Generation and Neuronal Differentiation of Self-Renewing Neuronal Progenitor Cell Lines As a Model to Investigate Synaptic Development and Functions in Patients Affected by the Phelan-Mcdermid Syndrome (PMS)

Background:

Phelan-McDermid syndrome is a developmental disorder characterized by severe expressive language and speech delay, hypotonia, global developmental delay, and autistic behaviour. Haploinsufficiency of the SHANK3/PROSAP2 gene is very likely to be an essential cause of the major neurological features associated with PMS. Shank3 is a large scaffolding protein enriched in the postsynaptic density (PSD) of neuronal synapses. When expressed in cultured hippocampal neurons, Shank3 promotes the maturation and enlargement of dendritic spines while Shank3 mouse mutants showed altered PSD protein composition, reduced size of dendritic spines and weaker basal synaptic transmission. Neuronal progenitor cell derived from human Induced pluripotent stem (iPS) represent an accessible and expandable source of disease specific cell types, offering an opportunity to study neuronal development and degeneration, circuit formation and function, and for generating new in vitro human models of brain diseases.

Objectives:

Generation and neuronal differentiation of self-renewing neuronal progenitor lines obtained from PMS patients and age-matched healthy individuals’ iPS cells (PMS-NP cells) to study the role of Shank3 in synapse formation and function.

Methods:

Fibroblasts from different affected children with the PMS were reprogrammed to iPS cells using the hSTEMCCA-loxP lentivirus. iPS cells were differentiate until obtain self-renewal human neuronal precursors (hNPs) from wild type and disease-related hiPSCs. hNPs were differentiated into functional cortical neurons through three different protocols: by coculturing them with rat primary neurons; glial cells; or simply by culturing them on matrigel. 

Results:  

The hNPs did not express markers of pluripotency such as OCT 3/4 and Tra-1-81 while expressing Nestin, which was progressively lost following the induction of terminal differentiation. The expression of Nestin however was maintained for up to 20 passages in all hNP cells. About 50% of the cells were also positive for the neuronal marker Tuj1 and only a minority of them was positive for the glial marker GFAP. We analyzed neuronal differentiation by using MAP2 for labeling dendrites, and VGLUT1 along with Synaptophysin for labeling synapses. hNP-derived neurons  had elaborate dendritic arbors and many Synaptophysin-positive puncta. VGLUT1 positive puncta were detected starting around day 50 of differentiation. We observed that the majority of the neurons generated from hNP cell cultures were MAP2+/VGLUT1+ excitatory neurons, and we basically did not detect MAP2+/GABA+ cells. However, after 60 days of differentiation of treated co-culture with 1μM retinoic acid, we detected GABAergic neurons and observed a MAP2+/GAD67+ population of cells. Additionally, hNP from patients affected by PMS presented a reduction in the levels of Shank3 compared to controls subjects.

Conclusions:

Here we describe the establishment of replicating neuronal-committed stem cell lines from human iPSCs. These cells can self-renew and specifically generate neuronal lineages that can be differentiated into mature neurons using different methodologies. This model presents a valuable approach to study PMS.