**8. Modulation of Hh signaling pathway in telencephalic cells generated from mouse and human ES cells**

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

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 specification. In addition, we developed a specific profile of marker genes, which was derived from *in vivo* studies.

by Hh pathway modulation, both in mES and hES cell-derived models. In particular, a very robust up-regulation of SHH by purmorphamine was observed in the human model, where SHH was not expressed in untreated controls. Shh was expressed in both progenitor cells and neurons in our cultures. This might be explained by the non-cell autonomous mecha‐ nisms recently described in the mouse embryonic telencephalon, where both Lhx8 and Lhx6 genes controlled the expression of Shh in the mantle zone of the MGE, corresponding to ear‐ ly-born neurons [118]. Thus, Lhx6 and Lhx8 appear to regulate MGE development by pro‐ moting Shh expression in MGE neurons, which, in turn, promotes the developmental

Telencephalic Neurogenesis Versus Telencephalic Differentiation of Pluripotent Stem Cells

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

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The activation of Hh signaling *via* Smo with purmorphamine converts the primitive dorsal telencephalic precursors to ventral progenitors. These progenitors differentiate into neuro‐

Our results provided evidence for the different default telencephalic differentiation of mouse and human ES cells: ventral and dorsal, respectively. Additionally, it proved the util‐ ity of the comparative system for optimizing the directed differentiation of human pluripo‐

Recent studies have shown that both mES cells and hES cells differentiate into region specif‐ ic progenitors, following the same developmental principles that have been identified by studying mouse CNS development. Together with previous findings, our own data support the model in which the human neural progenitors in culture develop a reverse default D/V phenotype compared with mouse. However, early human NE cells can be efficiently differ‐ entiated into dorsal and ventral telencephalic progenitors *via* modulating similar molecular

Therefore, mES and hES cell-derived models, directly compared in parallel experiments and temporally aligned to *in vivo* telencephalic development, offer a platform for testing the ef‐ fect of morphogens, growth factors, and pharmacological substances for the generation of

Additionally, telencephalic progenitors and neurons generated *in vitro* from human pluripo‐

Even more importantly, the telencephalic differentiation of human induced PS (IPS) cells

The application of optimized telencephalic differentiation protocols to IPS cell cultures de‐ rived from patients with neurodegenerative or neurogenetic diseases will provide unique new opportunities to develop *in vitro* models of human diseases such as Alzheimer's dis‐ ease, Huntington's disease, epilepsy, and neuropsychiatric disorders. These models, based on human neurons in culture, will critically complement existing animal models, which do not fully reflect important features specific for the normal and pathological human brain.

tent cells provide a unique paradigm to study the human telencephalic development.

nal subtypes including GABAergic and cholinergic neurons.

program of the dMGE.

tent stem cells.

**9. Conclusion**

pathways as described in rodents.

has recently been reported [119;120].

specific neuronal subtypes.

Our results demonstrated that neural differentiation took place in mES cell-derived cultures resulting in the generation of neural progenitors and neurons in a time-frame which mirrors telencephalic neurogenesis *in vivo*. The expression levels of telencephalic markers were com‐ parable between *in vivo* and *in vitro* differentiated populations. We demonstrated that the neural differentiation in human cells can be temporally aligned with mouse cells in the pro‐ posed neurogenic time-windows. Thus, our temporally aligned, comparative cell culture model offered a novel platform for analyzing the effect of signaling molecules on the gener‐ ation of specific telencephalic populations in mouse and human cell cultures.

**Figure 3.** Hh signaling for modulation of dorsal ventral patterning in mouse and human telencephalon in vivo and in vitro, in ES stem cell-derived cell cultures (modified from Nat et al 2012 [3]).

To exemplify the value of this approach we analyzed in greater detail a single process, the step of D/V telencephalic patterning. Thus, we monitored the effect of pharmacological modulators of the Hh signaling pathway, purmorphamine—an agonist and cyclopamine an antagonist acting on the Smoothened receptor (Smo), regarding the expression of regionspecific TFs and signaling molecules relevant for telencephalic development *in vivo*.

Purmorphamine strongly up-regulated the expression of telencephalic ventral markers Nkx2.1, Nkx6.2, Lhx6, and Lhx8 in mouse and human cells, thus reflecting the *in vivo* process of the MGE patterning and specification. Cyclopamine up-regulated the expression of telencephalic dorsal markers, but at lower levels in human compared with mouse cells. Interestingly, the modula‐ tion of Smo *in vitro* differentially affected the expression of molecules of the Hh pathway, espe‐ cially the Gli1 and Gli3 effectors and Ptch receptors, in mouse *vs* human cells.

We additionally examined how the SHH expression itself was modulated by Smo agonist or antagonist treatment. We reported that SHH expression is regulated in a very dynamic way by Hh pathway modulation, both in mES and hES cell-derived models. In particular, a very robust up-regulation of SHH by purmorphamine was observed in the human model, where SHH was not expressed in untreated controls. Shh was expressed in both progenitor cells and neurons in our cultures. This might be explained by the non-cell autonomous mecha‐ nisms recently described in the mouse embryonic telencephalon, where both Lhx8 and Lhx6 genes controlled the expression of Shh in the mantle zone of the MGE, corresponding to ear‐ ly-born neurons [118]. Thus, Lhx6 and Lhx8 appear to regulate MGE development by pro‐ moting Shh expression in MGE neurons, which, in turn, promotes the developmental program of the dMGE.

The activation of Hh signaling *via* Smo with purmorphamine converts the primitive dorsal telencephalic precursors to ventral progenitors. These progenitors differentiate into neuro‐ nal subtypes including GABAergic and cholinergic neurons.

Our results provided evidence for the different default telencephalic differentiation of mouse and human ES cells: ventral and dorsal, respectively. Additionally, it proved the util‐ ity of the comparative system for optimizing the directed differentiation of human pluripo‐ tent stem cells.
