The progress of induced pluripotent stem cells derived from pigs: a mini review of recent advances.

Pigs (Sus scrofa) are widely acknowledged as an important large mammalian animal model due to their similarity to human physiology, genetics, and immunology. Leveraging the full potential of this model presents significant opportunities for major advancements in the fields of comparative biology, disease modeling, and regenerative medicine. Thus, the derivation of pluripotent stem cells from this species can offer new tools for disease modeling and serve as a stepping stone to test future autologous or allogeneic cell-based therapies. Over the past few decades, great progress has been made in establishing porcine pluripotent stem cells (pPSCs), including embryonic stem cells (pESCs) derived from pre- and peri-implantation embryos, and porcine induced pluripotent stem cells (piPSCs) using a variety of cellular reprogramming strategies. However, the stabilization of pPSCs was not as straightforward as directly applying the culture conditions developed and optimized for murine or primate PSCs. Therefore, it has historically been challenging to establish stable pPSC lines that could pass stringent pluripotency tests. Here, we review recent advances in the establishment of stable porcine PSCs. We focus on the evolving derivation methods that eventually led to the establishment of pESCs and transgene-free piPSCs, as well as current challenges and opportunities in this rapidly advancing field.

JAK2/STAT3 Pathway Inhibition by AG490 Ameliorates Experimental Autoimmune Encephalomyelitis via Regulation of Th17 Cells and Autophagy.

Multiple sclerosis (MS) is an autoimmune inflammatory condition affecting the central nervous system, and experimental autoimmune encephalomyelitis (EAE) animal models have been extensively used to study it. T-helper 17 cells, which produce interleukin-17(IL-17), play crucial roles in MS pathogenesis, and the JAK2/STAT3 pathway has an essential function in their differentiation from naive CD4 + T cells. This study investigated the effects of the JAK2/STAT3 pathway inhibitor AG490 on EAE in vivo and in vitro, as well as the underlying mechanisms. AG490 ameliorated EAE severity and attenuated its typical symptoms by downregulating proteins associated with the JAK2/STAT3 pathway. Furthermore, it decreased T-helper 17 cell differentiation from naive CD4 + T cells by inactivating STAT3. In addition, it conferred protective effects against EAE by restoring autophagy. These findings indicate the potential of AG490 as a candidate anti-MS therapeutic.

Establishment of Transgene-Free Porcine Induced Pluripotent Stem Cells.

Although protocols to generate authentic transgene-free mouse and human induced pluripotent stem cells (iPSCs) are now well established, standard methods for reprogramming porcine somatic cells still suffer from low efficiency and transgene retention. The Basic Protocol describes reprogramming procedures to establish transgene-free porcine iPSCs (PiPSCs) from porcine fibroblasts. This method uses episomal plasmids encoding POU5F1, SOX2, NANOG, KLF4, SV40LT, c-MYC, LIN28A, and microRNA-302/367, combined with an optimized medium, to establish PiPSC lines. Support protocols describe the establishment and characterization of clonal PiPSC lines, as well as the preparation of feeder cells and EBNA1 mRNA. This optimized, step-by-step approach tailored to this species enables the efficient derivation of PiPSCs in ∼4 weeks. The establishment of transgene-free PiPSCs provides a new and valuable model for studies of larger mammalian species' development, disease, and regenerative biology. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Reprogramming of porcine fibroblasts with episomal plasmids Support Protocol 1: Preparation of mouse embryonic fibroblasts for feeder layer Support Protocol 2: Preparation of in vitro-transcribed EBNA1 mRNA Support Protocol 3: Establishment of clonal porcine induced pluripotent stem cell (PiPSC) lines Support Protocol 4: PiPSC characterization: Genomic DNA PCR and RT-PCR Support Protocol 5: PiPSC characterization: Immunostaining.

Chromosome level genome assembly of the Etruscan shrew Suncus etruscus.

Suncus etruscus is one of the world's smallest mammals, with an average body mass of about 2 grams. The Etruscan shrew's small body is accompanied by a very high energy demand and numerous metabolic adaptations. Here we report a chromosome-level genome assembly using PacBio long read sequencing, 10X Genomics linked short reads, optical mapping, and Hi-C linked reads. The assembly is partially phased, with the 2.472 Gbp primary pseudohaplotype and 1.515 Gbp alternate. We manually curated the primary assembly and identified 22 chromosomes, including X and Y sex chromosomes. The NCBI genome annotation pipeline identified 39,091 genes, 19,819 of them protein-coding. We also identified segmental duplications, inferred GO term annotations, and computed orthologs of human and mouse genes. This reference-quality genome will be an important resource for research on mammalian development, metabolism, and body size control.

