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Human cellular modelling and genetic dissection of the trisomy 21 effects on early brain development.

Objectives: To describe neuronal differentiation from the disomic (D21) and trisomic (T21) isogenic human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from persons with partial and full trisomy 21, and mosaic Down syndrome (DS). Methods: NSC were generated form the isogenic iPSCs following published protocol (1) and expanded in our laboratory. NSCs, between passage 5 and 10 were seeded on the commercially available mouse astrocytes and differentiated to the mature neurons 100 days in vitro (DIV) (2). Moreover, cerebral organoids were generated following published protocol (3) with our modification (4) and fixed at 30, 50, 70 and 100 DIV and analysed by immunohistochemistry. Results: In our previous work we published neuronal differentiation from EBs D3 and 47-1 (5). Here we find that Tubb3 positive cells show a specific neuronal phenotype. Disomic cells showed normal neuronal morphology, on the other hand trisomic cells showed a very specific cellular dysmorphology: neurons were smaller, shorter with thicker processes, and abnormal branching patterns. The same pattern we have observed in neurons derived form iPSCs in monolayer as well as in cerebral organoids. Both, neurons in monolayer, as well as in cerebral organoids expressed all neuronal markers: pan neuronal markers (Dcx, MAP2. Tubb3, SMI, 3R-Tau) and cortical layer specific markers (Reelen, Brn2, Ctip2, SATB2, TBR1 and TBR2). Conclusion: Trisomic cells could differentiate to mature, synaptic active neurons but morphology of trisomic cells shows a unique and specific dysmorphology pattern.

Human iPSC, Trisomy 21, Brain Development

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