Background:TheSCN5Agene encodes the α subunit of the cardiac voltage-gated sodium channel, NaV1.5. The missense mutation, D1275N, has been associated with a range of unusual phenotypes associated ...with reduced NaV1.5 function, including cardiac conduction disease and dilated cardiomyopathy. Curiously, the reported biophysical properties ofSCN5A-D1275N channels vary with experimental system.Methods and Results:First, using a human embryonic kidney (HEK) 293 cell-based heterologous expression system, theSCN5A-D1275N channels showed similar maximum sodium conductance but a significantly depolarizing shift of activation gate (+10 mV) compared to wild type. Second, we generated human-induced pluripotent stem cells (hiPSCs) from a 24-year-old female who carried heterozygousSCN5A-D1275N and analyzed the differentiated cardiomyocytes (CMs). AlthoughSCN5Atranscript levels were equivalent between D1275N and control hiPSC-CMs, both the total amount of NaV1.5 and the membrane fractions were reduced approximately half in the D1275N cells, which were rescued by the proteasome inhibitor MG132 treatment. Electrophysiological assays revealed that maximum sodium conductance was reduced to approximately half of that in control hiPSC-CMs in the D1275N cells, and maximum upstroke velocity of action potential was lower in D1275N, which was consistent with the reduced protein level of NaV1.5.Conclusions:This study successfully demonstrated diminished sodium currents resulting from lower NaV1.5 protein levels, which is dependent on proteasomal degradation, using a hiPSC-based model forSCN5A-D1275N-related sodium channelopathy.
Current differentiation protocols for human induced pluripotent stem cells (hiPSCs) produce heterogeneous cardiomyocytes (CMs). Although chamber-specific CM selection using cell surface antigens ...enhances biomedical applications, a cell surface marker that accurately distinguishes between hiPSC-derived atrial CMs (ACMs) and ventricular CMs (VCMs) has not yet been identified. We have developed an approach for obtaining functional hiPSC-ACMs and -VCMs based on CD151 expression. For ACM differentiation, we found that ACMs are enriched in the CD151
population and that CD151 expression is correlated with the expression of Notch4 and its ligands. Furthermore, Notch signaling inhibition followed by selecting the CD151
population during atrial differentiation leads to the highly efficient generation of ACMs as evidenced by gene expression and electrophysiology. In contrast, for VCM differentiation, VCMs exhibiting a ventricular-related gene signature and uniform action potentials are enriched in the CD151
population. Our findings enable the production of high-quality ACMs and VCMs appropriate for hiPSC-derived chamber-specific disease models and other applications.
Long QT syndrome type 3 (LQT3) is caused by gain-of-function mutations in the
gene, which encodes the α subunit of the cardiac voltage-gated sodium channel. LQT3 patients present bradycardia and ...lethal arrhythmias during rest or sleep. Further, the efficacy of β-blockers, the drug used for their treatment, is uncertain. Recently, a large multicenter LQT3 cohort study demonstrated that β-blocker therapy reduced the risk of life-threatening cardiac events in female patients; however, the detailed mechanism of action remains unclear.
This study aimed to establish LQT3-human induced pluripotent stem cells (hiPSCs) and to investigate the effect of propranolol in this model.
An hiPSCs cell line was established from peripheral blood mononuclear cells of a boy with LQT3 carrying the
-N1774D mutation. He had suffered from repetitive torsades de pointes (TdPs) with QT prolongation since birth (QTc 680 ms), which were effectively treated with propranolol, as it suppressed lethal arrhythmias. Furthermore, hiPSCs were differentiated into cardiomyocytes (CMs), on which electrophysiological functional assays were performed using the patch-clamp method.
N1774D-hiPSC-CMs exhibited significantly prolonged action potential durations (APDs) in comparison to those of the control cells (N1774D: 440 ± 37 ms vs. control: 272 ± 22 ms; at 1 Hz pacing;
< 0.01). Furthermore, N1774D-hiPSC-CMs presented gain-of-function features: a hyperpolarized shift of steady-state activation and increased late sodium current compared to those of the control cells. 5 μM propranolol shortened APDs and inhibited late sodium current in N1774D-hiPSC-CMs, but did not significantly affect in the control cells. In addition, even in the presence of intrapipette guanosine diphosphate βs (GDPβs), an inhibitor of G proteins, propranolol reduced late sodium current in N1774D cells. Therefore, these results suggested a unique inhibitory effect of propranolol on late sodium current unrelated to β-adrenergic receptor block in N1774D-hiPSC-CMs.
We successfully recapitulated the clinical phenotype of LQT3 using patient-derived hiPSC-CMs and determined that the mechanism, by which propranolol inhibited the late sodium current, was independent of β-adrenergic receptor signaling pathway.
The phenotypic changes in hematopoietic stem progenitor cells (HSPCs) with somatic mutations of malignancy‐related genes in patients with acquired aplastic anemia (AA) are poorly understood. As our ...initial study showed increased CXCR4 expression on HLA allele‐lacking (HLA−) HSPCs that solely support hematopoiesis in comparison to redundant HLA(+) HSPCs in AA patients, we screened the HSPCs of patients with various types of bone marrow (BM) failure to investigate their CXCR4 expression. In comparison to healthy individuals (n = 15, 12.3%–49.9%, median 43.2%), the median CXCR4+ cell percentages in the HSPCs of patients without somatic mutations were low: 29.3% (14.3%–37.3%) in the eight patients without HLA(−) granulocytes, 8.8% (4.1%–9.8%) in the five patients with HLA(−) cells accounting for >90% of granulocytes, and 7.8 (2.1%–8.7%) in the six patients with paroxysmal nocturnal hemoglobinuria. In contrast, the median percentage was much higher (78% 61.4%–88.7%) in the five AA patients without HLA(−) granulocytes possessing somatic mutations (c‐kit, t8;21, monosomy 7 one for each, ASXL1 n = 2), findings that were comparable to those (66.5%, 63.1%–88.9%) in the four patients with advanced myelodysplastic syndromes. The increased expression of CXCR4 may therefore reflect intrinsic abnormalities of HSPCs caused by somatic mutations that allow them to evade restriction by BM stromal cells.
