Spinal muscular atrophy (SMA) is an inherited neuromuscular disease primarily characterized by degeneration of spinal motor neurons, and caused by reduced levels of the SMN protein. Previous studies to understand the proteomic consequences of reduced SMN have mostly utilized patient fibroblasts and animal models. We have derived human motor neurons from type I SMA and healthy controls by creating their induced pluripotent stem cells (iPSCs). Quantitative mass spectrometry of these cells revealed increased expression of 63 proteins in control motor neurons compared to respective fibroblasts, whereas 30 proteins were increased in SMA motor neurons vs. their fibroblasts. Notably, UBA1 was significantly decreased in SMA motor neurons, supporting evidence for ubiquitin pathway defects. Subcellular distribution of UBA1 was predominantly cytoplasmic in SMA motor neurons in contrast to nuclear in control motor neurons; suggestive of neurodevelopmental abnormalities. Many of the proteins that were decreased in SMA motor neurons, including beta III-tubulin and UCHL1, were associated with neurodevelopment and differentiation. These neuron-specific consequences of SMN depletion were not evident in fibroblasts, highlighting the importance of iPSC technology. The proteomic profiles identified here provide a useful resource to explore the molecular consequences of reduced SMN in motor neurons, and for the identification of novel biomarker and therapeutic targets for SMA.
COVID-19 pandemic has infected more than 154 million people worldwide and caused more than 3.2 million deaths. It is transmitted by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and affects the respiratory tract as well as extra-pulmonary systems, including the pancreas, that express the virus entry receptor, Angiotensin-Converting Enzyme 2 (ACE2) receptor. Importantly, the endocrine and exocrine pancreas, the latter composed of ductal and acinar cells, express high levels of ACE2, which correlates to impaired functionality characterized as acute pancreatitis observed in some cases presenting with COVID-19. Since acute pancreatitis is already one of the most frequent gastrointestinal causes of hospitalization in the U.S. and the majority of studies investigating the effects of SARS-CoV-2 on the pancreas are clinical and observational, we utilized human iPSC technology to investigate the potential deleterious effects of SARS-CoV-2 infection on iPSC-derived pancreatic cultures containing endocrine and exocrine cells. Interestingly, iPSC-derived pancreatic cultures allow SARS-CoV-2 entry and establish infection, thus perturbing their normal molecular and cellular phenotypes. The infection increased a key cytokine, CXCL12, known to be involved in inflammatory responses in the pancreas. Transcriptome analysis of infected pancreatic cultures confirmed that SARS-CoV-2 hijacks the ribosomal machinery in these cells. Notably, the SARS-CoV-2 infectivity of the pancreas was confirmed in post-mortem tissues from COVID-19 patients, which showed co-localization of SARS-CoV-2 in pancreatic endocrine and exocrine cells and increased the expression of some pancreatic ductal stress response genes. Thus, we demonstrate that SARS-CoV-2 can directly infect human iPSC-derived pancreatic cells with strong supporting evidence of presence of the virus in post-mortem pancreatic tissue of confirmed COVID-19 human cases. This novel model of iPSC-derived pancreatic cultures will open new avenues for the comprehension of the SARS-CoV-2 infection and potentially establish a platform for endocrine and exocrine pancreas-specific antiviral drug screening.
The hypothalamus contains neurons that integrate hunger and satiety endocrine signals from the periphery and are implicated in the pathophysiology of obesity. The limited availability of human hypothalamic neurons hampers our understanding of obesity disease mechanisms. To address this, we generated human induced pluripotent stem cells (hiPSCs) from multiple normal body mass index (BMI; BMI ≤ 25) subjects and super-obese (OBS) donors (BMI ≥ 50) with polygenic coding variants in obesity-associated genes. We developed a method to reliably differentiate hiPSCs into hypothalamic-like neurons (iHTNs) capable of secreting orexigenic and anorexigenic neuropeptides. Transcriptomic profiling revealed that, although iHTNs maintain a fetal identity, they respond appropriately to metabolic hormones ghrelin and leptin. Notably, OBS iHTNs retained disease signatures and phenotypes of high BMI, exhibiting dysregulated respiratory function, ghrelin-leptin signaling, axonal guidance, glutamate receptors, and endoplasmic reticulum (ER) stress pathways. Thus, human iHTNs provide a powerful platform to study obesity and gene-environment interactions.
The utility of human induced pluripotent stem cells (hiPSCs) is contingent upon genomic integrity and stability. Recurrent genomic aberrations have been observed in human iPSC lines upon long-term culture, ~10-25% demonstrate karyotype abnormalities. We describe a new and reliable non-integrating episomal plasmid reprogramming method for fresh (unexpanded) peripheral blood mononuclear cells (PBMC) into iPSCs (PBMC-iPSCs). PBMC-iPSCs produced using this method have a superior chromosome-level karyotype stability rate (~5% abnormality rate for all chromosomes; 2.8% for autosomes). After extended culture PBMC-iPSCs maintain a low rate of abnormalities (2% for autosomes). Deep coverage whole genome sequencing in a subset of PBMC-iPSC lines showed no shared single nucleotide polymorphisms (SNPs) or structural variants are introduced during reprogramming and maintenance of PBMC-iPSCs. iPSCs reprogrammed from unexpanded PBMCs have consistently high cytogenetic stability and minimal genomic aberrations, suggesting this method is highly suited for iPSCs in research and therapeutic clinical applications.
Persistent exposure to man-made endocrine disrupting chemicals during fetal endocrine development may lead to disruption of metabolic homeostasis contributing to childhood obesity. Limited cellular platforms exist to test endocrine disrupting chemical-induced developmental abnormalities in human endocrine tissues. Here we use an human-induced pluripotent stem cell-based platform to demonstrate adverse impacts of obesogenic endocrine disrupting chemicals in the developing endocrine system. We delineate the effects upon physiological low-dose exposure to ubiquitous endocrine disrupting chemicals including, perfluoro-octanoic acid, tributyltin, and butylhydroxytoluene, in endocrine-active human-induced pluripotent stem cell-derived foregut epithelial cells and hypothalamic neurons. Endocrine disrupting chemicals induce endoplasmic reticulum stress, perturb NF-κB, and p53 signaling, and diminish mitochondrial respiratory gene expression, spare respiratory capacity, and ATP levels. As a result, normal production and secretion of appetite control hormones, PYY, α-MSH, and CART, are hampered. Blocking NF-κB rescues endocrine disrupting chemical-induced aberrant mitochondrial phenotypes and endocrine dysregulation, but not ER-stress and p53-phosphorylation changes.
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