A High-Quality Blue Whale Genome, Segmental Duplications, and Historical Demography.

The blue whale, Balaenoptera musculus, is the largest animal known to have ever existed, making it an important case study in longevity and resistance to cancer. To further this and other blue whale-related research, we report a reference-quality, long-read-based genome assembly of this fascinating species. We assembled the genome from PacBio long reads and utilized Illumina/10×, optical maps, and Hi-C data for scaffolding, polishing, and manual curation. We also provided long read RNA-seq data to facilitate the annotation of the assembly by NCBI and Ensembl. Additionally, we annotated both haplotypes using TOGA and measured the genome size by flow cytometry. We then compared the blue whale genome with other cetaceans and artiodactyls, including vaquita (Phocoena sinus), the world's smallest cetacean, to investigate blue whale's unique biological traits. We found a dramatic amplification of several genes in the blue whale genome resulting from a recent burst in segmental duplications, though the possible connection between this amplification and giant body size requires further study. We also discovered sites in the insulin-like growth factor-1 gene correlated with body size in cetaceans. Finally, using our assembly to examine the heterozygosity and historical demography of Pacific and Atlantic blue whale populations, we found that the genomes of both populations are highly heterozygous and that their genetic isolation dates to the last interglacial period. Taken together, these results indicate how a high-quality, annotated blue whale genome will serve as an important resource for biology, evolution, and conservation research.

Efficient derivation of transgene-free porcine induced pluripotent stem cells enables in vitro modeling of species-specific developmental timing.

Sus scrofa domesticus (pig) has served as a superb large mammalian model for biomedical studies because of its comparable physiology and organ size to humans. The derivation of transgene-free porcine induced pluripotent stem cells (PiPSCs) will, therefore, benefit the development of porcine-specific models for regenerative biology and its medical applications. In the past, this effort has been hampered by a lack of understanding of the signaling milieu that stabilizes the porcine pluripotent state in vitro. Here, we report that transgene-free PiPSCs can be efficiently derived from porcine fibroblasts by episomal vectors along with microRNA-302/367 using optimized protocols tailored for this species. PiPSCs can be differentiated into derivatives representing the primary germ layers in vitro and can form teratomas in immunocompromised mice. Furthermore, the transgene-free PiPSCs preserve intrinsic species-specific developmental timing in culture, known as developmental allochrony. This is demonstrated by establishing a porcine in vitro segmentation clock model that, for the first time, displays a specific periodicity at ∼3.7 h, a timescale recapitulating in vivo porcine somitogenesis. We conclude that the transgene-free PiPSCs can serve as a powerful tool for modeling development and disease and developing transplantation strategies. We also anticipate that they will provide insights into conserved and unique features on the regulations of mammalian pluripotency and developmental timing mechanisms.

Species-specific roles of the Notch ligands, receptors, and targets orchestrating the signaling landscape of the segmentation clock.

Somitogenesis is a hallmark feature of all vertebrates and some invertebrate species that involves the periodic formation of block-like structures called somites. Somites are transient embryonic segments that eventually establish the entire vertebral column. A highly conserved molecular oscillator called the segmentation clock underlies this periodic event and the pace of this clock regulates the pace of somite formation. Although conserved signaling pathways govern the clock in most vertebrates, the mechanisms underlying the species-specific divergence in various clock characteristics remain elusive. For example, the segmentation clock in classical model species such as zebrafish, chick, and mouse embryos tick with a periodicity of ∼30, ∼90, and ∼120 min respectively. This enables them to form the species-specific number of vertebrae during their overall timespan of somitogenesis. Here, we perform a systematic review of the species-specific features of the segmentation clock with a keen focus on mouse embryos. We perform this review using three different perspectives: Notch-responsive clock genes, ligand-receptor dynamics, and synchronization between neighboring oscillators. We further review reports that use non-classical model organisms and in vitro model systems that complement our current understanding of the segmentation clock. Our review highlights the importance of comparative developmental biology to further our understanding of this essential developmental process.

Cohort Study on the Effect of Psychological Education for Nurses in Psychiatric Department.

