Insulin regulates human pancreatic endocrine cell differentiation in vitro.

OBJECTIVE: The consequences of mutations in genes associated with monogenic forms of diabetes on human pancreas development cannot be studied in a time-resolved fashion in vivo. More specifically, if recessive mutations in the insulin gene influence human pancreatic endocrine lineage formation is still an unresolved question. METHODS: To model the extremely reduced insulin levels in patients with recessive insulin gene mutations, we generated a novel knock-in H2B-Cherry reporter human induced pluripotent stem cell (iPSC) line expressing no insulin upon differentiation to stem cell-derived (SC-) ß cells in vitro. Differentiation of iPSCs into the pancreatic and endocrine lineage, combined with immunostaining, Western blotting and proteomics analysis phenotypically characterized the insulin gene deficiency in SC-islets. Furthermore, we leveraged FACS analysis and confocal microscopy to explore the impact of insulin shortage on human endocrine cell induction, composition, differentiation and proliferation. RESULTS: Interestingly, insulin-deficient SC-islets exhibited low insulin receptor (IR) signaling when stimulated with glucose but displayed increased IR sensitivity upon treatment with exogenous insulin. Furthermore, insulin shortage did not alter neurogenin-3 (NGN3)-mediated endocrine lineage induction. Nevertheless, lack of insulin skewed the SC-islet cell composition with an increased number in SC-ß cell formation at the expense of SC-α cells. Finally, insulin deficiency reduced the rate of SC-ß cell proliferation but had no impact on the expansion of SC-α cells. CONCLUSIONS: Using iPSC disease modelling, we provide first evidence of insulin function in human pancreatic endocrine lineage formation. These findings help to better understand the phenotypic impact of recessive insulin gene mutations during pancreas development and shed light on insulin gene function beside its physiological role in blood glucose regulation.

Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Design and bioprinting for tissue interfaces.

Tissue interfaces include complex gradient structures formed by transitioning of biochemical and mechanical properties in micro-scale. This characteristic allows the communication and synchronistic functioning of two adjacent but distinct tissues. It is particularly challenging to restore the function of these complex structures by transplantation of scaffolds exclusively produced by conventional tissue engineering methods. Three-dimensional (3D) bioprinting technology has opened an unprecedented approach for precise and graded patterning of chemical, biological and mechanical cues in a single construct mimicking natural tissue interfaces. This paper reviews and highlights biochemical and biomechanical design for 3D bioprinting of various tissue interfaces, including cartilage-bone, muscle-tendon, tendon/ligament-bone, skin, and neuro-vascular/muscular interfaces. Future directions and translational challenges are also provided at the end of the paper.

Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

Phosphoproteomics and Organelle Proteomics in Pancreatic Islets.

Over the recent years, mass spectrometry (MS)-based proteomics has undergone dramatic advances in sample preparation, instrumentation, and computational methods. Here, we describe in detail, how a workflow quantifies global protein phosphorylation in pancreatic islets and characterizes intracellular organelle composition on protein level by MS-based proteomics.