Direct induction of human neurons from fibroblasts carrying the neuropsychiatric 22q11.2 microdeletion reveals transcriptome- and epigenome-wide alterations

Standard methods for the creation of neuronal cells via direct induction from primary tissue use perinatal fibroblasts, which hinders the important study of patient specific genetic lesions such as those underlying neuropsychiatric disorders. To address this we developed a novel method for the direct induction of neuronal cells (induced neuronal cells, iN cells) from adult human fibroblast cells. Reprogramming fibroblasts into iN cells via recombinant virus resulted in cells that stain for markers such as MAP2 and PSA-NCAM and exhibit electrophysiological properties such as action potentials and voltage dependent sodium- and potassium currents that reveal a neuronal phenotype. Transcriptome and chromatin analysis using RNA-Seq, microRNA-Seq and ATAC-Seq, respectively, further confirm neuronal character. 22q11.2 Deletion-Syndrome (22q11DS) is caused by a large 3 million base-pair heterozygous deletion on human chromosome 22 and is strongly associated with neurodevelopmental, neuropsychiatric phenotypes such as schizophrenia and autism. We leverage the direct-iN cell model for the study of genetic neurodevelopmental conditions by presenting gene-by-gene as well as network-wide effects of the 22q11DS deletion on gene expression in human neuronal cells, on several levels of functional genomics analysis. Some of the genes within the 22q11DS deletion boundary exhibit unexpected cell-type-specific changes in transcript levels, and genome-wide we can detect dysregulation of calcium channel subunit genes and other genes known to be involved in autism or schizophrenia, such as NRXN1, as well synaptic pathways. This genome-wide effect on gene expression can also be observed at the microRNA and chromatin levels, showing that the iN cells have indeed converted to a neuronal phenotype at several regulatory levels: chromatin, protein-coding RNAs and microRNAs, revealing relevant disease pathways and genes. We present this model of inducing neurons from fibroblasts as a useful general resource to study the genetic and molecular basis of normal and abnormal brain development and brain function.

Direct induction of hemogenic endothelial progenitors from hPSCs by defined factors revealed by single-cell transcriptome analysis

Transcription factors (TFs) play critical roles in stem cell maintenance and differentiation. Using single cell RNA sequencing, we investigated TFs expressed in hemogenic endothelial (HE) progenitors differentiated from human pluripotent stem cells (hPSCs) and identified upregulated expression of SOXF factors SOX7, SOX17, and SOX18 in the HE population. To test whether overexpression of these factors increases HE differentiation efficiency, we established inducible hPSC lines and found only SOX17 improved differentiation. Temporal expression analysis further revealed SOX17 was turned on immediately before VE-Cadherin, indicating SOX17 may be a causative factor for HE differentiation. Upon SOX17 knockdown via CRISPR-Cas13d, HE differentiation was significantly abrogated. Strikingly, we discovered SOX17 overexpression alone is sufficient to generate more than 50% CD34+VE-cadherin+CD73- cells that could be directed to hematopoietic progenitors, which emerged via an endothelial-to-hematopoietic transition and significantly upregulated definitive hematopoietic transcriptional programs. Functional assays showed that these progenitors can differentiate into blood cells from multiple lineages. Our analyses reveal an uncharacterized function of SOX17 in directing hPSCs differentiation towards HE cells.

