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2022 ◽  
Author(s):  
Asuka Eguchi ◽  
Sofía I. Torres-Bigio ◽  
Kassie Koleckar ◽  
Foster Birnbaum ◽  
Helen M. Blau

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart; however, these treatments do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that longer telomeres are protective against dilated cardiomyopathy. Here we investigated the role of telomeres as a target for therapy in DMD cardiomyocytes using human induced pluripotent stem cells (iPSCs) to model the disease. Compared to healthy controls, DMD cardiomyocytes exhibited reduced telomere lengths, cell size, nuclear size, and sarcomere density. The telomere-binding protein, TRF2, is a core component of the shelterin complex, which protects chromosome ends. TRF2 levels are reduced relative to healthy controls in DMD cardiomyocytes. We hypothesized that decreased TRF2 drives telomere attrition and subsequent cardiomyocyte death in the progression of dilated cardiomyopathy. Our data show that TRF2 overexpression prevented telomere attrition and also rescued deficits in cell size, nuclear size, sarcomere density, and calcium handling. These data highlight the benefits of TRF2 upregulation as a potential gene therapy to delay the onset of dilated cardiomyopathy.


2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Tobias Heinen ◽  
Stefano Secchia ◽  
James P. Reddington ◽  
Bingqing Zhao ◽  
Eileen E. M. Furlong ◽  
...  

AbstractWhile it is established that the functional impact of genetic variation can vary across cell types and states, capturing this diversity remains challenging. Current studies using bulk sequencing either ignore this heterogeneity or use sorted cell populations, reducing discovery and explanatory power. Here, we develop scDALI, a versatile computational framework that integrates information on cellular states with allelic quantifications of single-cell sequencing data to characterize cell-state-specific genetic effects. We apply scDALI to scATAC-seq profiles from developing F1 Drosophila embryos and scRNA-seq from differentiating human iPSCs, uncovering heterogeneous genetic effects in specific lineages, developmental stages, or cell types.


2022 ◽  
Vol 23 (1) ◽  
pp. 527
Author(s):  
Georg Lutter ◽  
Thomas Puehler ◽  
Lukas Cyganek ◽  
Jette Seiler ◽  
Anita Rogler ◽  
...  

Clinically used heart valve prostheses, despite their progress, are still associated with limitations. Biodegradable poly-ε-caprolactone (PCL) nanofiber scaffolds, as a matrix, were seeded with human endothelial colony-forming cells (ECFCs) and human induced-pluripotent stem cells-derived MSCs (iMSCs) for the generation of tissue-engineered heart valves. Cell adhesion, proliferation, and distribution, as well as the effects of coating PCL nanofibers, were analyzed by fluorescence microscopy and SEM. Mechanical properties of seeded PCL scaffolds were investigated under uniaxial loading. iPSCs were used to differentiate into iMSCs via mesoderm. The obtained iMSCs exhibited a comparable phenotype and surface marker expression to adult human MSCs and were capable of multilineage differentiation. EFCFs and MSCs showed good adhesion and distribution on PCL fibers, forming a closed cell cover. Coating of the fibers resulted in an increased cell number only at an early time point; from day 7 of colonization, there was no difference between cell numbers on coated and uncoated PCL fibers. The mechanical properties of PCL scaffolds under uniaxial loading were compared with native porcine pulmonary valve leaflets. The Young’s modulus and mean elongation at Fmax of unseeded PCL scaffolds were comparable to those of native leaflets (p = ns.). Colonization of PCL scaffolds with human ECFCs or iMSCs did not alter these properties (p = ns.). However, the native heart valves exhibited a maximum tensile stress at a force of 1.2 ± 0.5 N, whereas it was lower in the unseeded PCL scaffolds (0.6 ± 0.0 N, p < 0.05). A closed cell layer on PCL tissues did not change the values of Fmax (ECFCs: 0.6 ± 0.1 N; iMSCs: 0.7 ± 0.1 N). Here, a successful two-phase protocol, based on the timed use of differentiation factors for efficient differentiation of human iPSCs into iMSCs, was developed. Furthermore, we demonstrated the successful colonization of a biodegradable PCL nanofiber matrix with human ECFCs and iMSCs suitable for the generation of tissue-engineered heart valves. A closed cell cover was already evident after 14 days for ECFCs and 21 days for MSCs. The PCL tissue did not show major mechanical differences compared to native heart valves, which was not altered by short-term surface colonization with human cells in the absence of an extracellular matrix.


