Objective-Children with Hutchinson-Gilford progeria syndrome (HGPS) exhibit dramatically accelerated cardiovascular disease (CVD), causing death from myocardial infarction or stroke between the ages of 7 and 20 years. We undertook the first histological comparative evaluation between genetically confirmed HGPS and the CVD of aging. Methods and Results-We present structural and immunohistological analysis of cardiovascular tissues from 2 children with HGPS who died of myocardial infarction. Both had features classically associated with the atherosclerosis of aging, as well as arteriolosclerosis of small vessels. In addition, vessels exhibited prominent adventitial fibrosis, a previously undescribed feature of HGPS. Importantly, although progerin was detected at higher rates in the HGPS coronary arteries, it was also present in non-HGPS individuals. Between the ages of 1 month and 97 years, progerin staining increased an average of 3.34% per year (PϽ0.0001) in coronary arteries. Conclusion-We find concordance among many aspects of cardiovascular pathology in both HGPS and geriatric patients.HGPS generates a more prominent adventitial fibrosis than typical CVD. Vascular progerin generation in young non-HGPS individuals, which significantly increases throughout life, strongly suggests that progerin has a role in cardiovascular aging of the general population. Key Words: aging Ⅲ atherosclerosis Ⅲ pathology Ⅲ peripheral arterial disease Ⅲ progeria H utchinson-Gilford progeria syndrome (HGPS) is a rare, autosomal-dominant, fatal, progressive premature aging syndrome. Symptoms usually begin with failure to thrive or sclerodermatous skin changes, heralding generalized loss of subcutaneous fat, alopecia, osteopenia and acroosteolysis, and joint contracture. Death occurs at a mean age of 13 years because of myocardial infarction or stroke. 1 The majority of HGPS cases are caused by a single de novo nucleotide substitution at position 1824 (C3 T) in the LMNA gene. 2,3 The normal LMNA protein product, lamin A, is a key component of the inner nuclear lamina, which functions in nuclear structure, chromatin organization, and gene transcription. 4 The silent mutation in HGPS leads to alternative splicing at the 3Ј end of the LMNA mRNA and a 150-nucleotide deletion from the prelamin A transcript resulting in a mutant lamin A protein called progerin, which lacks 50 amino acids near its C-terminal end. 5 In non-HGPS individuals, there is convincing evidence that the HGPS splice site is functional and can lead to progerin accumulation over time, although to a lesser degree than in children with HGPS. 6 In HGPS, the cryptic donor splice site shares 6 of 7 bases with the consensus splice sequence, while non-HGPS individuals share 5 of 7 bases with the consensus splice sequence. Thus, non-HGPS individuals utilize the splice site less often. Progerin is not apparent in early passage non-HGPS cultured fibroblasts and skin biopsies, but it accumulates with increasing cell passage and donor age. 7,8 Thus, progerin is likely a previous...

This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (iPSCs). In this protocol, passaging one six-well or 10 cm plate of cells takes about 6–7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization, centrifugation, or drug treatment. It also allows for higher throughput, requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation, colony expansion, cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion, and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages, this procedure provides a consistent and universal approach to passaging human pluripotent stem cells in E8 medium.

␤-Amyloid peptide (A␤)1 is the essential component of senile plaques (1, 2), which are the major pathological hallmark of Alzheimer's disease (AD) (3). A␤ is derived from proteolytic processing of a ubiquitous transmembrane protein termed ␤-amyloid precursor protein (APP) (4). Although the factors governing production and deposition of A␤ are not fully understood, it has been shown that APP can undergo at least two post-translational processing pathways (3). In a nonamyloidogenic pathway APP is cleaved within the A␤ region by a proteinase activity known as ␣-secretase. This prevents generation of A␤ and gives rise to a soluble form of APP (sAPP␣), which is found in the extracellular milieu and constitutes a marker for nonamyloidogenic processing. Alternatively, APP can undergo proteolytic cleavage by ␤-and ␥-secretases to generate A␤. This is referred to as the amyloidogenic pathway of APP processing, which can take place while internalized cell surface APP trafficks through the endocytic pathway (5).Previous studies have demonstrated that the low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein (LDL) receptor family, binds and mediates the endocytosis of soluble (6) as well as cell surface APP (7) isoforms containing a Kunitz proteinase inhibitor (KPI) domain. LRP is a large, multifunctional endocytic receptor abundantly expressed in liver and brain that mediates the hepatic uptake of circulating chylomicron remnants (8), serpin-enzyme complexes (9), and proteinases of the fibrinolytic pathway (10, 11). Furthermore, LRP has been shown to regulate the cell surface levels of two receptors, the urokinase receptor (12) and tissue factor (13). LRP is also expressed in fibroblasts, macrophages, smooth muscle cells, neurons, and activated but not resting glial cells (14), suggesting that this receptor is involved in the binding and removal of interstitial ligands (e.g. proteinases and lipoproteins) produced by these cells. Targeted deletion of the LRP gene in mice leads to death of the embryo at day 13.5 (15), demonstrating that LRP plays a critical role during development. A 39-kDa receptor-associated protein (RAP) (16) binds reversibly to LRP and other members of the LDL receptor family such as gp330/megalin (17) and the very low density lipoprotein receptor (18) and inhibits ligand binding (19,20). RAP is found primarily in the endoplasmic reticulum, where it is thought to function as a molecular chaperone by assisting in receptor folding and processing and by preventing the association of newly synthesized receptors with endogenous ligands (21,22). Because of its high affinity for LRP and its ability to antagonize ligand binding, RAP constitutes a powerful tool to study LRP-mediated mechanisms.The objective of the present investigation was to test the hypothesis that LRP plays a role in the pathobiology of AD by facilitating delivery of cell surface APP to endosomal compartments, where the A␤ peptide can be generated. To this end, we employed two main experimental strateg...

Highlights d NGN2-BRN3A expression in human stem cells induces homogeneous sensory neurons (iSNs) d Three kinds of iSNs can be produced, including subtypes coexpressing TRPM8 and PIEZO2 d TRPM8 and PIEZO2 unexpectedly mark a subset of sensory neurons in humans, but not mice d iSNs from PIEZO2-deficient patients are insensitive to mechanical stimuli