With the deepening of the medical and health system, high-quality nursing service with the reform of nursing service model and the provision of holistic nursing care for patients as the core connotation is being comprehensively carried out. With the continuous improvement of medical quality, people's awareness of health has gradually increased. They have put forward higher requirements for medical quality, nursing service quality, and medical care safety. Under the influence of these conditions, the workload and work pressure of clinical nurses continue to increase, and mental health problems become increasingly prominent. According to relevant data, the detection rate of occupational stress among nurses was 100%, and the proportion of nurses who perceived considerable stress was 60.9%, which seriously affected their work efficiency and quality of life. However, the physical and mental health of the frontline nurses working in psychiatric hospitals is not optimistic. This study explored the effects of psychological education among nurses in the department of psychiatry. The results showed that psychological education intervention among nurses in the department of psychiatry could alleviate their professional tiredness, effectively improve their psychological elasticity and happiness index, and thus improve sleep quality and promote their physical and mental health development.

Rapid changes in chromatin structure during dedifferentiation of primary hepatocytes in vitro.

Primary hepatocytes are widely used in the pharmaceutical industry to screen drug candidates for hepatotoxicity, but hepatocytes quickly dedifferentiate and lose their mature metabolic function in culture. Attempts have been made to better recapitulate the in vivo liver environment in culture, but the full spectrum of signals required to maintain hepatocyte function ex vivo remains elusive. To elucidate molecular changes that accompany, and may contribute to dedifferentiation of hepatocytes ex vivo, we performed lineage tracing and comprehensive profiling of alterations in their gene expression profiles and chromatin landscape during culture. First, using genetically tagged hepatocytes we demonstrate that expression of the fetal gene alpha-fetoprotein in cultured hepatocytes comes from cells that previously expressed the mature gene albumin, and not from a population of albumin-negative precursor cells, proving mature hepatocytes undergo true dedifferentiation in culture. Next we studied the dedifferentiation process in detail through bulk RNA-sequencing of hepatocytes cultured over an extended period. We identified three distinct phases of dedifferentiation: an early phase, where mature hepatocyte genes are rapidly downregulated in a matter of hours; a middle phase, where fetal genes are activated; and a late phase, where initially rare contaminating non-parenchymal cells proliferate, taking over the culture. Lastly, to better understand the signaling events that result in the rapid downregulation of mature genes in hepatocytes, we examined changes in chromatin accessibility in these cells during the first 24 h of culture using Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). We find that drastic and rapid changes in chromatin accessibility occur immediately upon the start of culture. Using binding motif analysis of the areas of open chromatin sharing similar temporal profiles, we identify several candidate transcription factors potentially involved in the dedifferentiation of primary hepatocytes in culture.

Network inference with Granger causality ensembles on single-cell transcriptomics.

Cellular gene expression changes throughout a dynamic biological process, such as differentiation. Pseudotimes estimate cells' progress along a dynamic process based on their individual gene expression states. Ordering the expression data by pseudotime provides information about the underlying regulator-gene interactions. Because the pseudotime distribution is not uniform, many standard mathematical methods are inapplicable for analyzing the ordered gene expression states. Here we present single-cell inference of networks using Granger ensembles (SINGE), an algorithm for gene regulatory network inference from ordered single-cell gene expression data. SINGE uses kernel-based Granger causality regression to smooth irregular pseudotimes and missing expression values. It aggregates predictions from an ensemble of regression analyses to compile a ranked list of candidate interactions between transcriptional regulators and target genes. In two mouse embryonic stem cell differentiation datasets, SINGE outperforms other contemporary algorithms. However, a more detailed examination reveals caveats about poor performance for individual regulators and uninformative pseudotimes.

Enhancing biological signals and detection rates in single-cell RNA-seq experiments with cDNA library equalization.

Considerable effort has been devoted to refining experimental protocols to reduce levels of technical variability and artifacts in single-cell RNA-sequencing data (scRNA-seq). We here present evidence that equalizing the concentration of cDNA libraries prior to pooling, a step not consistently performed in single-cell experiments, improves gene detection rates, enhances biological signals, and reduces technical artifacts in scRNA-seq data. To evaluate the effect of equalization on various protocols, we developed Scaffold, a simulation framework that models each step of an scRNA-seq experiment. Numerical experiments demonstrate that equalization reduces variation in sequencing depth and gene-specific expression variability. We then performed a set of experiments in vitro with and without the equalization step and found that equalization increases the number of genes that are detected in every cell by 17-31%, improves discovery of biologically relevant genes, and reduces nuisance signals associated with cell cycle. Further support is provided in an analysis of publicly available data.

Reproducibility across single-cell RNA-seq protocols for spatial ordering analysis.