Author(s):  
David Sergeevichev ◽  
Victor Balashov ◽  
Victoria Kozyreva ◽  
Sophia Pavlova ◽  
Maria Vasiliyeva ◽  
...  

Different types of engineered cardiac constructs are being developed nowadays by many research groups. However, the immunological properties of such artificial tissues are not yet clearly understood. Previously, we have studied microfiber scaffolds carrying iPSC-derived cardiomyocytes. In this work, we evaluated the ability of these tissue-engineered constructs to activate the expression of CD28 and CTLA-4 proteins in T-lymphocytes which are early markers of the immune response. For this purpose electrospun PLA nanofibrous scaffolds were seeded with human iPSCs-CM and cultivated for 2 weeks. After, allogeneic mononuclear cells were co-cultured during 48 hours with 3 groups of samples that were tissue-engineered constructs, pure culture of cardiomyocytes and bare scaffolds followed by analysis of CD28/CTLA-4 expression on T-lymphocytes via flow cytometry. PLA scaffolds and concanavalin A (positive control) stimulation statistically significantly increased CD28 expression on CD4+ cells (up to 61.3% and 66.3%) and on CD8+ cells (up to 17.8% and 21.7%). CD28/CTLA-4 expression didn&rsquo;t increase during co-cultivation of T-lymphocytes with cardiac engineered constructs and iPSC-CM monolayers. Thus, iPSCs-CM in monolayers and on PLA nanofibrous scaffolds didn&rsquo;t cause T-cell activation, which allows us to expect that such cardiac constructs are not a cause of rejection after implantation.


Biomolecules ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 34
Author(s):  
Leticia Pérez-Sisqués ◽  
Júlia Solana-Balaguer ◽  
Genís Campoy-Campos ◽  
Núria Martín-Flores ◽  
Anna Sancho-Balsells ◽  
...  

RTP801/REDD1 is a stress-regulated protein whose levels are increased in several neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s diseases (HD). RTP801 downregulation ameliorates behavioral abnormalities in several mouse models of these disorders. In HD, RTP801 mediates mutant huntingtin (mhtt) toxicity in in vitro models and its levels are increased in human iPSCs, human postmortem putamen samples, and in striatal synaptosomes from mouse models of the disease. Here, we investigated the role of RTP801 in the hippocampal pathophysiology of HD. We found that RTP801 levels are increased in the hippocampus of HD patients in correlation with gliosis markers. Although RTP801 expression is not altered in the hippocampus of the R6/1 mouse model of HD, neuronal RTP801 silencing in the dorsal hippocampus with shRNA containing AAV particles ameliorates cognitive alterations. This recovery is associated with a partial rescue of synaptic markers and with a reduction in inflammatory events, especially microgliosis. Altogether, our results indicate that RTP801 could be a marker of hippocampal neuroinflammation in HD patients and a promising therapeutic target of the disease.


Biomolecules ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 15
Author(s):  
Grace A. Christopher ◽  
Rebecca J. Noort ◽  
Jessica L. Esseltine

