functional locomotor abilities was observed by Schira et al. af‐ ter transplantation of unrestricted UCB-MSCs into the surrounding area of a hemisection injury, accompanied by cell accumulation near the lesion, reduction in its size and en‐ hanced axon regrowth [107]. This study gives an example of what can be observed using naïve stem cells in cell therapy, without any pre-differentiation. In the same way, Sieber-Blum et al. transplanted EPI-NCSCs in the core of a spinal cord lesion, and observed βIIItubulin, GAD67, RIP and MBP positive cells among the grafted cells. Those cells were tightly close to endogenous neuritis, but did not show any sign of proliferation nor migra‐ tion in the tissue [109].

#### **6. Conclusions**

Mesenchymal stem cells (MSCs) and neural crest stem cells (NCSCs) are multipotent cells that are able to generate a wide range of cell types, including neural cells, which makes them incredibly interesting in restorative therapies for patients suffering from neurological diseases. A lot of induction protocols indicate that many signaling pathways may be involved in the neural fate of MSCs and NCSCs. Indeed, the signalization path‐ ways of cAMP, Retinoic acid, Hedgehog, Wnt and the neurotrophins-activated pathways have been implicated into the maturation of adult MSCs/NCSCs into neural-like cells. Af‐ ter an induction process consisting in various activators, lengths and conditions of cul‐ ture, treated cells adopt a neural morphology express markers (at the transcriptome level as well as at the protein level) that are usually described to characterize neurons at dif‐ ferent developmental stages [117, 118] in MSCs as well as in NCSCs. Despite the expres‐ sion of those specific neural markers, only a tiny number of *in vitro* protocols were able to provide convincing evidence for a neuron-specific electrophysiological signature of the differentiated cells.

During neural development, immature neural cells undergo a differentiation process to‐ wards functional neurons through different stages that are accurately defined by specific electrophysiological features. Briefly, the first currents that occur in the cell consist in voltage-dependent outward potassium currents. As maturation proceeds, voltage-depend‐ ent inward calcium and sodium currents arise sequentially. The ultimate step is finally characterized by the elicitation of action potential through the activity of several mature voltage-gated sodium channels: an important depolarization triggers intracellular modifi‐ cations, proteins activation, and vesicular trafficking that are required for proper synap‐ tic chemical and electrical function/transmission [119-121]. As clearly observed in Table 1, even if a few data attest of electrophysiological activity in MSCs/NCSCs-derived neuronlike cells (as showed by sodium and potassium currents), there is no sufficient evidence for action potential firings and for an appropriate neuronal function.

**Figure 3.** Percentages of differentiation protocols involving cAMP, RA or neurotrophins/MAPKs signaling pathways among the 31 detailed studies. Percentages are expressed in regard to the total number of studies. Numbers in red

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349

Overall, we tend to conclude that although the cells express neural-specific proteins and ex‐ hibit a preliminary electrical activity [122, 123], MSCs and NCSCs do not seem to be able to fully differentiate and generate functional neurons in order to reach the objective of cell-

However, several *in vivo* studies based on MSCs/NCSCs-derived neural cells demonstrate a significant improvement of symptoms and lesions in animal models of neurological disor‐ ders, such as Parkinson's disease or spinal cord injury [24]. In these studies, the transplanta‐ tion of differentiated MSCs/NCSCs leads to the limitation of the lesions and the recovering of motor functions. Regarding other successful cell therapy experiments based on the trans‐ plantation of non-differentiated MSCs/NCSCs, we can infer that the experimental enhance‐ ment is more likely to be due to the intrinsic properties of the grafted cells, and not to a genuine differentiation process nor to an authentic neuronal electrical activity. Indeed, even if their ability to generate neurons is present but quite limited, they still display important immunomodulatory and anti-inflammatory properties, they secrete a lot of growth and neu‐ rotrophic factors, they modulate apoptosis processes, and promote endogenous precursors

In conclusion, those observations confirmed the significance of MSCs and NCSCs use in cell therapy procedures to treat several neurological disorders, sustaining their high capacity to protect or restore neural tissue through many proceedings that are probably more owed to

indicate the percentages of studies describing significant electrophysiological recordings.

based therapy in human neurological treatments.

intrinsic abilities than to neuronal differentiation.

recruitment [124-128]

As showed by the diagram on the figure 3, most of the collected studies describe the cAMP signaling pathway to play a key role in the neural differentiation of MSCs/ NCSCs. On the other hand, neurotrophins are often used for neural differentiation, whereas MAPKs have been shown to be involved too. RA represents the third most used signaling molecule. We can see that the most part of studies showing significant electrophysiological recordings use RA treatment for differentiation, suggesting its note‐ worthy role in this process (16,1 % of the total number of studies (5/31) and 62,5 % of the number of studies showing significant electrophysiological recordings (5/8)). Addi‐ tionally, a major number of protocols were performed in association with cAMP path‐ way activation. On the other hand, 40% of the differentiation protocols using SHH signalization (2/5 studies) were able to induce changes in electrical activity (see Table 1). That presumably raises the question of a role for RA and SHH in the last stages of ma‐ turation of MSCs/NCSCs into neural-like cells.

