Differentiation of human telencephalic progenitor cells into MSNs by inducible expression of Gsx2 and Ebf1

Abstract

Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative disorder, Huntington's disease. Understanding how ventral neuroepithelial progenitors differentiate into MSNs is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells. Studies performed in murine models have identified some transcriptional determinants, including GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1). Here, we have generated human embryonic stem (hES) cell lines inducible for these transcription factors, with the aims of (i) studying their biological role in human neural progenitors and (ii) incorporating TF conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 delays cell-cycle exit and reduces Pax6 expression, whereas Ebf1 promotes neuronal differentiation. Moreover, we found that Gsx2 and Ebf1 combined overexpression in hES cells achieves high yields of MSNs, expressing Darpp32 and Ctip2, in vitro as well in vivo after transplantation. We show that hES-derived striatal progenitors can be transplanted in animal models and can differentiate and integrate into the host, extending fibers over a long distance.he striatum is the largest component of the basal ganglia, it is the hub of converging excitatory connections from the cortex and thalamus, and it originates the direct and indirect pathways, which are distinct basal ganglia circuits involved in motor control (1). In humans, the degeneration of the principal striatal neuronal population, the medium spiny neurons (MSNs), causes a severe neurodegenerative condition, Huntington's disease (HD). A main goal in the field is the study of the mechanisms underlying neuronal specification and degeneration. A large number of studies performed in model organisms, such as the mouse model organism, have provided fundamental insights into brain development, shedding light on genes, signaling pathways, and general rules of brain formation. It is not incidental to point out that obvious speciesspecific differences exist in many aspects between mice and humans (gestation, morphology, and gene expression regulation in time and space). Thus, additional model systems are needed to uncover specific functions of a gene in human development (2, 3). This task is also driven by the need to investigate neurological diseases, such as HD, in a model that more closely resembles human biology.Here, we decided to take advantage of human embryonic stem (hES) cells to develop a model to study the roles of selected transcription factors (TFs) in human striatal development and as a strategy to increase recovery of authentic MSNs for transplantation purposes. During brain development, a set of TFs are expressed in different regions and times and cooperate to establish a dorsalventral and medial-lateral positional identity in progenitor cells and to specify neuronal terminal differentiation. In particular, in...

Active intermixing of indirect and direct neurons builds the striatal mosaic

“…While both SPN derive from the LGE 19,22-24,49 , producing fully differentiated dSPN or iSPN in vitro [77][78][79][80][81] has remained a challenge. While there was increasing evidence that distinct transcriptional programs underlie the specification of both subtypes 23, 41,45,46 , in vitro studies raised questions regarding the mechanisms controlling SPN differentiation.…”

Regenerative Approaches in Huntington’s Disease: From Mechanistic Insights to Therapeutic Protocols

“…The majority of recent transplantation studies were performed using the Quinolinic Acid (QA) excitotoxic lesion model, as it induces a selective loss of striatal MSNs with a relative preservation of interneurons, largely resembling the neuropathological features of human HD ( Beal et al, 1991 ). In these studies human progenitor cells, either hESCs or hiPSCs were, prior to their transplantation, in vitro differentiated to striatal progenitors or immature MSNs, either through directed differentiation protocols modulating the levels of extrinsic developmental signals, such as BMP/TGFβ ( Carri et al, 2013 ), Sonic Hedgehog (SHH) and Activin A ( Arber et al, 2015 ) or by forced expression of transcription factors (TFs) involved in MSNs differentiation, such as GSX2 and EBF1 ( Faedo et al, 2017 ). In these studies, transplantation of the enriched populations of striatal progenitors resulted in their functional integration into the lesioned striatum, a subpopulation of which differentiated to DARPP-32 + MSNs ( Arber et al, 2015 ), extended fibers over a long distance ( Faedo et al, 2017 ), projected to the substantia nigra and received GABAergic and glutamatergic inputs, leading to restoration of apomorphine-induced rotational behavior ( Ma et al, 2012 ).…”

“…In these studies human progenitor cells, either hESCs or hiPSCs were, prior to their transplantation, in vitro differentiated to striatal progenitors or immature MSNs, either through directed differentiation protocols modulating the levels of extrinsic developmental signals, such as BMP/TGFβ ( Carri et al, 2013 ), Sonic Hedgehog (SHH) and Activin A ( Arber et al, 2015 ) or by forced expression of transcription factors (TFs) involved in MSNs differentiation, such as GSX2 and EBF1 ( Faedo et al, 2017 ). In these studies, transplantation of the enriched populations of striatal progenitors resulted in their functional integration into the lesioned striatum, a subpopulation of which differentiated to DARPP-32 + MSNs ( Arber et al, 2015 ), extended fibers over a long distance ( Faedo et al, 2017 ), projected to the substantia nigra and received GABAergic and glutamatergic inputs, leading to restoration of apomorphine-induced rotational behavior ( Ma et al, 2012 ). In a very recent study, a hydrogel scaffold has been used for the more effective, rapid and scalable directed differentiation of human iPSCs to striatal progenitors in three-dimensional (3D) organoid-like structures ( Adil et al, 2018 ).…”

Concise Review: The Use of Stem Cells for Understanding and Treating Huntington's Disease

“…To increase lineage specificity and encourage differentiation into MSNs, several groups differentiated hESCs into lateral ganglionic eminence progenitors or striatal precursors, although using a range of different protocols (Table ). All of these studies reported the generation of MSNs following transplantation into the QA‐lesioned striatum demonstrating the requirement to drive hESCs to a lineage‐specific progenitor fate before transplant.…”