Epigenetic Control of Endocrine Pancreas Differentiation in vitro: Current Knowledge and Future Perspectives

The raising worldwide prevalence of Type 1 and Type 2 diabetes mellitus (T1DM and T2DM) solicits the derivation of in vitro methods yielding mature and fully functional β-cells to be used in regenerative medicine. Several protocols to differentiate human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into human pancreatic β-like cells have recently been developed. These methods, coupled with a bioengineering approach using biocompatible encapsulating devices, have recently led to experimental clinical trials showing great promises to ultimately end the battle of diabetic patients for managing hyperglycemia. However, in vitro differentiation protocols face the challenge of achieving homogenous population of mono-hormonal insulin-secreting mature β-cells. Major epigenetic events such as DNA methylation, post-translational modification of histones and non-coding RNAs expression, orchestrate physiological endocrine pancreas specification into α-, β-, γ-, and δ-cells, both in vivo and in vitro. The dysregulation of such epigenetic processes is associated to multiple pancreatic disorders including diabetes. Understanding the epigenomic and transcriptomic landscape underlying endocrine pancreas development could, therefore, improve in vitro differentiation methods. In this review, we summarize the most effective protocols for in vitro differentiation of hESCs/hiPSCs toward pancreatic β-cells and we discuss the current limitations in the derivation of functional glucose-responsive, insulin-releasing β-cells. Moreover, we focus on the main transcriptional and epigenetic events leading to pancreatic specification and on the applicative potential of novel epigenetic drugs for the establishment of innovative pharmacological therapeutic approaches

Loss of Either Rac1 or Rac3 GTPase Differentially Affects the Behavior of Mutant Mice and the Development of Functional GABAergic Networks

Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons

Fine-tuned KDM1A alternative splicing regulates human cardiomyogenesis through an enzymatic-independent mechanism

The histone demethylase KDM1A is a multi- faceted regulator of vital developmental processes, including mesodermal and cardiac tube formation during gastrulation. However, it is unknown whether the fine-tuning of KDM1A splicing isoforms, already shown to regulate neuronal maturation, is crucial for the specification and maintenance of cell identity during cardiogenesis. Here, we discovered a temporal modulation of ubKDM1A and KDM1A+2a during human and mice fetal cardiac development and evaluated their impact on the regulation of cardiac differentiation. We revealed a severely impaired cardiac differentiation in KDM1A(-/-) hESCs that can be rescued by re-expressing ubKDM1A or catalytically impaired ubKDM1A-K661A, but not by KDM1A+2a or KDM1A+2a-K661A. Conversely, KDM1A+2a(-/-) hESCs give rise to functional cardiac cells, displaying increased beating amplitude and frequency and enhanced expression of critical cardiogenic markers. Our findings prove the existence of a divergent scaffolding role of KDM1A splice variants, independent of their enzymatic activity, during hESC differentiation into cardiac cells

Protocol to measure calcium spikes in cardiomyocytes obtained from human pluripotent stem cells using a ready-to-use media

Summary: The derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) is a powerful tool to investigate early cardiogenesis and model diseases in vitro. Here, we present an optimized protocol to obtain contracting hPSCs-derived cardiomyocytes using a ready-to-use kit. We describe steps for hPSC culture and differentiation to cardiomyocytes including the identification of key parameters such as starting cell confluency and temperature. We then detail immunofluorescence, flow cytometry, and the quantification of cardiomyocytes' calcium spikes using live imaging.For complete details on the use and execution of this protocol, please refer to Astro et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Effects of the scaffold proteins liprin-α1, β1 and β2 on invasion by breast cancer cells

Background InformationThe expression of the scaffold protein liprin-1 is upregulated in human breast cancer. This protein is part of a molecular network that is important for tumour cell invasion in vitro. Liprin-1 promotes invasion by supporting the protrusive activity at the leading edge of the migrating tumour cell and the degradation of the extracellular matrix by invadopodia. In this study, we have addressed the role of liprin-1 in the invasive process in vivo and of liprin-proteins in tumor cell motility.ResultsThe human tumour cell line MDA-MB-231 expresses liprin-1 and is able to promote the formation of metastasis in mice. Liprin- proteins may hetero-oligomerize with the members of the subfamily of the liprin- adaptor proteins. Analysis of the role of liprin-1 and liprin-2 has shown that while liprin-1 contributes positively to tumour cell motility in vitro; liprin-2 has a negative effect on both cell motility and invasion. Interestingly, we also observed differential effects on the ability of tumour cells to degrade the extracellular matrix, which is required for efficient invasion by tumour cells. In addition, analysis of the formation of lung metastases in vivo revealed that while the overexpression of liprin-1 in MDA-MB-231 cells did not evidently affect the metastatic process, silencing of the endogenous protein strongly impaired the formation of metastases by two independent invasion assays, without inhibiting the growth of primary tumours.ConclusionsOur data support an important role of distinct liprin family members in the regulation of tumour cell invasion, highlighting pro-invasive and anti-invasive effects by liprin-1 and liprin-2, respectively.SignificanceOur results indicate the importance of liprins in breast cancer cell invasion, and are expected to lead to future investigations on the mechanisms underlying the effects of distinct liprin proteins in different processes linked to tumor cell migration and invasion

Hyperphosphorylation of GIT1-N by Src and pervanadate does not affect its binding <i>in vitro</i> to full length GIT1 proteins.

