**7. Telencephalic neurons generated from human ES cells**

Neural induction takes place in human ES (hES) cells similar to mES cells, however with a different timing. Under serum-free conditions, without adding known morpho‐ gens [3;101;105-110], by co-culture with stromal cells [111;112] or by using a recently defined protocol with dual-SMAD-inhibition [113], it has been demonstrated that hES cells differentiate into a synchronized population of NE cells organized into neuraltube-like rosettes within 2 weeks, a time corresponding to the development of the neu‐ ral plate/tube in a human embryo. Around day 8-10, the primitive NE cells express PAX6, and the neural fate is consolidated by SOX1 expression around day 14 [3;100;105;106;108-110].

#### **7.1. Early anterior/posterior patterning**

In the absence of exogenous morphogens, hES cells differentiate into progenitors that uni‐ formly expressed anterior TF, including FOXG1, OTX2, SIX3, LHX2 but not posterior TF [3;107;106;109]. Even when the neural induction is performed by co-culturing with stromal cells, a large population of neural progenitors exhibit anterior characteristics [112]. FOXG1 appears at approximately day 10 and it is still expressed at day 24 and even in postmitotic neurons, 1 month after differentiation. PAX6 is co-expressed in nearly all of the FOXG1 tele‐ ncephalic progenitors (95% of all cells) one month after differentiation, in the absence of growth factors or morphogens [3;106;114].

#### **7.2. Dorsal/ventral patterning**

WNT proteins and their downstream molecules have been found to be highly expressed right after the generation of NE cells. In minimal medium and in the absence of known morphogens, hES cell-derived telencephalic progenitors exhibit a dorsal telencephalic trait, which has been attributed to endogenous WNT signaling [115]. The activation of SHH and/or inhibition of WNT permitts the specification of ventral telencephalic progenitors.

At the lower dosage, SHH reduces the PAX6-expressing cell population and increases the NKX2.1 cell population. At the higher dosage, SHH almost completely eliminates the PAX6 expressing cells, while increasing the NKX2.1 ventral progenitors. The combination of DKK1 and SHH at the lower dosage significantly increases NKX2.1 and decreases PAX6 and GLI3 expression. High concentrations of SHH significantly inhibits GLI3 expression compared with low concentrations of SHH. The specification of dorsal-ventral progenitors by WNT and/or SHH has been partially achieved through differentially regulating the expression of 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 have found a significant up-regulation of the LGE markers GSH2 and DLX2 [111].

#### **7.3. Neuronal specification**

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

Neural induction takes place in human ES (hES) cells similar to mES cells, however with a different timing. Under serum-free conditions, without adding known morpho‐ gens [3;101;105-110], by co-culture with stromal cells [111;112] or by using a recently defined protocol with dual-SMAD-inhibition [113], it has been demonstrated that hES cells differentiate into a synchronized population of NE cells organized into neuraltube-like rosettes within 2 weeks, a time corresponding to the development of the neu‐ ral plate/tube in a human embryo. Around day 8-10, the primitive NE cells express PAX6, and the neural fate is consolidated by SOX1 expression around day 14

In the absence of exogenous morphogens, hES cells differentiate into progenitors that uni‐ formly expressed anterior TF, including FOXG1, OTX2, SIX3, LHX2 but not posterior TF [3;107;106;109]. Even when the neural induction is performed by co-culturing with stromal cells, a large population of neural progenitors exhibit anterior characteristics [112]. FOXG1 appears at approximately day 10 and it is still expressed at day 24 and even in postmitotic neurons, 1 month after differentiation. PAX6 is co-expressed in nearly all of the FOXG1 tele‐ ncephalic progenitors (95% of all cells) one month after differentiation, in the absence of

WNT proteins and their downstream molecules have been found to be highly expressed right after the generation of NE cells. In minimal medium and in the absence of known morphogens, hES cell-derived telencephalic progenitors exhibit a dorsal telencephalic trait, which has been attributed to endogenous WNT signaling [115]. The activation of SHH and/or inhibition of WNT permitts the specification of ventral telencephalic progenitors.

At the lower dosage, SHH reduces the PAX6-expressing cell population and increases the NKX2.1 cell population. At the higher dosage, SHH almost completely eliminates the PAX6 expressing cells, while increasing the NKX2.1 ventral progenitors. The combination of DKK1 and SHH at the lower dosage significantly increases NKX2.1 and decreases PAX6 and GLI3 expression. High concentrations of SHH significantly inhibits GLI3 expression compared with low concentrations of SHH. The specification of dorsal-ventral progenitors by WNT and/or SHH has been partially achieved through differentially regulating the expression of

**7. Telencephalic neurons generated from human ES cells**

cortical interneuron characteristics [100].

226 Trends in Cell Signaling Pathways in Neuronal Fate Decision

[3;100;105;106;108-110].

**7.1. Early anterior/posterior patterning**

growth factors or morphogens [3;106;114].

**7.2. Dorsal/ventral patterning**

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

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 expressed TBR1 and CTIP2 [96, 115].

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

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 telencephalon after transplantation [117].
