Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient‐specific neural cells

Abstract

Standardization of culture methods for human pluripotent stem cell (PSC) neural differentiation can greatly contribute to the development of novel clinical advancements through the comprehension of neurodevelopmental diseases. Here, we report an approach that reproduces neural commitment from human induced pluripotent stem cells using dual-SMAD inhibition under defined conditions in a vitronectin-based monolayer system. By employing this method it was possible to obtain neurons derived from both control and Rett syndrome patients' pluripotent cells. During differentiation mutated cells displayed alterations in the number of neuronal projections, and production of Tuj1 and MAP2-positive neurons. Although investigation of a broader number of patients would be required, these observations are in accordance with previous studies showing impaired differentiation of these cells. Consequently, our experimental methodology was proved useful not only for the generation of neural cells, but also made possible to compare neural differentiation behavior of different cell lines under defined culture conditions. This study thus expects to contribute with an optimized approach to study the neural commitment of human PSCs, and to produce patient-specific neural cells that can be used to gain a better understanding of disease mechanisms.

Transcriptomic signatures of neuronal differentiation and their association with risk genes for autism spectrum and related neuropsychiatric disorders

“…Yet, it should be acknowledged that embryonic stem cell, iPSC and human NPC models are expensive, and culturing and differentiating of these cells in a reproducible manner is far more difficult than for SH-SY5Y cells. 10 , 11 Recognising the limitations of all model systems, it therefore is important to understand how the optimal protocol for using the efficient SH-SY5Y model system recapitulates in vivo development, which will be helpful in guiding this and future studies. In summary, we focused on improving neuronal differentiation of SH-SY5Y cells because of feasibility, reproducibility, the broad use of this model in previous publications and the large data resources available.…”

Stem Cell and Biologic Scaffold Engineering

“…For E6 differentiation, after overnight growth, the medium was changed to E6 as previously described by Lippmann et al [36]. For dual SMAD Inhibition-based neural induction, after cultures were nearly confluent, the medium was changed to 1:1 N2/B27 media supplemented with 10 μM SB431542 (Stemgent, Cambridge, MA, USA) and 100 nM LDN193189 (Stemgent, Cambridge, MA, USA), as previously described [37,38]. For both protocols, the medium was changed daily, and cells were collected and analyzed at day 12 of differentiation.…”

Towards Multi-Organoid Systems for Drug Screening Applications

“…The same occurs in human pluripotent stem cells (hPSCs), which highlights the ability of the cells to regulate self-organization processes [ 7 ]. hiPSCs also provide the capacity to generate patient-specific cells that allow disease modeling, development of personalized medicine approaches and bypassing rejection in cell therapy settings [ 8 , 9 , 10 ].…”