MULTI-FACTOR EXPERIMENTAL RESEARCH OF THE GRAIN DRYING PROCESS BY INDUCTION HEATERS

This is a method of drying grain related to agriculture and can be used as a grain dryer on an elevator. Increasing the efficiency of the process of heating the grain material and reducing energy costs is due to the fact that there is a uniform distribution of moisture in the dried product. The induction drying method has the advantage that it does not have heat transfer from the heater. Ensuring the quality of the heat treatment process, in compliance with operational and technological requirements with minimal energy costs, an idealized contact type installation for heat treatment of grain should include the following main elements in its structural composition. For the study, the entire design of the grain dryer with direct induction heaters was assembled. The installation of a cylindrical shape with a helical surface auger includes all the above elements – this is the supply of grain to the hopper, the method of heat supply, the determination of the distance. Inside this cylinder is a fixed helical surface, on which the grain from the hopper is filled from the top. Due to the gravitational forces, the grain moves down the helical surface. There is one feature here – the helical surface must have a variable pitch, decreasing from the top to the bottom. This is due to the fact that as the grain dries, the coefficient of friction of the grain on the material of the helical surface decreases. High-frequency grain dryers have been designed, tested, and even put into practice. The grain dried in such installations meets all technological requirements, and in some cases surpassed the grain dried in the traditional, convective way in quality. Economic changes in our country have revealed the demand for miniaturization of plants, mini grain dryers have been created, mobile grain dryers with relatively low power consumption, highly economical, easy to use and maintain, and most importantly-quite cheap.

In vitro dexamethasone treatment does not induce alternative ATM transcripts in cells from Ataxia–Telangiectasia patients

Short term treatment with low doses of glucocorticoid analogues has been shown to ameliorate neurological symptoms in Ataxia–Telangiectasia (A–T), a rare autosomal recessive multisystem disease that mainly affects the cerebellum, immune system, and lungs. Molecular mechanisms underlying this clinical observation are unclear. We aimed at evaluating the effect of dexamethasone on the induction of alternative ATM transcripts (ATMdexa1). We showed that dexamethasone cannot induce an alternative ATM transcript in control and A–T lymphoblasts and primary fibroblasts, or in an ATM-knockout HeLa cell line. We also demonstrated that some of the reported readouts associated with ATMdexa1 are due to cellular artifacts and the direct induction of γH2AX by dexamethasone via DNA-PK. Finally, we suggest caution in interpreting dexamethasone effects in vitro for the results to be translated into a rational use of the drug in A–T patients.

Direct conversion of human endothelial cells to hepatic progenitor cells

This protocol describes direct lineage reprogramming of human endothelial cells isolated from the umbilical vein and peripheral blood into hepatic progenitor cells. These induced human hepatic progenitor cells (hiHepPCs) proliferate in long-term culture and give rise to hepatocytes and cholangiocytes as descendants. To induce hiHepPCs from endothelial cells, we first established an efficient culture condition, enabling hiHepPC generation and propagation in a monolayer culture. Then, we confirmed the ability of the hiHepPCs to differentiate into mature hepatocytes by formation of cell aggregates in each well of ultra-low attachment 96-well plates. Furthermore, upon culturing in Matrigel, the hiHepPCs differentiated into cholangiocytes and formed cystic epithelial spheroids, similar to human liver-derived cholangiocytes. Direct induction of the expandable and bipotential human hepatic progenitor cells provides a possibility for generating cells such as hepatocytes and cholangiocytes, which will be useful for developing therapeutic strategies for human liver diseases.

Anacardic acid induces IL-33 and promotes remyelination in CNS

Given the known neuroreparative actions of IL-33 in experimental models of central nervous system (CNS) injury, we predicted that compounds which induce IL-33 are likely to promote remyelination. We found anacardic acid as a candidate molecule to serve as a therapeutic agent to promote remyelination. Addition of anacardic acid to cultured oligodendrocyte precursor cells (OPCs) rapidly increased expression of myelin genes and myelin proteins, suggesting a direct induction of genes involved in myelination by anacardic acid. Also, when added to OPCs, anacardic acid resulted in the induction of IL-33. In vivo, treatment of with anacardic acid in doses which ranged from 0.025 mg/kg to 2.5 mg/kg,improved pathologic scores in experimental allergic encephalitis (EAE) and in the cuprizone model of demyelination/remyelination. Electron microscopic studies performed in mice fed with cuprizone and treated with anacardic acid showed lower g-ratio scores when compared to controls, suggesting increased remyelination of axons. In EAE, improvement in paralytic scores was seen when the drug was given prior to or following the onset of paralytic signs. In EAE and in the cuprizone model, areas of myelin loss, which are likely to remyelinate, was associated with a greater recruitment of IL-33–expressing OPCs in mice which received anacardic acid when compared to controls.