**6. Telencephalic neurons generated from mouse ES cells**

Mouse ES (mES) cells have emerged as a powerful tool for developmental biology. Several studies have focused on mES-derived telencephalic progenitors and the specific neuronal populations they generate *in vitro* and *in vivo* [3;96-101].

#### **6.1. Early A/P patterning**

After the first generation of defined telencephalic precursors from mES cells based on studies of forebrain development [101], significant improvements regarding the success of neural induction and telencephalic patterning have been reported during the follow‐ ing years [96;100].

Blocking the WNTs and BMPs pathways by applying the antagonists DKK1 and BMPR1A-FC, respectively, cause neural induction in more than 90% of mES cells and maintained around 35% of the progenitors as telencephalic, expressing FoxG1 [101]. Oth‐ er following studies has reported a massive FoxG1 expressing telencephalic progenitor generation in serum-free, RA-free conditions [96-98;100], while using a RA treatment, FoxG1 expression has not been detected [98].

#### **6.2. Dorsal/ventral patterning**

Mash1 is the main neurogenic TF in the ventral telencephalon and is involved also in the neurotransmitter identity specification, being a selective instructor of **GABAergic** identity [70;76-82]. Olig1/2 can promote both neuronal and oligodendroglial fates while inhibiting

Dlx genes (Dlx1, 2, 5 and 6) are expressed in ventral progenitors and neurons in MGE, LGE

Interestingly, MGE, LGE and CGE progenitor domains do not give rise to homogeneous populations of neurons, which is most likely due to a further subdivision of these domains

There is also a temporal control of the specification of various neuronal subtypes; as a gener‐ al pattern, the earlier- born ventral cells give rise to projection neurons while the more dor‐

MGE is characterized by the early production of cholinergic projection neurons from its ventral part, followed by the late production of GABAergic and cholinergic interneurons from the dorsal domains. Two TF are detected at E12.5 exclusively in the MGE: Lhx6 and Lhx8 (or Lhx7) [89], suggesting a role in the specification of MGE-derived neurons. Lhx6*-*expressing neurons have the characteristics of proto-GABAergic neurons with dual differentiation potential, while Lhx8 seems to be involved in the differentiation of specif‐ ic cholinergic neurons [15;90]. The differentiation of a common proto-GABAergic precur‐ sor into mature subtypes is regulated by the combinatorial activity of the Lhx6, Lhx8 and Isl1. Those proto-GABAergic neurons that maintain the expression of Lhx6 differen‐ tiate into mature GABAergic striatal interneurons. By contrast, induction of Isl1 and the combined activity of Lhx7/8 and Isl1 results in down-regulation of Lhx6 and commit‐ ment along the cholinergic interneuron sublineage [90-92]. Thus, it appears that a LIM HD transcriptional code determines cell-fate specification and neurotransmitter identity

Ventral LGE generate GABAergic projection neurons that also express Isl1 during early specification, followed by the expression of other striatal-specific TF such as FoxP1, FoxP2, and Ctip2 [93-95]. Later in development, dLGE generates interneurons that migrate to the

Mouse ES (mES) cells have emerged as a powerful tool for developmental biology. Several studies have focused on mES-derived telencephalic progenitors and the specific neuronal

After the first generation of defined telencephalic precursors from mES cells based on studies of forebrain development [101], significant improvements regarding the success

and CGE, and are likely to play a role in neural specification [78;84;85].

into regions with spatially restricted expression of specific TF.

in neuronal subpopulations of the ventral telencephalon.

populations they generate *in vitro* and *in vivo* [3;96-101].

**6. Telencephalic neurons generated from mouse ES cells**

sally positioned later-born cells generate interneurons [24;82;86-88].

astrogliogeneis [83].

224 Trends in Cell Signaling Pathways in Neuronal Fate Decision

olfactory bulbs [13;95].

**6.1. Early A/P patterning**

SHH or a Hh agonist treatment on telencephalic progenitors suppresses the dorsal marker Pax6 and induced the ventral marker Nkx2.1. SHH application does not cause substantial difference in the level of Gsh2 expression [100;101].

The majority of mES cell-derived progenitors exhibit by default a ventral phenotype which has been attributed to the high level of endogenous SHH signaling. Blocking the Hh signaling converts most of the ventral telencephalic precursors into dorsal progeni‐ tors, with the majority expressing typical markers of the dorsal telencephalon*:* Pax6 and Emx1/2 [3;97;99].

