The retina is a widely profiled tissue in multiple species by single-cell RNA sequencing studies. However, integrative research of the retina across species is lacking. Here, we construct the first ...single-cell atlas of the human and porcine ocular compartments and study inter-species differences in the retina. In addition to that, we identify putative adult stem cells present in the iris tissue. We also create a disease map of genes involved in eye disorders across compartments of the eye. Furthermore, we probe the regulons of different cell populations, which include transcription factors and receptor-ligand interactions and reveal unique directional signalling between ocular cell types. In addition, we study conservation of regulons across vertebrates and zebrafish to identify common core factors. Here, we show perturbation of KLF7 gene expression during retinal ganglion cells differentiation and conclude that it plays a significant role in the maturation of retinal ganglion cells.
Blood reprogramming, in which induced pluripotent stem cells (iPSCs) are derived from haematopoietic lineages, has rapidly advanced over the past decade. Since the first report using human blood, ...haematopoietic cell types from various sources, such as the peripheral bone marrow and cord blood, have been successfully reprogrammed. The volume of blood required has also decreased, from around tens of millilitres to a single finger‐prick drop. Besides, while early studies were limited to reprogramming methods relying on viral integration, nonintegrating reprogramming systems for blood lineages have been subsequently established. Together, these improvements have made feasible the future clinical applications of blood‐derived iPSCs. Here, we review the progress in blood reprogramming from various perspectives, including the starting materials and subsequent reprogramming strategies. We also discuss the downstream applications of blood‐derived iPSCs, highlighting their clinical value in terms of disease modelling and therapeutic development.
Human embryonic stem (ES) cell lines that have the ability to self-renew and differentiate into specific cell types have been established. The molecular mechanisms for self-renewal and ...differentiation, however, are poorly understood. We determined the transcriptome profiles for two proprietary human ES cell lines (HES3 and HES4, ES Cell International), and compared them with murine ES cells and other human tissues. Human and mouse ES cells appear to share a number of expressed gene products although there are numerous notable differences, including an inactive leukemia inhibitory factor pathway and the high preponderance of several important genes like POU5F1 and SOX2 in human ES cells. We have established a list of genes comprised of known ES-specific genes and new candidates that can serve as markers for human ES cells and may also contribute to the "stemness" phenotype. Of particular interest was the downregulation of DNMT3B and LIN28 mRNAs during ES cell differentiation. The overlapping similarities and differences in gene expression profiles of human and mouse ES cells provide a foundation for a detailed and concerted dissection of the molecular and cellular mechanisms governing their pluripotency, directed differentiation into specific cell types, and extended ability for self-renewal.
In Vivo Genome Editing as a Therapeutic Approach Ho, Beatrice Xuan; Loh, Sharon Jia Hui; Chan, Woon Khiong ...
International journal of molecular sciences,
09/2018, Letnik:
19, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Genome editing has been well established as a genome engineering tool that enables researchers to establish causal linkages between genetic mutation and biological phenotypes, providing further ...understanding of the genetic manifestation of many debilitating diseases. More recently, the paradigm of genome editing technologies has evolved to include the correction of mutations that cause diseases via the use of nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and more recently, Cas9 nuclease. With the aim of reversing disease phenotypes, which arise from somatic gene mutations, current research focuses on the clinical translatability of correcting human genetic diseases in vivo, to provide long-term therapeutic benefits and potentially circumvent the limitations of in vivo cell replacement therapy. In this review, in addition to providing an overview of the various genome editing techniques available, we have also summarized several in vivo
engineering strategies that have successfully demonstrated disease correction via in vivo genome editing. The various benefits and challenges faced in applying in vivo genome editing in humans will also be discussed.
The human umbilical cord that originates from the embryo is an extra-embryonic membrane and the Wharton’s jelly within it is a rich source of stem cells (hWJSCs). It is not definitely known whether ...these cells behave as human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSC) or both. They have the unique properties of high proliferation rates, wide multipotency, hypoimmunogenicity, do not induce teratomas and have anticancer properties. These advantages are important considerations for their use in cell based therapies and treatment of cancers. In a search for properties that confer these advantages we compared a detailed transcriptome profiling of hWJSCs using DNA microarrays with that of a panel of known hESCs, hMSCs and stromal cells. hWJSCs expressed low levels of the pluripotent embryonic stem cell markers including POUF1, NANOG, SOX2 and LIN28, thus explaining why they do not produce teratomas. Several cytokines were significantly upregulated in hWJSCs including IL12A which is associated with the induction of apoptosis, thus explaining their anticancer properties. When GO Biological Process analysis was compared between the various stem cell types, hWJSCs showed an increased expression of genes associated with the immune system, chemotaxis and cell death. The ability to modulate immune responses makes hWJSCs an important compatible stem cell source for transplantation therapy in allogeneic settings without immunorejection. The data in the present study which is the first detailed report on hWJSC transcriptomes provide a foundation for future functional studies where the exact mechanisms of these unique properties of hWJSCs can be confirmed.
Human embryonic stem (hES) cells are typically derived and serially propagated on inactivated murine embryonic fibroblast (MEF) feeders. The use of MEFs and other components of animal origin in the ...culture media for hES cell support substantially elevates the risk of contaminating these cell lines with infectious agents of animal origin thereby severely limiting their potential for clinical application. We have previously shown that it is possible to derive and establish new hES cell lines in a xeno-free culture system using human fetal muscle fibroblast feeders. In this report, we have comparatively evaluated a panel of 11 different human adult, fetal, and neonatal feeders for hES cell support and have ranked them as supportive and non-supportive. We report that two adult skin fibroblast cell lines established in-house from abdominal skin biopsies supported prolonged undifferentiated hES cell growth for over 30 weekly passages in culture. Furthermore, hES cell lines cultured on adult skin fibroblast feeders retain hES cell morphology and remain pluripotent. Also, differences in feeder support exist between human cell types and sources. The use of human adult skin feeders is convenient for hES cell support given the ease of obtaining skin biopsies.