**6. Conclusions**

348 Trends in Cell Signaling Pathways in Neuronal Fate Decision

differentiated cells.

Mesenchymal stem cells (MSCs) and neural crest stem cells (NCSCs) are multipotent cells that are able to generate a wide range of cell types, including neural cells, which makes them incredibly interesting in restorative therapies for patients suffering from neurological diseases. A lot of induction protocols indicate that many signaling pathways may be involved in the neural fate of MSCs and NCSCs. Indeed, the signalization path‐ ways of cAMP, Retinoic acid, Hedgehog, Wnt and the neurotrophins-activated pathways have been implicated into the maturation of adult MSCs/NCSCs into neural-like cells. Af‐ ter an induction process consisting in various activators, lengths and conditions of cul‐ ture, treated cells adopt a neural morphology express markers (at the transcriptome level as well as at the protein level) that are usually described to characterize neurons at dif‐ ferent developmental stages [117, 118] in MSCs as well as in NCSCs. Despite the expres‐ sion of those specific neural markers, only a tiny number of *in vitro* protocols were able to provide convincing evidence for a neuron-specific electrophysiological signature of the

During neural development, immature neural cells undergo a differentiation process to‐ wards functional neurons through different stages that are accurately defined by specific electrophysiological features. Briefly, the first currents that occur in the cell consist in voltage-dependent outward potassium currents. As maturation proceeds, voltage-depend‐ ent inward calcium and sodium currents arise sequentially. The ultimate step is finally characterized by the elicitation of action potential through the activity of several mature voltage-gated sodium channels: an important depolarization triggers intracellular modifi‐ cations, proteins activation, and vesicular trafficking that are required for proper synap‐ tic chemical and electrical function/transmission [119-121]. As clearly observed in Table 1, even if a few data attest of electrophysiological activity in MSCs/NCSCs-derived neuronlike cells (as showed by sodium and potassium currents), there is no sufficient evidence

As showed by the diagram on the figure 3, most of the collected studies describe the cAMP signaling pathway to play a key role in the neural differentiation of MSCs/ NCSCs. On the other hand, neurotrophins are often used for neural differentiation, whereas MAPKs have been shown to be involved too. RA represents the third most used signaling molecule. We can see that the most part of studies showing significant electrophysiological recordings use RA treatment for differentiation, suggesting its note‐ worthy role in this process (16,1 % of the total number of studies (5/31) and 62,5 % of the number of studies showing significant electrophysiological recordings (5/8)). Addi‐ tionally, a major number of protocols were performed in association with cAMP path‐ way activation. On the other hand, 40% of the differentiation protocols using SHH signalization (2/5 studies) were able to induce changes in electrical activity (see Table 1). That presumably raises the question of a role for RA and SHH in the last stages of ma‐

for action potential firings and for an appropriate neuronal function.

turation of MSCs/NCSCs into neural-like cells.

**Figure 3.** Percentages of differentiation protocols involving cAMP, RA or neurotrophins/MAPKs signaling pathways among the 31 detailed studies. Percentages are expressed in regard to the total number of studies. Numbers in red indicate the percentages of studies describing significant electrophysiological recordings.

Overall, we tend to conclude that although the cells express neural-specific proteins and ex‐ hibit a preliminary electrical activity [122, 123], MSCs and NCSCs do not seem to be able to fully differentiate and generate functional neurons in order to reach the objective of cellbased therapy in human neurological treatments.

However, several *in vivo* studies based on MSCs/NCSCs-derived neural cells demonstrate a significant improvement of symptoms and lesions in animal models of neurological disor‐ ders, such as Parkinson's disease or spinal cord injury [24]. In these studies, the transplanta‐ tion of differentiated MSCs/NCSCs leads to the limitation of the lesions and the recovering of motor functions. Regarding other successful cell therapy experiments based on the trans‐ plantation of non-differentiated MSCs/NCSCs, we can infer that the experimental enhance‐ ment is more likely to be due to the intrinsic properties of the grafted cells, and not to a genuine differentiation process nor to an authentic neuronal electrical activity. Indeed, even if their ability to generate neurons is present but quite limited, they still display important immunomodulatory and anti-inflammatory properties, they secrete a lot of growth and neu‐ rotrophic factors, they modulate apoptosis processes, and promote endogenous precursors recruitment [124-128]

In conclusion, those observations confirmed the significance of MSCs and NCSCs use in cell therapy procedures to treat several neurological disorders, sustaining their high capacity to protect or restore neural tissue through many proceedings that are probably more owed to intrinsic abilities than to neuronal differentiation.

### **Author details**

Virginie Neirinckx1 , Cécile Coste1 , Bernard Rogister1,2,3 and Sabine Wislet-Gendebien1\* [11] Jiang, Y., et al., Pluripotency of mesenchymal stem cells derived from adult marrow.

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351

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**Author details**

Virginie Neirinckx1

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p. 83-92.

791-2.

, Cécile Coste1

\*Address all correspondence to: s.wislet@ulg.ac.be

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3 Neurology Department, University of Liège, Belgium

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