generation-scaffold-free-three-dimensional-insulin-expressing

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors <em>In Vitro</em>

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Baylor College of Medicine

During postnatal development the heart undergoes a rapid and dramatic transition to adult function through transcriptional and post-transcriptional mechanisms, including alternative splicing (AS). Here we perform deep RNA-sequencing on RNA from cardiomyocytes and cardiac fibroblasts to conduct a high-resolution analysis of transcriptome changes during postnatal mouse heart development. We reveal extensive changes in gene expression and AS that occur primarily between postnatal days 1 and 28. Cardiomyocytes and cardiac fibroblasts show reciprocal regulation of gene expression reflecting differences in proliferative capacity, cell adhesion functions and mitochondrial metabolism. We further demonstrate that AS plays a role in vesicular trafficking and membrane organization. These AS transitions are enriched among targets of two RNA-binding proteins, Celf1 and Mbnl1, which undergo developmentally regulated changes in expression. Vesicular trafficking genes affected by AS during normal development (when Celf1 is downregulated) show a reversion to neonatal splicing patterns after Celf1 re-expression in adults. Short-term Celf1 induction in adult animals results in disrupted transverse tubule organization and calcium handling. These results identify potential roles for AS in multiple aspects of postnatal heart maturation, including vesicular trafficking and intracellular membrane dynamics. 

Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development.

Alternative splicing plays important regulatory roles during periods of physiological change. During development, a large number of genes coordinately express protein isoform transitions regulated by alternative splicing; however, the mechanisms that coordinate splicing and the functional integration of the resultant tissue-specific protein isoforms are typically unknown. Here we show that the conserved Rbfox2 RNA binding protein regulates 30% of the splicing transitions observed during myogenesis and is required for the specific step of myoblast fusion. Integration of Rbfox2-dependent splicing outcomes from RNA-seq with Rbfox2 iCLIP data identified Mef2d and Rock2 as Rbfox2 splicing targets. Restored activities of Mef2d and Rock2 rescued myoblast fusion in Rbfox2-depleted cultures, demonstrating functional cooperation of protein isoforms generated by coordinated alterative splicing. The results demonstrate that coordinated alternative splicing by a single RNA binding protein modulates transcription (Mef2d) and cell signaling (Rock2) programs to drive tissue-specific functions (cell fusion) to promote a developmental transition. 

Rbfox2-coordinated alternative splicing of Mef2d and Rock2 controls myoblast fusion during myogenesis.

Efficient gene editing is essential to fully utilize human pluripotent stem cells (hPSCs) in regenerative medicine. Custom endonuclease-based gene targeting involves two mechanisms of DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR is the preferred mechanism for common applications such knock-in, knock-out or precise mutagenesis, but remains inefficient in hPSCs. Here, we demonstrate that synchronizing synchronizing hPSCs in G2/M with ABT phase increases on-target gene editing, defined as correct targeting cassette integration, 3 to 6 fold. We observed improved efficiency using ZFNs, TALENs, two CRISPR/Cas9, and CRISPR/Cas9 nickase to target five genes in three hPSC lines: three human embryonic stem cell lines, neural progenitors and diabetic iPSCs. neural progenitors and diabetic iPSCs. Reversible synchronization has no effect on pluripotency or differentiation. The increase in on-target gene editing is locus-independent and specific to the cell cycle phase as G2/M phase enriched cells show a 6-fold increase in targeting efficiency compared to cells in G1 phase. Concurrently inhibiting NHEJ with SCR7 does not increase HDR or improve gene targeting efficiency further, indicating that HR is the major DNA repair mechanism after G2/M phase arrest. The approach outlined here makes gene editing in hPSCs a more viable tool for disease modeling, regenerative medicine and cell-based therapies. 

Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases.

Editing genetics, stem cells are prophetic, what's the best way to model cells of diabetics?

