Generation of cardiomyocytes from human-induced pluripotent stem cells resembling atrial cells with ability to respond to adrenoceptor agonists

Atrial fibrillation (AF) is the most common chronic arrhythmia presenting a heavy disease burden. We report a new approach for generating cardiomyocytes (CMs) resembling atrial cells from human-induced pluripotent stem cells (hiPSCs) using a combination of Gremlin 2 and retinoic acid treatment. More than 40% of myocytes showed rod-shaped morphology, expression of CM proteins (including ryanodine receptor 2, α-actinin-2 and F-actin) and striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes (AMs). Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an AM profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately −70 mV. Single-cell RNA sequence analysis showed a high level of expression of several atrial-specific transcripts including NPPA, MYL7, HOXA3, SLN, KCNJ4, KCNJ5 and KCNA5. Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of α-adrenoceptors (activated by phenylephrine and blocked by prazosin) or β-adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Our new approach provides human AMs with mature characteristics from hiPSCs which will facilitate drug discovery by enabling the study of human atrial cell signalling pathways and AF. This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’

Sustained levels of FGF2 maintain undifferentiated stem cell cultures with biweekly feeding.

An essential aspect of stem cell culture is the successful maintenance of the undifferentiated state. Many types of stem cells are FGF2 dependent, and pluripotent stem cells are maintained by replacing FGF2-containing media daily, while tissue-specific stem cells are typically fed every 3rd day. Frequent feeding, however, results in significant variation in growth factor levels due to FGF2 instability, which limits effective maintenance due to spontaneous differentiation. We report that stabilization of FGF2 levels using controlled release PLGA microspheres improves expression of stem cell markers, increases stem cell numbers and decreases spontaneous differentiation. The controlled release FGF2 additive reduces the frequency of media changes needed to maintain stem cell cultures, so that human embryonic stem cells and induced pluripotent stem cells can be maintained successfully with biweekly feedings

Intermediate filament protein accumulation in motor neurons derived from giant axonal neuropathy iPSCs rescued by restoration of gigaxonin

Giant axonal neuropathy (GAN) is a progressive neurodegenerative disease caused by autosomal recessive mutations in the GAN gene resulting in a loss of a ubiquitously expressed protein, gigaxonin. Gene replacement therapy is a promising strategy for treatment of the disease; however, the effectiveness and safety of gigaxonin reintroduction have not been tested in human GAN nerve cells. Here we report the derivation of induced pluripotent stem cells (iPSCs) from three GAN patients with different GAN mutations. Motor neurons differentiated from GAN iPSCs exhibit accumulation of neurofilament (NF-L) and peripherin (PRPH) protein and formation of PRPH aggregates, the key pathological phenotypes observed in patients. Introduction of gigaxonin either using a lentiviral vector or as a stable transgene resulted in normalization of NEFL and PRPH levels in GAN neurons and disappearance of PRPH aggregates. Importantly, overexpression of gigaxonin had no adverse effect on survival of GAN neurons, supporting the feasibility of gene replacement therapy. Our findings demonstrate that GAN iPSCs provide a novel model for studying human GAN neuropathologies and for the development and testing of new therapies in relevant cell types

Culture with FGF2 beads and biweekly feeding produces a more undifferentiated hESC culture than traditional daily feeding.

<p>(A) hESCs grown one week on MEF feeders exhibit a similar FACS stem cell profile when fed with FGF2beads in medium every third day compared to soluble FGF2 in medium daily. However, the expression of Nanog was significantly increased and the differentiation markers SOX17 and Brachyury were significantly reduced, by qRT-PCR (left panel). (B) A similar but more pronounced effect was seen for hESCs grown for one month. (C) hESCs grown one week on matrigel exhibit a similar FACS stem cell profile when fed with medium+FGF2 beads every third day compared to medium+soluble FGF2 added daily. A similar effect was seen for hESCs grown for one month. After one month of growth, the pluripotency markers OCT4 and Nanog can be visualised by immunocytochemisty in both conditions. In addition, the expression of differentiation markers PAX6, SOX17 and Brachyury were significantly less by qRT-PCR. (D) Normal Karyotypes for hESCs were assessed before expansion and then after one month of expansion in either soluble FGF2 or FGF2 beads, and neither showed abnormalities.</p

Sustained levels of FGF2 in stem cell culture improve activation of the MAPK pathway.

<p>(A) hESCs were fed with medium containing soluble FGF2 or FGF2 beads, 25 ugs of protein were loaded for each sample and pERK2 levels were assessed over the subsequent 48 hours by western blot (left panel), quantified by densitometry (right panel). FGF2 beads more efficiently activated the MAPK pathway, seen as early as 15 minutes post-addition, with activity still higher than baseline at 48 hours. (B) For hESCs, a global view of expression of 85 genes involved in the MAPK pathway reveals a very different signature in cells treated with FGF2 beads compared to soluble FGF2 at 48 hours. (C) qRT-PCR of specific genes in the pathway shows cell cycle inhibitors such as CDKN2B and CDKN2C were downregulated, while positive MAPK regulators MAPK1 and CCNE1 were upregulated in FGF2 bead culture compared to soluble FGF2. Genes listed were significantly different compared to soluble FGF2 at 48 hours.</p

FGF2 maintain iPSC and increase differentiation potential of hESC.

<p>(A) iPSC lines were derived and assessed for pluripotency markers (B) iPSCs grown for two weeks on matrigel exhibit a similar FACS stem cell profile when fed with medium+FGF2 beads every third day compared to conventional medium+soluble FGF2 daily feeding. Scale bar = 50 microns. (C) Using directed differentiation protocols, hESC differentiation potential was assessed for all three germ lineages. From left to right panels, hESCs grown using FGF2 beads gave rise to neural, mesodermal, and endodermal progeny, assessed by FACS for relevant markers. (D) By qRT-PCR, FGF2 Beads expanded hESCs expressed higher levels of lineage markers after differentiation to all lineages.</p

Sustained levels of FGF2 are achieved using PLGA microspheres.

<p>(A) A FACS based assay was used to measure FGF2 levels in culture. This method was sensitive at a range of concentrations and specific to FGF2. On the y-axis is Mean Fluorescence Intensity (MFI), the x-axis is concentrations of FGF2. (B) Standard daily medium changes in hESC culture lead to drastic fluctuations in FGF2 levels due to poor FGF2 stability. For mNSCs, the standard every 3rd day medium change leaves cultures with little FGF2 after the first 24 hours. (C) FGF2 beads visualized in cultures (arrows) have an average diameter of 14.5 microns scale bar = 100 microns. (D) Using PLGA microspheres, FGF2 is released at a constant rate over a 3 day period, sustaining FGF2 levels.</p

FGF2 beads produce a more undifferentiated, mouse and human neural stem cell culture.

<p>(A) Mouse NSCs grown for one week in FGF2beads show increased progenitor cells (Nestin+) and decreased neuronal differentiation (TUJ1+) compared to no FGF2 and soluble FGF2, data quantified in (B). (C) hESC-derived hNSCs grown for two weeks in the presence of FGF2 beads show higher levels of Nestin and lower levels of TUJ1 than those grown with soluble FGF2 (scale bars = 50 microns).</p

Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting

<div><p>Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13^NEGSSEA4^POSTra-1-60^POS on day 7–10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and “contaminating” partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential <i>in vitro</i> and <i>in vivo</i>. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.</p> </div

Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, interlaboratory variability of MEF preparation, and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used, chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151