Scalable in vitro production of defined mouse erythroblasts

Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.

Intermittent compressive force induces cell cycling and reduces apoptosis in embryoid bodies of mouse induced pluripotent stem cells

In vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor β1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-β inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-β signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication.

Optogenetic Stimulation of Gi Signaling Enables Instantaneous Modulation of Cardiomyocyte Pacemaking

G-protein signaling pathways are central in the regulation of cardiac function in physiological and pathophysiological conditions. Their functional analysis through optogenetic techniques with selective expression of opsin proteins and activation by specific wavelengths allows high spatial and temporal precision. Here, we present the application of long wavelength-sensitive cone opsin (LWO) in cardiomyocytes for activation of the Gi signaling pathway by red light. Murine embryonic stem (ES) cells expressing LWO were generated and differentiated into beating cardiomyocytes in embryoid bodies (EBs). Illumination with red light (625 nm) led to an instantaneous decrease up to complete inhibition (84–99% effectivity) of spontaneous beating, but had no effect on control EBs. By using increasing light intensities with 10 s pulses, we determined a half maximal effective light intensity of 2.4 μW/mm2 and a maximum effect at 100 μW/mm2. Pre-incubation of LWO EBs with pertussis toxin completely inhibited the light effect proving the specificity for Gi signaling. Frequency reduction was mainly due to the activation of GIRK channels because the specific channel blocker tertiapin reduced the light effect by ~80%. Compared with pharmacological stimulation of M2 receptors with carbachol with slow kinetics (>30 s), illumination of LWO had an identical efficacy, but much faster kinetics (<1 s) in the activation and deactivation demonstrating the temporal advantage of optogenetic stimulation. Thus, LWO is an effective optogenetic tool for selective stimulation of the Gi signaling cascade in cardiomyocytes with red light, providing high temporal precision.

Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies

The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×10 3 cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×10 3 cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×10 3 and 11×10 3 and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages

Single-cell quantification of a broad RNA spectrum reveals unique noncoding patterns associated with cell types and states

The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current single-cell RNA-sequencing (RNA-seq) methods are unable to simultaneously monitor all forms of RNA transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and noncoding RNA from a single cell. Smart-seq-total does not require splitting the RNA content of a cell and allows the incorporation of unique molecular identifiers into short and long RNA molecules for absolute quantification. It outperforms current poly(A)-independent total RNA-seq protocols by capturing transcripts of a broad size range, thus enabling simultaneous analysis of protein-coding, long-noncoding, microRNA, and other noncoding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T, and MCF7 cells, as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. By analyzing the coexpression patterns of both noncoding RNA and mRNA from the same cell, we were able to discover new roles of noncoding RNA throughout essential processes, such as cell cycle and lineage commitment during embryonic development. Moreover, we show that independent classes of short-noncoding RNA can be used to determine cell-type identity.

Functional and structural phenotyping of cardiomyocytes in the 3D organization of embryoid bodies exposed to arsenic trioxide

Chronic exposure to environmental pollutants threatens human health. Arsenic, a world-wide diffused toxicant, is associated to cardiac pathology in the adult and to congenital heart defects in the foetus. Poorly known are its effects on perinatal cardiomyocytes. Here, bioinformatic image-analysis tools were coupled with cellular and molecular analyses to obtain functional and structural quantitative metrics of the impairment induced by 0.1, 0.5 or 1.0 µM arsenic trioxide exposure on the perinatal-like cardiomyocyte component of mouse embryoid bodies, within their 3D complex cell organization. With this approach, we quantified alterations to the (a) beating activity; (b) sarcomere organization (texture, edge, repetitiveness, height and width of the Z bands); (c) cardiomyocyte size and shape; (d) volume occupied by cardiomyocytes within the EBs. Sarcomere organization and cell morphology impairment are paralleled by differential expression of sarcomeric α-actin and Tropomyosin proteins and of acta2, myh6 and myh7 genes. Also, significant increase of Cx40, Cx43 and Cx45 connexin genes and of Cx43 protein expression profiles is paralleled by large Cx43 immunofluorescence signals. These results provide new insights into the role of arsenic in impairing cytoskeletal components of perinatal-like cardiomyocytes which, in turn, affect cell size, shape and beating capacity.

