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Generation of Striatal Neurons from Mouse and Human Embryonic Stem Cells: its Relevance for Regenerative Medicine in Huntington's Disease and for Studying Striatal Development

Final Report Summary - HESC DIFFERENTIATION (Generation of Striatal Neurons from Mouse and Human Embryonic Stem Cells: its Relevance for Regenerative Medicine in Huntington's Disease and for Studying Striatal Development.)

The coordination of embryonic human brain development is a process involving multiple players, among which transcription factors (TFs) play a fundamental role. Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative condition, Huntington's disease. Understanding how the human ventral telencephalon drives neuroepithelial progenitors towards this specific neuronal lineage is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells.
Studies performed in murine models have identified a handful of transcriptional determinants, including Gsx2 and Ebf1. Here, we have generated human embryonic stem (hES) cell lines inducible for these TFs, with the aim (i) of studying their biological role in human neural progenitors, and (ii) of incorporating TFs conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 promotes cell cycle retention, while Ebf1 co-expression reverses this phenotype. Moreover, we show that the inducible Gsx2-Ebf1 combined expression in a specific temporal window drives MSNs differentiation from hES cells.
We have previously demonstrated that human ventral telencephalic progenitors can be generated from hES cells by using a Shh-treatment coupled with Wnt-inhibition. These progenitors eventually differentiate into mature, electro-physiologically mature neurons. However, Gsx2 expression showed an inconsistent expression across lines, and the protocol yielded cultures containing Darpp32+-Ctip2+ cells never exceeding 10-15%.
We therefore wished to establish a hES cell-based inducible gain-of-function (iGOF) model system whereby transcription factors expressed in the developing striatum can be harnessed to improve MSNs differentiation and to study human striatal development. We uncovered novel roles for Gsx2 and Ebf1 during human striatal specification and differentiation, in particular in cell cycle regulation. Moreover, we report that a specific temporal window of Gsx2 and Ebf1 over-expression in hES cells achieves high yields of MSNs, expressing Darpp32 and Ctip2.

We decided to develop an inducible over-expression system in hES H9 cells. To this goal, we modified a commercially available TetON system (Clontech) by moving the TetON cassette into a chicken beta-actin promoter with CMV enhancer-based plasmid (pCAG), in order to avoid silencing effects. This construct was introduced by nucleofection in hES H9 cells (p40-p70) along with a linear construct encoding for puromycin. After selection, several stable hES cell clones were picked, amplified, and tested for inducibility first by using a pTRE-Luciferase construct: we selected four clones that showed no basal Luciferase activity and high induction after 48hrs of doxycycline treatment. Next, we performed transient transfections in these four clones using a pTRE responsive vector where the Gsx2 gene was cloned (pTRE-Gsx2). After 48 hours of doxycycline treatment the cells were fixed and analyzed by immunofluorescence for Gsx2 expression. The clones showed a doxycycline response very similar to the Luciferase assay, with no basal Gsx2 expression. We amplified and characterized two clones that showed the highest Gsx2 and Luciferase expression after transient transfections. Next, we constructed a second conditional vector with Gsx2 expressed alongside with Ebf1 by means of an IRES2 sequence (pTRE-Gsx2-Ebf1). Using the pTRE-Gsx2 and pTRE-Gsx2-Ebf1 constructs, we carried out nucleofections in the hES inducible clones. After selection, several stable hES cell clones were picked, amplified, and tested for Gsx2 and Gsx2-Ebf1 expression. Four Gsx2 and one Gsx2-Ebf1 over-expressing clones were chosen for the next experiments. We next examined transgenes expression by a time-course analysis, in order to study doxycycline activation timing. We treated the cells with doxycycline for 8, 24, and 48 hours in Gsx2 and Gsx2-Ebf1 inducible lines, finding a peak of expression at around 48-72 hours. Quantification of Gsx2+ cells after 24hrs of doxycycline induction in hES cells showed 43.82±2.67% and 46.34±1.93% of Gsx2 expression in Gsx2 and Gsx2-Ebf1 iGOF lines, respectively.
Next, we studied if the two generated clonal lines responded appropriately to a specific neuronal differentiation protocol previously characterized in hES H9 cells. We found that at day40 both cell lines expressed specific neuronal markers as Map2, Calbindin, Foxp2, Ctip2, and Gaba. Thus, the process of genetic modification and selection did not alter the capacity of these H9 sub-clones to differentiate toward a neuronal lineage. Therefore, we used these two inducible lines to study hES cells differentiation in striatal cells, in order to gain insights into human striatal development, and to increase MSNs differentiation.

First, we investigated the patterning activity of Gsx2 at early-mid stages of neuronal differentiation in hES cells. The data indicated that in hES cells that are undergoing neuronal conversion Gsx2 and Gsx2-Ebf1 iGOF instruct a ventral identity by suppressing the dorsal marker Pax6 (Figure 1) while maintaining typical neuroepithelial markers (Otx2, N-Cadherin). We next investigated if Gsx2 and Gsx2-Ebf1 expression modified cell proliferation.
The data showed that Gsx2 has a prominent role in inhibiting cell cycle progression and cell differentiation, and that other TFs (like Ebf1) should be turned on sequentially in order to promote neurogenesis (Figure 2). Gsx2 iGOF showed a marked reduction of Map2+ cells, and this phenotype was rescued in the Gsx2-Ebf1 iGOF line, in agreement with the cell cycle analysis data and further suggesting that Gsx2+ cells could not exit the cell cycle, and needed a post-mitotic inducer as Ebf1 to progress through cell cycle.
We next examined the striatal differentiation potential of the Gsx2-Ebf1 iGOF line. Interestingly, we found a more consistent MSNs differentiation efficiency: in four out of five experiments that reached day 80 we observed a higher efficiency of Darpp32+/Ctip2+ neurons compared both to the control line (from 3.78±3.08 in basal condition to 38.76±13.70 in iGOF, and to the Gsx2 iGOF line (from 16.64±3.08% to 38.76±13.70%). Also for this line we confirmed that Darpp32+ cells expressed Map2.
This study aimed to achieve two goals: (1) to study Gsx2 and Ebf1function in human ventral telencephalic development, and (2) to improve MSNs differentiation from hES cells by transcriptional specification. In each of these efforts, we have succeeded in applying an iGOF system for forcing TFs expression in defined temporal windows, and in combining this approach with a morphogens-driven ventral telencephalic specification. To our knowledge, this is the first manipulation of telencephalic determinants in hES-derived neural progenitors.