#### **6.3. Neuronal specification**

The telencephalic progenitors derived from mES cells can be directed to neurons that express the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter GABA [3;102-106].

The dorsal progenitors have been shown to produce mature neurons with many features of cortical pyramidal neurons in a temporal manner similar with *in vivo* corticogenesis. The first neurons generated in these cultures are reelin-positive Cajal-Retzius-like and subplatelike neurons expressing Tbr1, followed by the glutamatergic neurons generated in an insidefirst, outside-last manner. The majority of the cells generated by Gaspard *et al*. express markers of deep cortical layer V and VI neurons, like Tbr1, Otx1, Ctip2 and FoxP2 [102]. Eir‐ aku *et al.* have also generated deep layer neurons, positive for Ctip2 and Emx1 in the first 9 days of neuronal differentiation of dorsalized progenitors [96].

By transplantation of dorsally patterned progenitors into postnatal murine cerebral cortex, the production of cortical projection neurons with the correct morphology and axonal con‐ nectivity has been demonstrated [98]. The pyramidal neurons express Otx1, Emx1, and Ctip2, corresponding to deep layers neurons. They integrate and appropriately project longdistance axons to subcortical targets, without forming tumors [98].

Regarding the protocols for ventral neuronal specification, it has been shown that Nkx2.1 and Gsh2 expressing progenitors give rise both to GABAergic and cholinergic neurons [3;100;101].

Furthermore, the use of Lhx6::GFP ES cells has allowed the isolation of cells with potential for developing into ventral telencephalic subpopulations and their follow-up during trans‐ plantation into the postnatal brain. Transplanted Lhx6::GFP cells demonstrate the ability to retain migratory capacity and neuronal commitment without forming tumors and exhibit cortical interneuron characteristics [100].

active and repressive forms of GLI3. Using of a low concentration of SHH alone results in the differentiation of both LGE and MGE progenitors, whereas additional WNT inhibition (by DKK1) further ventralizes the human neural progenitors, resulting in a predominant population of NKX2.1 expressing MGE progenitors [115]. Aubry *et al*. have patterned the tel‐ encephalic progenitors by SHH and DKK1 treatment for 12 days in adherent culture and

Telencephalic Neurogenesis Versus Telencephalic Differentiation of Pluripotent Stem Cells

http://dx.doi.org/10.5772/54251

227

The regionalized dorsal and ventral human telencephalic progenitors further differentiate into functional cortical glutamatergic neurons and telencephalic GABAergic neurons, re‐

Cortical glutamatergic neurons have been efficiently generated in the absence of morpho‐ gens. This indicates the intrinsic tendency of hES cells to generate cortical neural cells [3;109]. Neurons differentiated from dorsal progenitors in the absence of exogenous morph‐ ogens for 6 weeks exhibits a pyramidal morphology, with extensive neurite outgrowth, and

Ventral telencephalic GABAergic neurons have been induced with a high concentration of SHH, a low dose of SHH together with WNT inhibitors [115] or by using SHH agonists [3].

Additionally, Aubry *et al*. have focused to the characterization of the striatal-like progeni‐ tors, showing that more than a half of the specified neurons were DARPP32 and Calr posi‐ tive, exhibiting phenotypic features of MSN. Transplantation of the LGE-patterned progenitors in quinolinic acid–lesioned rats (a model for Huntington disease) has confirmed

Human ES cell-derived telencephalic glutamatergic and GABAergic neurons have been re‐ ported to be electro-physiologically active [111;115;116] and also to integrate in the mature

**8. Modulation of Hh signaling pathway in telencephalic cells generated**

We recently proposed a novel model system in which the *in vitro* differentiation of hES and mES cells are temporally aligned to each other and compared with mouse telencephalic neu‐ rogenesis *in vivo*. In this comparative model system, we tested the *in vitro* role of Hh signal‐

Neural differentiation of 2 lines of mES cells and 2 lines of hES cells was studied under identical, defined conditions, but following different time-schedules for mouse and hu‐ man cell cultures. The *in vitro* time schedules were based on data from *in vivo* develop‐ ment as a reference for the stages of neural induction, neural patterning, and neuronal

have found a significant up-regulation of the LGE markers GSH2 and DLX2 [111].

**7.3. Neuronal specification**

expressed TBR1 and CTIP2 [96, 115].

the *in vivo* specification toward striatal MSN [111].

telencephalon after transplantation [117].

**from mouse and human ES cells**

ing for ES cell-derived telencephalic differentiation (Figure 3) [3].

spectively.