Replacement of lost beta cells in patients with diabetes has the potential to alleviate them of their disease, yet current protocols to make beta cells are inadequate for therapy. In vitro screens can reveal the signals necessary for endocrine maturation to improve beta cell production, however the complexities of in vivo development that lead to beta cell formation are lost in two-dimensional systems. Here, we create three-dimensional organotypic pancreatic cultures, named pancreatoids, composed of embryonic day 10.5 murine epithelial progenitors and native mesenchyme. These progenitors assemble in scaffold-free, floating conditions and, with the inclusion of native mesenchyme, develop into pancreatoids expressing markers of different pancreatic lineages including endocrine-like cells. Treatment of pancreatoids with (-)-Indolactam-V or phorbol 12-myristate 13-acetate, two protein kinase C activators, leads to altered morphology which otherwise would be overlooked in two-dimensional systems. Protein kinase C activation also led to fewer Insulin+ cells, decreased Ins1 and Ins2 mRNA levels, and increased Pdx1 and Hes1 mRNA levels with a high number of DBA+ cells. Thus, organotypic pancreatoids provide a useful tool for developmental studies, and can further be used for disease modeling, small molecules and genetic screens, or applied to human pluripotent stem cell differentiation for beta-like cell formation.

Organotypic pancreatoids with native mesenchyme develop Insulin producing endocrine cells.

Regulatory T cells (Tregs) use a distinct TCR repertoire and are more self-reactive compared with conventional T cells. However, the extent to which TCR affinity regulates the function of self-reactive Tregs is largely unknown. In this study, we used a two-TCR model to assess the role of TCR affinity in Treg function during autoimmunity. We observed that high- and low-affinity Tregs were recruited to the pancreas and contributed to protection from autoimmune diabetes. Interestingly, high-affinity cells preferentially upregulated the TCR-dependent Treg functional mediators IL-10, TIGIT, GITR, and CTLA4, whereas low-affinity cells displayed increased transcripts for  and , suggesting distinct functional profiles. The results of this study suggest mechanistically distinct and potentially nonredundant roles for high- and low-affinity Tregs in controlling autoimmunity.

Cutting Edge: Low-Affinity TCRs Support Regulatory T Cell Function in Autoimmunity.

T cell receptor (TCR) affinity is a critical factor of Treg lineage commitment, but whether self-reactivity is a determining factor in peripheral Treg function remains unknown. Here, we report that a high degree of self-reactivity is crucial for tissue-specific Treg function in autoimmunity. Based on high expression of CD5, we identified a subset of self-reactive Tregs expressing elevated levels of T-bet, GITR, CTLA-4, and ICOS, which imparted significant protection from autoimmune diabetes. We observed that T-bet expression in Tregs, necessary for control of Th1 autoimmunity, could be induced in an IFNγ-independent fashion and, unlike in conventional T cells (Tconv), was strongly correlated with the strength of TCR signaling. The level of CD5 similarly identified human Tregs with an increased functional profile, suggesting that CD5hi Tregs may constitute an efficacious subpopulation appropriate for use in adoptive Treg therapies for treatment of inflammatory conditions. Overall, this work establishes an instrumental role of high TCR self-reactivity in driving Treg function.

Article	Year
Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development. Nature communications\| PubMed ID: 24752171	2014
Rbfox2-coordinated alternative splicing of Mef2d and Rock2 controls myoblast fusion during myogenesis. Molecular cell\| PubMed ID: 25087874	2014
Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Scientific reports\| PubMed ID: 26887909	2016
Editing genetics, stem cells are prophetic, what's the best way to model cells of diabetics? Stem cell investigation\| PubMed ID: 28066783	2016
Organotypic pancreatoids with native mesenchyme develop Insulin producing endocrine cells. Scientific reports\| PubMed ID: 28883507	2017
Cutting Edge: Low-Affinity TCRs Support Regulatory T Cell Function in Autoimmunity. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 29282307	2018
High self-reactivity drives T-bet and potentiates Treg function in tissue-specific autoimmunity. JCI insight\| PubMed ID: 29367462	2018

Marissa A. Scavuzzo

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