Loss of PRC2 subunits primes lineage choice during exit of pluripotency

Polycomb Repressive Complex 2 (PRC2) is crucial for the coordinated expression of genes during early embryonic development, catalyzing histone H3 lysine 27 trimethylation. Two distinct PRC2 complexes, PRC2.1 and PRC2.2, contain respectively MTF2 and JARID2 in embryonic stem cells (ESCs). In this study, we explored their roles in lineage specification and commitment, using single-cell transcriptomics and mouse embryoid bodies derived from Mtf2 and Jarid2 null ESCs. We observe that the loss of Mtf2 results in enhanced and faster differentiation towards cell fates from all germ layers, while the Jarid2 null cells are predominantly directed towards early differentiating precursors, with reduced efficiency towards mesendodermal lineages. These effects are caused by derepression of developmental regulators that are poised for activation in pluripotent cells and gain H3K4me3 at their promoters in the absence of PRC2 repression. Upon lineage commitment, the differentiation trajectories are relatively similar to those of wild-type cells. Together, our results uncover a major role for MTF2-containing PRC2.1 in balancing poised lineage-specific gene activation, whereas the contribution of JARID2-containing PRC2 is more selective in nature compared to MTF2. These data explain how PRC2 imposes thresholds for lineage choice during the exit of pluripotency.

Single-cell profiling of transcriptome and histone modifications with EpiDamID

Recent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, including various combinations of transcriptome, genome and epigenome. However, comprehensive profiling of the histone post-translational modifications that influence gene expression at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of genetically engineered proteins. By fusing Dam to single-chain variable fragment antibodies, engineered chromatin reader domains, or endogenous chromatin-binding proteins, we render the DamID technology and all its implementations compatible with the genome-wide identification of histone post-translational modifications. Importantly, this enables the joint analysis of chromatin marks and transcriptome in a variety of biological systems at the single-cell level. In this study, we use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. We further demonstrate the applicability of this method to in vivo systems by mapping H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specifically in the notochord. Overall, EpiDamID is a new addition to a vast existing toolbox for obtaining systematic insights into the role of chromatin states during dynamic cellular processes.

The Use of Induced Pluripotent Stem Cells to Study the Effects of Adenosine Deaminase Deficiency on Human Neutrophil Development

Inherited defects that abrogate the function of the adenosine deaminase (ADA) enzyme and consequently lead to the accumulation of toxic purine metabolites cause profound lymphopenia and severe combined immune deficiency. Additionally, neutropenia and impaired neutrophil function have been reported among ADA-deficient patients. However, due to the rarity of the disorder, the neutrophil developmental abnormalities and the mechanisms contributing to them have not been characterized. Induced pluripotent stem cells (iPSC) generated from two unrelated ADA-deficient patients and from healthy controls were differentiated through embryoid bodies into neutrophils. ADA deficiency led to a significant reduction in the number of all early multipotent hematopoietic progenitors. At later stages of differentiation, ADA deficiency impeded the formation of granulocyte colonies in methylcellulose cultures, leading to a significant decrease in the number of neutrophils generated from ADA-deficient iPSCs. The viability and apoptosis of ADA-deficient neutrophils isolated from methylcellulose cultures were unaffected, suggesting that the abnormal purine homeostasis in this condition interferes with differentiation or proliferation. Additionally, there was a significant increase in the percentage of hyperlobular ADA-deficient neutrophils, and these neutrophils demonstrated significantly reduced ability to phagocytize fluorescent microspheres. Supplementing iPSCs and methylcellulose cultures with exogenous ADA, which can correct adenosine metabolism, reversed all abnormalities, cementing the critical role of ADA in neutrophil development. Moreover, chemical inhibition of the ribonucleotide reductase (RNR) enzyme, using hydroxyurea or a combination of nicotinamide and trichostatin A in iPSCs from healthy controls, led to abnormal neutrophil differentiation similar to that observed in ADA deficiency, implicating RNR inhibition as a potential mechanism for the neutrophil abnormalities. In conclusion, the findings presented here demonstrate the important role of ADA in the development and function of neutrophils while clarifying the mechanisms responsible for the neutrophil abnormalities in ADA-deficient patients.