DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show ...that these "replicon clusters" coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call "replication domains," separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) ...cells with normal numbers of poorly functioning B cells (T–B+NK–). Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, we demonstrate a complete block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores normal T cell development, including the production of mature T cell populations with a broad T cell receptor (TCR) repertoire. Whole-genome sequencing of corrected cells demonstrates no CRISPR/Cas9 off-target modifications. These studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies.
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•iPSC modeling of SCID•CRISPR/Cas-enhanced gene correction•Broad T cell receptor repertoire•No off-target modifications
Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, Chang et al. define a block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores normal T cell development.
We show that knockdown of KLF1 in human and mouse adult erythroid progenitors markedly reduces BCL11A levels and increases human γ-globin/β-globin expression ratios. These results suggest that KLF1 ...controls globin gene switching by directly activating β-globin and indirectly repressing γ-globin gene expression. Controlled knockdown of KLF1 in adult erythroid progenitors may provide a method to activate fetal hemoglobin expression in individuals with β-thalassemia or sickle cell disease.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Background We aim to generate a line of "universal donor" human induced pluripotent stem cells (hi PSC s) that are nonimmunogenic and, therefore, can be used to derive cell products suitable for ...allogeneic transplantation. Methods and Results hi PSC s carrying knockout mutations for 2 key components (β2 microglobulin and class II major histocompatibility class transactivator) of major histocompatibility complexes I and II (ie, human leukocyte antigen HLA I/ II knockout hi PSC s) were generated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene-editing system and differentiated into cardiomyocytes. Pluripotency-gene expression and telomerase activity in wild-type ( WT ) and HLAI / II knockout hi PSC s, cardiomyocyte marker expression in WT and HLAI / II knockout hi PSC -derived cardiomyocytes, and assessments of electrophysiological properties (eg, conduction velocity, action-potential and calcium transient half-decay times, and calcium transient increase times) in spheroid-fusions composed of WT and HLAI / II knockout cardiomyocytes, were similar. However, the rates of T-cell activation before (≈21%) and after (≈24%) exposure to HLAI / II knockout hi PSC -derived cardiomyocytes were nearly indistinguishable and dramatically lower than after exposure to WT hi PSC -derived cardiomyocytes (≈75%), and when WT and HLAI / II knockout hi PSC -derived cardiomyocyte spheroids were cultured with human peripheral blood mononuclear cells, the WT hi PSC -derived cardiomyocyte spheroids were smaller and displayed contractile irregularities. Finally, expression of HLA -E and HLA -F was inhibited in HLAI / II knockout cardiomyocyte spheroids after coculture with human peripheral blood mononuclear cells, although HLA -G was not inhibited; these results are consistent with the essential role of class II major histocompatibility class transactivator in transcriptional activation of the HLA -E and HLA-F genes, but not the HLA -G gene. Expression of HLA -G is known to inhibit natural killer cell recognition and killing of cells that lack other HLAs. Conclusions HLAI / II knockout hi PSC s can be differentiated into cardiomyocytes that induce little or no activity in human immune cells and, consequently, are suitable for allogeneic transplantation.
To assess the genetic consequences of induced pluripotent stem cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, ...and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. These data suggest that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which “captures” their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method.
► iPSC clones contain hundreds of SNVs that are unique to each clone ► Most iPSC genomes do not contain recurrently mutated genes or pathways ► Reprogramming can select for rare cells with shared genetic variants ► Most SNVs are probably preexisting mutations “captured” by cloning
Hemolytic diseases are characterized by enhanced intravascular hemolysis resulting in heme-catalyzed reactive oxygen species generation, which leads to endothelial dysfunction and oxidative damage. ...Hemopexin (Hx) is a plasma heme scavenger able to prevent endothelial damage and tissue congestion in a model of heme overload. Here, we tested whether Hx could be used as a therapeutic tool to counteract heme toxic effects on the cardiovascular system in hemolytic diseases.
By using a model of heme overload in Hx-null mice, we demonstrated that heme excess in plasma, if not bound to Hx, promoted the production of reactive oxygen species and the induction of adhesion molecules and caused the reduction of nitric oxide availability. Then, we used β-thalassemia and sickle cell disease mice as models of hemolytic diseases to evaluate the efficacy of an Hx-based therapy in the treatment of vascular dysfunction related to heme overload. Our data demonstrated that Hx prevented heme-iron loading in the cardiovascular system, thus limiting the production of reactive oxygen species, the induction of adhesion molecules, and the oxidative inactivation of nitric oxide synthase/nitric oxide, and promoted heme recovery and detoxification by the liver mainly through the induction of heme oxygenase activity. Moreover, we showed that in sickle cell disease mice, endothelial activation and oxidation were associated with increased blood pressure and altered cardiac function, and the administration of exogenous Hx was found to almost completely normalize these parameters.
Hemopexin treatment is a promising novel therapy to protect against heme-induced cardiovascular dysfunction in hemolytic disorders.
A major unmet need in the cystic fibrosis (CF) therapeutic landscape is the lack of effective treatments for nonsense CFTR mutations, which affect approximately 10% of CF patients. Correction of ...nonsense CFTR mutations via genomic editing represents a promising therapeutic approach. In this study, we tested whether prime editing, a novel CRISPR-based genomic editing method, can be a potential therapeutic modality to correct nonsense CFTR mutations. We generated iPSCs from a CF patient homozygous for the CFTR W1282X mutation. We demonstrated that prime editing corrected one mutant allele in iPSCs, which effectively restored CFTR function in iPSC-derived airway epithelial cells and organoids. We further demonstrated that prime editing may directly repair mutations in iPSC-derived airway epithelial cells when the prime editing machinery is efficiently delivered by helper-dependent adenovirus (HDAd). Together, our data demonstrated that prime editing may potentially be applied to correct CFTR mutations such as W1282X.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Sickle cell disease (SCD)-associated nephropathy is a major source of morbidity and mortality in patients because of the lack of efficacious treatments targeting renal manifestations of the disease. ...Here, we describe a long-term treatment strategy with the selective endothelin-A receptor (ET
) antagonist, ambrisentan, designed to interfere with the development of nephropathy in a humanized mouse model of SCD. Ambrisentan preserved GFR at the level of nondisease controls and prevented the development of proteinuria, albuminuria, and nephrinuria. Microscopy studies demonstrated prevention of podocyte loss and structural alterations, the absence of vascular congestion, and attenuation of glomerulosclerosis in treated mice. Studies in isolated glomeruli showed that treatment reduced inflammation and oxidative stress. At the level of renal tubules, ambrisentan treatment prevented the increased excretion of urinary tubular injury biomarkers. Additionally, the treatment strategy prevented tubular brush border loss, diminished tubular iron deposition, blocked the development of interstitial fibrosis, and prevented immune cell infiltration. Furthermore, the prevention of albuminuria in treated mice was associated with preservation of cortical megalin expression. In a separate series of identical experiments, combined ET
and ET
receptor antagonism provided only some of the protection observed with ambrisentan, highlighting the importance of exclusively targeting the ET
receptor in SCD. Our results demonstrate that ambrisentan treatment provides robust protection from diverse renal pathologies in SCD mice, and suggest that long-term ET
receptor antagonism may provide a strategy for the prevention of renal complications of SCD.
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