X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who ...are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.
•A patient-derived iPSC model recapitulates defective erythroid maturation in female XLSA.•Azacitidine reactivates the silent wild-type ALAS2 allele and ameliorates inefficient erythropoiesis in iPSC-derived HPCs from female XLSA.
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Primary large B‐cell lymphoma of the bone marrow Hishizawa, Masakatsu; Okamoto, Kohei; Chonabayashi, Kazuhisa ...
British journal of haematology,
February 2007, Letnik:
136, Številka:
3
Journal Article
Calmodulin is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1-3. Recently, mutations in CALM1-3 have been reported to be associated with severe early-onset long-QT ...syndrome (LQTS). However, the underlying mechanism through which heterozygous calmodulin mutations lead to severe LQTS remains unknown, particularly in human cardiomyocytes. We aimed to establish an LQTS disease model associated with a CALM2 mutation (LQT15) using human induced pluripotent stem cells (hiPSCs) and to assess mutant allele-specific ablation by genome editing for the treatment of LQT15. We generated LQT15-hiPSCs from a 12-year-old boy with LQTS carrying a CALM2-N98S mutation and differentiated these hiPSCs into cardiomyocytes (LQT15-hiPSC-CMs). Action potentials (APs) and L-type Ca2+ channel (LTCC) currents in hiPSC-CMs were analyzed by the patch-clamp technique and compared with those of healthy controls. Furthermore, we performed mutant allele-specific knockout using a CRISPR-Cas9 system and analyzed electrophysiological properties. Electrophysiological analyses revealed that LQT15-hiPSC-CMs exhibited significantly lower beating rates, prolonged AP durations, and impaired inactivation of LTCC currents compared with control cells, consistent with clinical phenotypes. Notably, ablation of the mutant allele rescued the electrophysiological abnormalities of LQT15-hiPSC-CMs, indicating that the mutant allele caused dominant-negative suppression of LTCC inactivation, resulting in prolonged AP duration. We successfully recapitulated the disease phenotypes of LQT15 and revealed that inactivation of LTCC currents was impaired in CALM2-N98S hiPSC model. Additionally, allele-specific ablation using the latest genome-editing technology provided important insights into a promising therapeutic approach for inherited cardiac diseases.
BackgroundAlthough studies have demonstrated the feasibility of in vivo cardiac transplantation of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) using large animals, it requires large ...quantities of purified PSC-CMs. Moreover, genetic modification and contamination of non-CMs are inappropriate for clinical application. Using antibodies on the surface of the transplanted cells is one of the useful methods, but can be immunogenic and cause local inflammation or graft failure. We have shown the synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by microRNAs (miRNAs) expressed in specific cell types can efficiently detect and purify the particular cell populations. Using a miRNA switch and magnetic-activated cell sorting (MACS), we evaluated the efficiency of purification of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in a large scale.MethodsWe used CD4 as a selection marker for MACS and miR-208a as a specific mRNA of CMs. We synthesized CD4 mRNA and transfected it into differentiated cells from iPSCs to confirm CD4 expressing on the surface of the transfected cells. We also synthesized a miRNA switch encoding CD4 tagged with sequences targeted by miR-208a (CD4-208a switch) and transfected it into differentiated cells to demonstrate transfected non-CMs expressing CD4 and transfected CMs non-expressing CD4. Finally, we transfected the CD4-208a switch and puromycin resistance mRNA simultaneously, and purified iPSC-CMs by eliminating CD4+ cells using MACS and untransfected cells using puromycin. Purified cells were transplanted into NOG mouse hearts with myocardial infarction by direct injections into the myocardium.ResultsAfter transfecting CD4 mRNA into differentiated cells from iPSCs, 78±5% expressed CD4 on the surface. We also confirmed that the CD4-208a switch separated CMs and non-CMs. Using MACS and puromycin selection, we purified iPS-CMs 69±5% to 97±2% assessed by troponin T. Purified cells were also engrafted as CMs in mouse hearts.ConclusionsWe demonstrated that CD4-208a switch purifies iPSC-CMs efficiently in a large scale. Synthetic microRNA switches can apply for many studies of stem cell-based cell replacement therapy for clinical application.
Differential diagnosis of juvenile hemochromatosis along with hemolytic anemia is often difficult. We report a 23-year-old woman with macrocytic hemolytic anemia with iron overload. The patient ...showed high serum ferritin and transferrin saturation and low serum transferrin and ceruloplasmin. We also noticed stomatocytes in her blood smear, which was confirmed by scanning electron microscopy. Target gene sequencing identified a mutation in PIEZO1 (heterozygous c.6008C>A: p.A2003D). This mutation was reported previously in a family with dehydrated hereditary stomatocytosis (DHS1, OMIM 194380), but in the current case, it was identified to be a de novo mutation. We underscore DHS1 in the differential diagnosis of iron overload associated with non-transfused hemolytic anemia in children and young adults.
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