Transcriptional programs regulating neuronal differentiation are disrupted in DLG2 knockout human embryonic stem cells and enriched for schizophrenia and related disorders risk variants

Coordinated programs of gene expression drive brain development. It is unclear which transcriptional programs, in which cell-types, are affected in neuropsychiatric disorders such as schizophrenia. Here we integrate human genetics with transcriptomic data from differentiation of human embryonic stem cells into cortical excitatory neurons. We identify transcriptional programs expressed during early neurogenesis in vitro and in human foetal cortex that are down-regulated in DLG2−/− lines. Down-regulation impacted neuronal differentiation and maturation, impairing migration, morphology and action potential generation. Genetic variation in these programs is associated with neuropsychiatric disorders and cognitive function, with associated variants predominantly concentrated in loss-of-function intolerant genes. Neurogenic programs also overlap schizophrenia GWAS enrichment previously identified in mature excitatory neurons, suggesting that pathways active during prenatal cortical development may also be associated with mature neuronal dysfunction. Our data from human embryonic stem cells, when combined with analysis of available foetal cortical gene expression data, de novo rare variants and GWAS statistics for neuropsychiatric disorders and cognition, reveal a convergence on transcriptional programs regulating excitatory cortical neurogenesis.

A novel RNA-mediated mechanism causing down-regulation of insulating promoter interactions in human embryonic stem cells

The genome-wide promoter interactome is primarily maintained and regulated by architectural proteins such as CTCF and cohesin. However, some studies suggest a role for non-coding RNAs (ncRNAs) in this process. We aimed to characterise the regulatory role of RNA-mediated promoter interactions in the control of gene expression. We integrated genome-wide datasets of RNA-chromatin and promoter-genome interactions in human embryonic stem cells (hESCs) to identify putative RNA-mediated promoter interactions. We discovered that CTCF sites were enriched in RNA-PIRs (promoter interacting regions co-localising with RNA-chromatin interaction sites) and genes interacting with RNA-PIRs containing CTCF sites showed higher expression levels. One of the long noncoding RNAs (lncRNAs) expressed in hESCs, Syntaxin 18-Antisense 1 (STX18-AS1), appeared to be involved in an insulating promoter interaction with the neighbouring gene, MSX1. By knocking down STX18-AS1, the MSX1 promoter-PIR interaction was intensified and the target gene (MSX1) expression was down-regulated. Conversely, reduced MSX1 promoter-PIR interactions, resulting from CRISPR-Cas9 deletion of the PIR, increased the expression of MSX1. We conclude that STX18-AS1 RNA antagonised local CTCF-mediated insulating promoter interactions to augment gene expression. Such down-regulation of the insulating promoter interactions by this novel mechanism may explain the higher expression of genes interacting with RNA-PIRs linked to CTCF sites.

Delayed DNA replication in haploid human embryonic stem cells

Haploid human embryonic stem cells (ESCs) provide a powerful genetic system but diploidize at high rates. We hypothesized that diploidization results from aberrant DNA replication. To test this, we profiled DNA replication timing in isogenic haploid and diploid ESCs. The greatest difference was the earlier replication of the X Chromosome in haploids, consistent with the lack of X-Chromosome inactivation. We also identified 21 autosomal regions that had delayed replication in haploids, extending beyond the normal S phase and into G2/M. Haploid-delays comprised a unique set of quiescent genomic regions that are also underreplicated in polyploid placental cells. The same delays were observed in female ESCs with two active X Chromosomes, suggesting that increased X-Chromosome dosage may cause delayed autosomal replication. We propose that incomplete replication at the onset of mitosis could prevent cell division and result in re-entry into the cell cycle and whole genome duplication.

Transplantation of human embryonic stem cells alleviates motor dysfunction in AAV2-Htt171-82Q transfected rat model of Huntington’s disease

Background Human embryonic stem cells (hESCs) transplantation had shown to provide a potential source of cells in neurodegenerative disease studies and lead to behavioral recovery in lentivirus transfected or, toxin-induced Huntington's disease (HD) rodent model. Here, we aimed to observe if transplantation of superparamagnetic iron oxide nanoparticle (SPION)-labeled hESCs could migrate in the neural degenerated area and improve motor dysfunction in an AAV2-Htt171-82Q transfected Huntington rat model. Methods All animals were randomly allocated into three groups at first: HD group, sham group, and control group. After six weeks, the animals of the HD group and sham group were again divided into two subgroups depending on animals receiving either ipsilateral or contralateral hESCs transplantation. We performed cylinder test and stepping test every two weeks after AAV2-Htt171-82Q injection and hESCs transplantation. Stem cell tracking was performed once per two weeks using T2 and T2*-weighted images at 4.7 Tesla MRI. We also performed immunohistochemistry and immunofluorescence staining to detect the presence of hESCs markers, huntingtin protein aggregations, and iron in the striatum. Results After hESCs transplantation, the Htt virus-injected rats exhibited significant behavioral improvement in behavioral tests. SPION labeled hESCs showed migration with hypointense signal in MRI. The cells were positive with βIII-tubulin, GABA, and DARPP32. Conclusion Collectively, our results suggested that hESCs transplantation can be a potential treatment for motor dysfunction of Huntington's disease.

Colloidal Self-Assembled Patterns Maintain the Pluripotency and Promote the Hemopoietic Potential of Human Embryonic Stem Cells

The generation of blood cells in a significant amount for clinical uses is still challenging. Human pluripotent stem cells-derived hemopoietic cells (hPSC-HCs) are a promising cell source to generate blood cells. Previously, it has been shown that the attached substrates are crucial in the maintenance or differentiation of hPSCs. In this study, a new family of artificial extracellular matrix (ECM) called colloidal self-assembled patterns (cSAPs: #1–#5) was used for the expansion of mouse and human PSCs. The optimized cSAP (i.e., #4 and #5) was selected for subsequent hemopoietic differentiation of human embryonic stem cells (hESCs). Results showed that the hematopoietic potential of hESCs was enhanced approx 3–4 folds on cSAP #5 compared to the flat control. The cell population of hematopoietic progenitors (i.e., CD34+CD43+ cells) and erythroid progenitors (i.e., CD71+GPA+ cells) were enhanced 4 folds at day 8 and 3 folds at day 14. RNA sequencing analysis of cSAP-derived hESCs showed that there were 300 genes up-regulated and 627 genes down-regulated compared to the flat control. The enriched signaling pathways, including up-regulation (i.e., Toll-like receptor, HIF-1a, and Notch) or down-regulation (i.e., FAs, MAPK, JAK/STAT, and TGF-β) were classic in the maintenance of hESC phenotype Real time PCR confirmed that the expression of focal adhesion (PTK2, VCL, and CXCL14) and MAPK signaling (CAV1) related genes was down-regulated 2-3 folds compared to the flat control. Altogether, cSAP enhances the pluripotency and the hematopoietic potential of hESCs that subsequently generates more blood-like cells. This study reveals the potential of cSAPs on the expansion and early-stage blood cell lineage differentiation of hPSCs.