Seeding Induced Pluripotent Stem Cell-Derived Neurons onto 384-Well Plates.

Induced pluripotent stem cell (iPSC) derived neurons are an excellent in vitro model of neurological diseases that are often used in early stage drug discovery projects. Thus far, the use of iPSC-derived cells in small molecule drug screening has been limited, and one of the reasons for this has been the challenge of miniaturization of iPSC culture and differentiation in low volume microwell plate formats. Here we describe a method of seeding iPSC-derived neurons into 384-well plates towards the end of the differentiation procedure. This method covers coating the plates with substrates to aid attachment, dissociation of the cells into a single cell suspension, and seeding onto 384-well plates to give an even distribution of neurons. This method facilitates the use of iPSC-derived neurons for high-content imaging, whole-well assays, and small-molecule drug screening.

High-Content Autophagy Analysis in iPSC-Derived Neurons Using Immunofluorescence.

Autophagy is the process by which cellular proteins and organelles are degraded and recycled and is essential to the survival of cells. Defective autophagic degradation has been linked to many neurodegenerative diseases and in particular lysosomal storage diseases. Here we describe a high-content assay to detect defects in the autophagy pathway in induced pluripotent stem cell-derived neurons. This assay utilizes immunofluorescence to stain autophagosomes and uses automated image analysis to measure changes in autophagosome levels in response to modulators of autophagy.

High-Content Analysis of Mitochondrial Function in iPSC-Derived Neurons.

Mitochondrial dysfunction is linked to many neurological diseases; therefore, the ability to measure mitochondrial function is of great use for researching disease and testing potential therapeutics. Here we describe a high-content assay to simultaneously measure mitochondrial membrane potential, morphology and cell viability in iPSC-derived neurons. Neurons are seeded into plates suitable for fluorescent microscopy, stained with the mitochondrial membrane potential-dependent dye TMRM, cytoplasmic dye Calcein AM, and nuclear stain Hoechst 33342. Images are acquired in live cells and analyzed using automated image analysis software.

A single cell high content assay detects mitochondrial dysfunction in iPSC-derived neurons with mutations in SNCA.

Mitochondrial dysfunction is implicated in many neurodegenerative diseases including Parkinson's disease (PD). Induced pluripotent stem cells (iPSCs) provide a unique cell model for studying neurological diseases. We have established a high-content assay that can simultaneously measure mitochondrial function, morphology and cell viability in iPSC-derived dopaminergic neurons. iPSCs from PD patients with mutations in SNCA and unaffected controls were differentiated into dopaminergic neurons, seeded in 384-well plates and stained with the mitochondrial membrane potential dependent dye TMRM, alongside Hoechst-33342 and Calcein-AM. Images were acquired using an automated confocal screening microscope and single cells were analysed using automated image analysis software. PD neurons displayed reduced mitochondrial membrane potential and altered mitochondrial morphology compared to control neurons. This assay demonstrates that high content screening techniques can be applied to the analysis of mitochondria in iPSC-derived neurons. This technique could form part of a drug discovery platform to test potential new therapeutics for PD and other neurodegenerative diseases.

Mouse decellularised liver scaffold improves human embryonic and induced pluripotent stem cells differentiation into hepatocyte-like cells.

Liver transplantation is the definitive treatment of liver failure but donor organ shortage limits its availability. Stem cells are highly expandable and have the potential to differentiate into any specialist cell. Use of patient-derived induced Pluripotent Stem Cells (hiPSCs) has the additional advantage for organ regeneration therapies by removing the need for immunosuppression. We compared hepatocyte differentiation of human embryonic stem cells (hESCs) and hiPSCs in a mouse decellularised liver scaffold (3D) with standard in vitro protocol (2D). Mouse livers were decellularised preserving micro-architecture, blood vessel network and extracellular matrix. hESCs and hiPSCs were primed towards the definitive endoderm. Cells were then seeded either in 3D or 2D cultures and the hepatocyte differentiation was continued. Both hESCs and hiPSCs differentiated more efficiently in 3D than in 2D, with higher and earlier expression of mature hepatocyte marker albumin, lipid and glycogen synthesis associated with a decrease in expression of fetal hepatocyte marker alpha-fetoprotein. Thus we conclude that stem cell hepatocyte differentiation in 3D culture promotes faster cell maturation. This finding suggests that optimised 3D protocols could allow generation of mature liver cells not achieved so far in standard 2D conditions and lead to improvement in cell models of liver disease and regenerative medicine applications.

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

NuRD-dependent DNA methylation prevents ES cells from accessing a trophectoderm fate.

Embryonic Stem (ES) cells are able to give rise to the three germ layers of the embryo but are prevented from contributing to the trophoblast. The molecular nature of this barrier between embryonic and trophectodermal cell fates is not clear, but is known to involve DNA methylation. Here we demonstrate that the Nucleosome Remodeling and Deacetylation (NuRD) co-repressor complex maintains the developmental barrier between embryonic and trophectodermal cell fates by maintaining transcriptional silencing of trophectoderm determinant genes in ES cells. We further show that NuRD activity facilitates DNA methylation of several of its target promoters, where it acts non-redundantly with DNA methylation to enforce transcriptional silencing. NuRD-deficient ES cells fail to completely silence expression of the trophectoderm determinant genes Elf5 and Eomes, but this alone is not sufficient to induce transdifferentiation towards the trophectoderm fate. Rather this leaves ES cells capable of activating expression of trophectoderm-specific genes in response to appropriate extracellular signals, enabling them to commit to a trophectodermal cell fate. Our findings clarify the molecular nature of the developmental barrier between the embryonic and trophoblast cell fates, and establish a role for NuRD activity in specifying sites for de novo DNA methylation.

NuRD suppresses pluripotency gene expression to promote transcriptional heterogeneity and lineage commitment.

Transcriptional heterogeneity within embryonic stem cell (ESC) populations has been suggested as a mechanism by which a seemingly homogeneous cell population can initiate differentiation into an array of different cell types. Chromatin remodeling proteins have been shown to control transcriptional variability in yeast and to be important for mammalian ESC lineage commitment. Here we show that the Nucleosome Remodeling and Deacetylation (NuRD) complex, which is required for ESC lineage commitment, modulates both transcriptional heterogeneity and the dynamic range of a set of pluripotency genes in ESCs. In self-renewing conditions, the influence of NuRD at these genes is balanced by the opposing action of self-renewal factors. Upon loss of self-renewal factors, the action of NuRD is sufficient to silence transcription of these pluripotency genes, allowing cells to exit self-renewal. We propose that modulation of transcription levels by NuRD is key to maintaining the differentiation responsiveness of pluripotent cells.