Transcriptional networks identify synaptotagmin-like 3 as a regulator of cortical neuronal migration during early neurodevelopment.

Human brain development is a complex process involving neural proliferation, differentiation, and migration that are directed by many essential cellular factors and drivers. Here, using the NetBID2 algorithm and developing human brain RNA sequencing dataset, we identify synaptotagmin-like 3 (SYTL3) as one of the top drivers of early human brain development. Interestingly, SYTL3 exhibits high activity but low expression in both early developmental human cortex and human embryonic stem cell (hESC)-derived neurons. Knockout of SYTL3 (SYTL3-KO) in human neurons or knockdown of Sytl3 in embryonic mouse cortex markedly promotes neuronal migration. SYTL3-KO causes an abnormal distribution of deep-layer neurons in brain organoids and reduces presynaptic neurotransmitter release in hESC-derived neurons. We further demonstrate that SYTL3-KO-accelerated neuronal migration is modulated by high expression of matrix metalloproteinases. Together, based on bioinformatics and biological experiments, we identify SYTL3 as a regulator of cortical neuronal migration in human and mouse developing brains.

Generation of an induced pluripotent stem cell line from an Alström Syndrome patient with ALMS1 mutation (c.3902C > A, c.6436C > T) and a gene correction isogenic iPSC line.

To develop a disease model for the human Alström Syndrome (AS), we used the episomal reprogramming system and CRISPR/Cas9 technology to generate an induced pluripotent stem cell (iPSC) line with the compound heterozygous patient mutation (ALMS1 c.3902C > A, c.6436C > T) along with an isogenic gene-corrected control iPSC line. Both iPSC lines showed normal karyotype, expressed pluripotent markers, and differentiated into cells of three embryonic germ layer. These AS mutant and isogenic iPSC control line will be of great use in investigating the disease mechanisms, drug screening and treatment in patients.

Mutations of CNTNAP1 led to defects in neuronal development.

Mutations of CNTNAP1 were associated with myelination disorders, suggesting the role of CNTNAP1 in myelination processes. Whether CNTNAP1 may have a role in early cortical neuronal development is largely unknown. In this study, we identified 4 compound heterozygous mutations of CNTNAP1 in 2 Chinese families. Using mouse models, we found that CNTNAP1 is highly expressed in neurons and is located predominantly in MAP2+ neurons during the early developmental stage. Importantly, Cntnap1 deficiency results in aberrant dendritic growth and spine development in vitro and in vivo, and it delayed migration of cortical neurons during early development. Finally, we found that the number of parvalbumin+ neurons in the cortex and hippocampus of Cntnap1-/- mice is strikingly increased by P15, suggesting that excitation/inhibition balance is impaired. Together, this evidence elucidates a critical function of CNTNAP1 in cortical development, providing insights underlying molecular and circuit mechanisms of CNTNAP1-related disease.

JMJD3 acts in tandem with KLF4 to facilitate reprogramming to pluripotency.

The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3's catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.

Generation of two induced pluripotent stem cell (iPSC) lines from human breast milk using episomal reprogramming system.

Human breast milk epithelial cells (BMECs) can be isolated and cultured with high purity. Induced pluripotent stem cells (iPSCs) were generated from BMECs with Yamanaka factors (OCT4, SOX2, c-MYC, KLF4) using episomal system. Pluripotency of breast milk-derived iPSCs (BM-iPSCs) was confirmed by the expression of pluripotent markers with immunocytochemistry and spontaneous differentiation of three germ layers in vitro and teratoma formation assay in vivo. Besides, the iPSC lines displayed normal karyotype. Breast milk is a non-invasive and easily accessible cell source, we can obtain BM-iPSCs from BMECs with low costs in a transgene-free episomal system.