Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.

Arginine methylation of METTL14 promotes RNA N6-methyladenosine modification and endoderm differentiation of mouse embryonic stem cells

RNA N6-methyladenosine (m6A), the most abundant internal modification of mRNAs, plays key roles in human development and health. Post-translational methylation of proteins is often critical for the dynamic regulation of enzymatic activity. However, the role of methylation of the core methyltransferase METTL3/METTL14 in m6A regulation remains elusive. We find by mass spectrometry that METTL14 arginine 255 (R255) is methylated (R255me). Global mRNA m6A levels are greatly decreased in METTL14 R255K mutant mouse embryonic stem cells (mESCs). We further find that R255me greatly enhances the interaction of METTL3/METTL14 with WTAP and promotes the binding of the complex to substrate RNA. We show that protein arginine N-methyltransferases 1 (PRMT1) interacts with and methylates METTL14 at R255, and consistent with this, loss of PRMT1 reduces mRNA m6A modification globally. Lastly, we find that loss of R255me preferentially affects endoderm differentiation in mESCs. Collectively, our findings show that arginine methylation of METTL14 stabilizes the binding of the m6A methyltransferase complex to its substrate RNA, thereby promoting global m6A modification and mESC endoderm differentiation. This work highlights the crosstalk between protein methylation and RNA methylation in gene expression.

Co-development of central and peripheral neurons with trunk mesendoderm in human elongating multi-lineage organized gastruloids

Stem cell technologies including self-assembling 3D tissue models provide access to early human neurodevelopment and fundamental insights into neuropathologies. Gastruloid models have not been used to investigate co-developing central and peripheral neuronal systems with trunk mesendoderm which we achieve here in elongating multi-lineage organized (EMLO) gastruloids. We evaluate EMLOs over a forty-day period, applying immunofluorescence of multi-lineage and functional biomarkers, including day 16 single-cell RNA-Seq, and evaluation of ectodermal and non-ectodermal neural crest cells (NCCs). We identify NCCs that differentiate to form peripheral neurons integrated with an upstream spinal cord region after day 8. This follows initial EMLO polarization events that coordinate with endoderm differentiation and primitive gut tube formation during multicellular spatial reorganization. This combined human central-peripheral nervous system model of early organogenesis highlights developmental events of mesendoderm and neuromuscular trunk regions and enables systemic studies of tissue interactions and innervation of neuromuscular, enteric and cardiac relevance.

Sustained intrinsic WNT and BMP4 activation impairs hESC differentiation to definitive endoderm and drives the cells towards extra-embryonic mesoderm

We identified a human embryonic stem cell subline that fails to respond to the differentiation cues needed to obtain endoderm derivatives, differentiating instead into extra-embryonic mesoderm. RNA-sequencing analysis showed that the subline has hyperactivation of the WNT and BMP4 signalling. Modulation of these pathways with small molecules confirmed them as the cause of the differentiation impairment. While activation of WNT and BMP4 in control cells resulted in a loss of endoderm differentiation and induction of extra-embryonic mesoderm markers, inhibition of these pathways in the subline restored its ability to differentiate. Karyotyping and exome sequencing analysis did not identify any changes in the genome that could account for the pathway deregulation. These findings add to the increasing evidence that different responses of stem cell lines to differentiation protocols are based on genetic and epigenetic factors, inherent to the line or acquired during cell culture.

Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

ABSTRACTAlthough many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.

Automated optimization of endoderm differentiation on chip

Here we developed an automated mLSI chip platform with general analytical workflow for 3D stem cell cultures offers the optimization of in vitro generation of various cell types for cell replacement therapies.