<p>(<b>A–C</b>) COS7 cells were transfected with full length GFP-GIT1 or GIT1-N constructs (WT, FF, or EE), or with wildtype or mutant FLAG-GIT1-N fragments, alone or together with c-Src. The cells cotransfected with c-Src were incubated 20 min at 37°C with 1 mM pervanadate before lysis (pV). Aliquots of the lysates (200 μg of protein) were immunoprecipitated (IP) with anti-FLAG (<b>A,C</b>) or anti-GFP (<b>B</b>) antibodies. For pulldowns shown in (<b>A</b>) and (<b>C</b>): FLAG-immunoprecipitates were washed and incubated for 2 h at 4°C with lysates (250 μg of protein) from cells transfected with the indicated full length GFP-GIT1 constructs. Equal amounts of lysates (25 μg of protein), and the pulldowns performed with GIT1-N without (<b>A</b>) or with c-Src and pervanadate treatment (<b>C</b>) were blotted for the detection of the indicated antigens. In (<b>B</b>) the immunoprecipitations with anti-GFP antibody (right) were immunoblotted to detect the levels of GFP-GIT1-N protein (upper filter) and of its tyrosine phosphorylation (lower filter). (<b>D</b>) COS7 cells were transfected to express the indicated GFP-GIT1 mutants, or transfected with the HA-GIT1-N fragment alone or together with c-Src. The cells co-transfected with c-Src were treated as in (<b>A,C</b>). Aliquots of the lysates from cells expressing GFP-GIT1 mutants (250 μg of protein) were immunoprecipitated with anti-GFP. Pulldowns: GFP-immunoprecipitates were washed and incubated for 2 h at 4°C with lysates (400 μg of protein) from cells transfected with HA-GIT1-N alone, or together with c-Src. Equal amounts of lysates (25 μg of protein), and the pulldowns were blotted for the detection of the indicated antigens.</p

Mutation of tyrosines 246 and 293 does not affect the binding of GIT1 to βPIX.

<p>(<b>A</b>) Lysates from mock-transfected cells or from cells transfected with the indicated GIT1 constructs were immunoprecipitated for GIT1 with anti-GFP antibody. Filters with immunoprecipitates (IP, from 200 μg of protein lysate), and lysates (25 μg) were blotted with anti-GFP (for GIT1), anti-paxillin, and anti-βPIX antibodies. (<b>B</b>) Binding of GIT1-N to GIT1-Y246E/Y293E does not affect the interaction of GIT1-Y246E/Y293E to βPIX. Lysates from COS7 cells co-transfected to express the HA-GIT1-N fragment and the full length GFP-GIT1-WT or GIT1-Y246E/Y293E proteins were immunoprecipitated (IP, from 175 μg of protein lysate) with anti-GFP. Lysates (25 μg, oƒn the lef) and IP (right) were blotted to reveal the full length proteins (anti-GFP), the HA-GIT1-N fragment (anti-HA mAb 12CA5), and endogenous βPIX. (<b>C</b>) Our data support the hypothesis that the tyrosine 293 of the SHD domain of GIT1 is required for the intramolecular interaction, but not for the interaction of the SHD domain of GIT1 with βPIX.</p

Efficient binding of the amino-terminal portion of GIT1 to the carboxy-terminal portion requires the inclusion of the first Spa2 region of the SHD.

<p>(<b>A</b>) Scheme of the constructs of avian GIT1 used in the following experiment. To be noted that tyrosine 284 of avian GIT1 corresponds to tyrosine 293 of human GIT1. (<b>B</b>) COS7 cells were cotransfected with the indicated combination of HA-tagged amino-terminal and FLAG-tagged carboxy-terminal fragments of GIT1. Lysates were immunoprecipitated with the anti-FLAG M2-conjugated beads. Filters with immunoprecipitates (IP, from 250 μg of protein lysate) and lysates (50 μg) were incubated with anti-HA or anti-FLAG antibodies. Amino-terminal fragments in the lysates are indicated by asterisks.</p

Tyrosines 246 and 293 are required to hold GIT1 in a closed conformation.

<p>(<b>A,C,D,F</b>) COS7 cells were cotransfected with the indicated constructs, lysed, and immunoprecipitated with anti-GFP (<b>A,C,F</b>) or anti-FLAG antibodies (<b>D</b>). Filters with immunoprecipitates (IP) and lysates were blotted to reveal the full length GFP-tagged constructs (with anti-GFP), HA-GIT1-N (with mouse 12CA5 mAb), or GIT1-C2-LZ fragment (with anti-FLAG). (<b>B,E,G</b>) Schemes of the interactions proposed from the experiments presented in (<b>A</b>), (<b>C,D</b>), and (<b>F</b>), respectively. The asterisks in (<b>E</b>) indicate the monomeric mutated carboxy-terminal fragment that can not dimerize. See the Results for details.</p