As newer single-cell protocols generate increasingly more cells at reduced sequencing depths, the value of a higher read depth may be overlooked. Using data from three different single-cell RNA-seq protocols that lend themselves to having either higher read depth (Smart-seq) or many cells (MARS-seq and 10X), we evaluate their ability to recapitulate biological signals in the context of spatial reconstruction. Overall, we find gene expression profiles after spatial reconstruction analysis are highly reproducible between datasets despite being generated by different protocols and using different computational algorithms. While UMI-based protocols such as 10X and MARS-seq allow for capturing more cells, Smart-seq's higher sensitivity and read-depth allow for analysis of lower expressed genes and isoforms. Additionally, we evaluate trade-offs for each protocol by performing subsampling analyses and find that optimizing the balance between sequencing depth and number of cells within a protocol is necessary for efficient use of resources. Our analysis emphasizes the importance of selecting a protocol based on the biological questions and features of interest.

An In Vitro Human Segmentation Clock Model Derived from Embryonic Stem Cells.

Defects in somitogenesis result in vertebral malformations at birth known as spondylocostal dysostosis (SCDO). Somites are formed with a species-specific periodicity controlled by the "segmentation clock," which comprises a group of oscillatory genes in the presomitic mesoderm. Here, we report that a segmentation clock model derived from human embryonic stem cells shows many hallmarks of the mammalian segmentation clock in vivo, including a dependence on the NOTCH and WNT signaling pathways. The gene expression oscillations are highly synchronized, displaying a periodicity specific to the human clock. Introduction of a point of mutation into HES7, a specific mutation previously associated with clinical SCDO, eliminated clock gene oscillations, successfully reproducing the defects in the segmentation clock. Thus, we provide a model for studying the previously inaccessible human segmentation clock to better understand the mechanisms contributing to congenital skeletal defects.

Epithelial DNA methyltransferase-1 regulates cell survival, growth and maturation in developing prostatic buds.

DNA methyltransferase 1 (DNMT1) is required for embryogenesis but roles in late forming organ systems including the prostate, which emerges from the urethral epithelium, have not been fully examined. We used a targeted genetic approach involving a Shhcre recombinase to demonstrate requirement of epithelial DNA methyltransferase-1 (Dnmt1) in mouse prostate morphogenesis. Dnmt1 mutant urethral cells exhibit DNA hypomethylation, DNA damage, p53 accumulation and undergo cell cycle arrest and apoptosis. Urethral epithelial cells are disorganized in Dnmt1 mutants, leading to impaired prostate growth and maturation and failed glandular development. We evaluated oriented cell division as a mechanism of bud elongation and widening by demonstrating that mitotic spindle axes typically form parallel or perpendicular to prostatic bud elongation axes. We then deployed a ShhcreERT allele to delete Dnmt1 from a subset of urethral epithelial cells, creating mosaic mutants with which to interrogate the requirement for cell division in specific prostatic bud epithelial populations. DNMT1- cell distribution within prostatic buds is not random as would be expected in a process where DNMT1 was not required. Instead, replication competent DNMT1 + cells primarily accumulate in prostatic bud margins and tips while replication impeded DNMT1- cells accumulate in prostatic bud cores. Together, these results highlight the role of DNMT1 in regulating epithelial bud formation by maintaining cell cycle progression and survival of rapidly dividing urethral epithelial cells, which can be extended to the study of other developing epithelial organs. In addition, our results show that prostatic buds consist of two epithelial cell populations with distinct molecular and functional characteristics that could potentially contribute to specialized lineages in the adult prostate.

A folic acid-enriched diet attenuates prostate involution in response to androgen deprivation.

BACKGROUND: Serum folate concentrations in the United States have risen since dietary folic acid fortification was first mandated in 1998. Although maternal folic acid offers protection against neural tube defects in conceptuses, its impact on other organ systems and life stages have not been fully examined. Here, we used a mouse model to investigate the impact of a Folic acid (FA) enriched diet on prostate homeostasis and response to androgen deprivation. METHODS: Male mice were fed a control diet (4 mg FA/kg feed) or a folic acid supplemented diet (24 mg FA/kg feed) beginning at conception and continuing through early adulthood, when mice were castrated. RESULTS: We made the surprising observation that dietary FA supplementation confers partial resistance to castration-mediated prostate involution. At 3, 10, and 14 days post-castration, FA enriched diet fed mice had larger prostates as assessed by wet weight, taller prostatic luminal epithelial cells, and more abundant RNAs encoding prostate secretory proteins than castrated control diet fed mice. Diet did not significantly affect prostate weights of intact mice or serum testosterone concentrations of castrated mice. RNA-Seq analysis revealed that the FA enriched diet was associated with a unique prostate gene expression signature, affecting several signaling and metabolic pathways. CONCLUSIONS: Continuous exposure to a FA enriched diet slows prostate involution in response to androgen deprivation. Prostates from FA diet mice have increased secretory gene expression and increased luminal cell heights. The influence of dietary FA supplementation on the prostate response to androgen deprivation raises a future need to consider how dietary folic acid supplementation affects efficacy of androgen-reducing therapies for treating prostate disease.

