A Novel Human Pluripotent Stem Cell-Derived Neural Crest Model of Treacher Collins Syndrome Shows Defects in Cell Death and Migration

Abstract

The neural crest (NC) is a transient multipotent cell population present during embryonic development. The NC can give rise to multiple cell types and is involved in a number of different diseases. Therefore, the development of new strategies to model NC in vitro enables investigations into the mechanisms involved in NC development and disease. In this study, we report a simple and efficient protocol to differentiate human pluripotent stem cells (HPSC) into NC using a chemically defined media, with basic fibroblast growth factor 2 (FGF2) and the transforming growth factor-β inhibitor SB-431542. The cell population generated expresses a range of NC markers, including P75, TWIST1, SOX10, and TFAP2A. NC purification was achieved in vitro through serial passaging of the population, recreating the developmental stages of NC differentiation. The generated NC cells are highly proliferative, capable of differentiating to their derivatives in vitro and engraft in vivo to NC specific locations. In addition, these cells could be frozen for storage and thawed with no loss of NC properties, nor the ability to generate cellular derivatives. We assessed the potential of the derived NC population to model the neurocristopathy, Treacher Collins Syndrome (TCS), using small interfering RNA (siRNA) knockdown of TCOF1 and by creating different TCOF1+/− HPSC lines through CRISPR/Cas9 technology. The NC cells derived from TCOF1+/− HPSC recapitulate the phenotype of the reported TCS murine model. We also report for the first time an impairment of migration in TCOF1+/− NC and mesenchymal stem cells. In conclusion, the developed protocol permits the generation of the large number of NC cells required for developmental studies, disease modeling, and for drug discovery platforms in vitro.

Genomic profiling of human vascular cells identifies TWIST1 as a causal gene for common vascular diseases

“…To determine a possible role of TWIST1 in vascular disease, we next investigated TWIST1 abundance and function in vascular smooth muscle cells. Using a human pluripotent stem cell derived neural crest model of in vitro lineage-specific differentiation of vascular smooth muscle cells [39], we found that TWIST1 expression was highest at the SMC progenitor stage, particularly in neural crest progenitor cells which selectively contribute to formation of the aortic arch ( Fig 5A). TWIST1 expression was decreased in the differentiated SMCs arising from these progenitors.…”

MAPRE2 mutations result in altered human cranial neural crest migration, underlying craniofacial malformations in CSC-KT syndrome

“…Neural crest cells were first discovered in chicken embryos and later their unique features have been revealed in other avian and amphibian embryos 32 – 34 . Chicken embryos have been used as a model to study human stem cell potency 35 , 36 and are nowadays used to study neurocristopathies 37 , 38 . In order to further assess the cranial migration capacity of our MAPRE2 haploinsufficient cell lines, we performed orthotopic xenotransplantation of embryoid bodies (EB) generated from eGFP labelled cells (Fig.…”

AN1-type zinc finger protein 3 (ZFAND3) is a transcriptional regulator that drives Glioblastoma invasion

“…7j). Of interest, these three proteins have been previously involved in cell motility in cancer or during development [41][42][43] . Binding sites for ZF containing DNA-binding proteins are enriched in the promoter regions of COL6A2, NRCAM, or FN1, which were previously described in specific invasive signatures in GBM 44,45 .…”