Tissue organoids generated from human pluripotent stem cells are valuable tools for disease modelling and to understand developmental processes. While recent progress in human cardiac organoids ...revealed the ability of these stem cell-derived organoids to self-organize and intrinsically formed chamber-like structure containing a central cavity, it remained unclear the processes involved that enabled such chamber formation.
Chambered cardiac organoids (CCOs) differentiated from human embryonic stem cells (H7) were generated by modulation of Wnt/ß-catenin signalling under fully defined conditions, and several growth factors essential for cardiac progenitor expansion. Transcriptomic profiling of day 8, day 14 and day 21 CCOs was performed by quantitative PCR and single-cell RNA sequencing. Endothelin-1 (EDN1) known to induce oxidative stress in cardiomyocytes was used to induce cardiac hypertrophy in CCOs in vitro. Functional characterization of cardiomyocyte contractile machinery was performed by immunofluorescence staining and analysis of brightfield and fluorescent video recordings. Quantitative PCR values between groups were compared using two-tailed Student's t tests. Cardiac organoid parameters comparison between groups was performed using two-tailed Mann-Whitney U test when sample size is small; otherwise, Welch's t test was used. Comparison of calcium kinetics parameters derived from the fluorescent data was performed using two-tailed Student's t tests.
Importantly, we demonstrated that a threshold number of cardiac progenitor was essential to line the circumference of the inner cavity to ensure proper formation of a chamber within the organoid. Single-cell RNA sequencing revealed improved maturation over a time course, as evidenced from increased mRNA expression of cardiomyocyte maturation genes, ion channel genes and a metabolic shift from glycolysis to fatty acid ß-oxidation. Functionally, CCOs recapitulated clinical cardiac hypertrophy by exhibiting thickened chamber walls, reduced fractional shortening, and increased myofibrillar disarray upon treatment with EDN1. Furthermore, electrophysiological assessment of calcium transients displayed tachyarrhythmic phenotype observed as a consequence of rapid depolarization occurring prior to a complete repolarization.
Our findings shed novel insights into the role of progenitors in CCO formation and pave the way for the robust generation of cardiac organoids, as a platform for future applications in disease modelling and drug screening in vitro.
The increasing use of silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) in consumer products and their inevitable seepage into the environment prompted us to investigate their potential ...toxicity to a fish cell line (BF-2) and zebrafish embryos under dark and Simulated Solar Light (SSL) exposure conditions. Using high throughput screening (HTS) platforms, we showed that the oxidative stress-dependent cytotoxicity and embryonic toxicity of NPs were significantly increased upon exposure to SSL. While, the toxicity of TiO2 NPs under SSL exposure could be explained by hydroxyl radical generation, the enhanced toxicity of Ag NPs under SSL exposure was due to surface oxidation and physicochemical modification of Ag NPs and shedding of Ag+, leading to an increased bioavailability of silver. Our observations that solar light could induce physicochemical transformation of TiO2 and Ag NPs and enhance their toxic potential emphasizes the need for conducting future toxicity studies under environmentally relevant exposure conditions to guide decision making on the safe handling of NPs.
Medical research in the recent years has achieved significant progress due to the increasing prominence of organoid technology. Various developed tissue organoids bridge the limitations of ...conventional 2D cell culture and animal models by recapitulating
cellular complexity. Current 3D cardiac organoid cultures have shown their utility in modelling key developmental hallmarks of heart organogenesis, but the complexity of the organ demands a more versatile model that can investigate more fundamental parameters, such as structure, organization and compartmentalization of a functioning heart. This review will cover the prominence of cardiac organoids in recent research, unpack current
3D models of the developing heart and look into the prospect of developing physiologically appropriate cardiac organoids with translational applicability. In addition, we discuss some of the limitations of existing cardiac organoid models in modelling embryonic development of the heart and manifestation of cardiac diseases.
Human embryonic stem cells (hESCs) promise to revolutionize reparative medicine through their potential in developing cell replacement therapies for diseases like diabetes and parkinsonism. Most of ...the existing hESC lines available for research, including all National Institutes of Health-registered lines, have been derived and maintained on mouse embryonic fibroblast feeders in the presence of xenoproteins. For future clinical application, many more hESC lines derived and grown in current good manufacturing practice, good tissue culture practice, and xeno-free conditions need to be developed. Concurrently, effective cryopreservation methods that prevent or limit the accidental contact of hESCs with nonsterile liquid nitrogen during periods of long-term storage have to be formulated. We describe a safe, xeno-free cryopreservation protocol for hESCs involving vitrification in closed sealed straws using human serum albumin as opposed to fetal calf serum as the main protein source in the cryoprotectant and long-term storage in the vapor phase of liquid nitrogen. After thaw, hESCs exhibited high thaw-survival rates and low differentiation rates, remained pluripotent, and maintained normal diploid karyotypes throughout extended passage. The cryopreservation technique we describe here should complement xeno-free culture conditions for hESCs already in refinement and will prove very useful for the setting up of hESC banks throughout the world.
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