Trendy: segmented regression analysis of expression dynamics in high-throughput ordered profiling experiments.

BACKGROUND: High-throughput expression profiling experiments with ordered conditions (e.g. time-course or spatial-course) are becoming more common for studying detailed differentiation processes or spatial patterns. Identifying dynamic changes at both the individual gene and whole transcriptome level can provide important insights about genes, pathways, and critical time points. RESULTS: We present an R package, Trendy, which utilizes segmented regression models to simultaneously characterize each gene's expression pattern and summarize overall dynamic activity in ordered condition experiments. For each gene, Trendy finds the optimal segmented regression model and provides the location and direction of dynamic changes in expression. We demonstrate the utility of Trendy to provide biologically relevant results on both microarray and RNA-sequencing (RNA-seq) datasets. CONCLUSIONS: Trendy is a flexible R package which characterizes gene-specific expression patterns and summarizes changes of global dynamics over ordered conditions. Trendy is freely available on Bioconductor with a full vignette at https://bioconductor.org/packages/release/bioc/html/Trendy.html .

In vivo replacement of damaged bladder urothelium by Wolffian duct epithelial cells.

The bladder's remarkable regenerative capacity had been thought to derive exclusively from its own progenitors. While examining consequences of DNA methyltransferase 1 (Dnmt1) inactivation in mouse embryonic bladder epithelium, we made the surprising discovery that Wolffian duct epithelial cells can support bladder regeneration. Conditional Dnmt1 inactivation in mouse urethral and bladder epithelium triggers widespread apoptosis, depletes basal and intermediate bladder cells, and disrupts uroplakin protein expression. These events coincide with Wolffian duct epithelial cell recruitment into Dnmt1 mutant urethra and bladder where they are reprogrammed to express bladder markers, including FOXA1, keratin 5, P63, and uroplakin. This is evidence that Wolffian duct epithelial cells are summoned in vivo to replace damaged bladder epithelium and function as a reservoir of cells for bladder regeneration.

Spatial patterns of gene expression are unveiled in the chick primitive streak by ordering single-cell transcriptomes.

During vertebrate development, progenitor cells give rise to tissues and organs through a complex choreography that commences at gastrulation. A hallmark event of gastrulation is the formation of the primitive streak, a linear assembly of cells along the anterior-posterior (AP) axis of the developing organism. To examine the primitive streak at a single-cell resolution, we measured the transcriptomes of individual chick cells from the streak or the surrounding tissue (the rest of the area pellucida) in Hamburger-Hamilton stage 4 embryos. The single-cell transcriptomes were then ordered by the statistical method Wave-Crest to deduce both the relative position along the AP axis and the prospective lineage of single cells. The ordered transcriptomes reveal intricate patterns of gene expression along the primitive streak.

A Novel Approach to Single Cell RNA-Sequence Analysis Facilitates In Silico Gene Reporting of Human Pluripotent Stem Cell-Derived Retinal Cell Types.

Cell type-specific investigations commonly use gene reporters or single-cell analytical techniques. However, reporter line development is arduous and generally limited to a single gene of interest, while single-cell RNA (scRNA)-sequencing (seq) frequently yields equivocal results that preclude definitive cell identification. To examine gene expression profiles of multiple retinal cell types derived from human pluripotent stem cells (hPSCs), we performed scRNA-seq on optic vesicle (OV)-like structures cultured under cGMP-compatible conditions. However, efforts to apply traditional scRNA-seq analytical methods based on unbiased algorithms were unrevealing. Therefore, we developed a simple, versatile, and universally applicable approach that generates gene expression data akin to those obtained from reporter lines. This method ranks single cells by expression level of a bait gene and searches the transcriptome for genes whose cell-to-cell rank order expression most closely matches that of the bait. Moreover, multiple bait genes can be combined to refine datasets. Using this approach, we provide further evidence for the authenticity of hPSC-derived retinal cell types. Stem Cells 2018;36:313-324.

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells.

Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.