During embryonic germ layer development, cells communicate with each other and their environment to ensure proper lineage specification and tissue development. Connexin (Cx) proteins facilitate direct cell–cell communication through gap junction channels. While previous reports suggest that gap junctional intercellular communication may contribute to germ layer formation, there have been limited comprehensive expression analyses or genetic ablation studies on Cxs during human pluripotent stem cell (PSC) germ lineage specification. We screened the mRNA profile and protein expression patterns of select human Cx isoforms in undifferentiated human induced pluripotent stem cells (iPSCs), and after directed differentiation into the three embryonic germ lineages: ectoderm, definitive endoderm, and mesoderm. Transcript analyses by qPCR revealed upregulation of Cx45 and Cx62 in iPSC-derived ectoderm; Cx45 in mesoderm; and Cx30.3, Cx31, Cx32, Cx36, Cx37, and Cx40 in endoderm relative to control human iPSCs. Generated Cx43 (GJA1) CRISPR-Cas9 knockout iPSCs successfully differentiated into cells of all three germ layers, suggesting that Cx43 is dispensable during directed iPSC lineage specification. Furthermore, qPCR screening of select Cx transcripts in our GJA1-/- iPSCs showed no significant Cx upregulation in response to the loss of Cx43 protein. Future studies will reveal possible compensation by additional Cxs, suggesting targets for future CRISPR-Cas9 ablation studies in human iPSC lineage specification.


2021 ◽  
Author(s):  
Olivia Cypris ◽  
Julia Franzen ◽  
Joana Frobel ◽  
Philipp Glueck ◽  
Chao-Chung Kuo ◽  
...  

DNA methyltransferase 3A (DNMT3A) is a frequently mutated gene in many hematological malignancies, indicating that it may be essential for hematopoietic differentiation. Here, we addressed the functional relevance of DNMT3A for differentiation of human induced pluripotent stem cells (iPSCs) by knocking out exon 2, 19, or 23. Exon 19-/- and 23-/- lines revealed absence of almost the entire de novo DNA methylation during mesenchymal and hematopoietic differentiation. Yet, differentiation was only slightly reduced in exon 19-/- and increased in exon 23-/- lines, whereas there was no significant impact on gene expression in hematopoietic progenitors (iHPCs). Notably, DNMT3A-/- iHPCs recapitulate some DNA methylation differences of acute myeloid leukemia with DNMT3A mutations. Furthermore, multicolor genetic barcoding revealed competitive growth advantage of exon 23-/- iHPCs. Our results demonstrate that de novo DNA methylation during hematopoietic differentiation of iPSCs is almost entirely dependent on DNMT3A and exon 23-/- iHPCs even gained growth advantage.


2021 ◽  
Author(s):  
Vincent Soubannier ◽  
Mathilde Chaineau ◽  
Lale Gursu ◽  
Ghazal Haghi ◽  
Anna Kristyna Franco Flores ◽  
...  

Astrocytes play important roles in the function and survival of neuronal cells. Dysfunctions of astrocytes are associated with numerous disorders and diseases of the nervous system, including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Human induced pluripotent stem cell (iPSC)-based approaches are becoming increasingly important for the study of the mechanisms underlying the involvement of astrocytes in non-cell autonomous processes of motor neuron degeneration in ALS. These studies must account for the molecular and functional diversity among astrocytes in different regions of the brain and spinal cord. It is essential that the most pathologically-relevant astrocyte preparations are used when investigating non-cell autonomous mechanisms of either upper or lower motor neuron degeneration in ALS. In this context, the main aim of this study was to establish conditions enabling rapid and robust generation of physiologically-relevant ventral spinal cord-like astrocytes that would provide an enhanced experimental model for the study of lower motor neuron degeneration in ALS. Neural progenitor cells with validated caudal and ventral features were derived from human iPSCs and differentiated into astrocytes, which were then characterized by examining morphology, markers of ventral spinal cord astrocytes, spontaneous and induced calcium transients, and astrogliosis markers. Efficient and streamlined generation of human iPSC-derived astrocytes with molecular and biological properties similar to physiological astrocytes in the ventral spinal cord was achieved. These induced astrocytes express markers of mature ventral spinal cord astrocytes, exhibit spontaneous and ATP-induced calcium transients, and lack signs of overt activation. Human iPSC-derived astrocytes with ventral spinal features offer advantages over more generic astrocyte preparations for the study of both ventral spinal cord astrocyte biology and the involvement of astrocytes in mechanisms of lower motor neuron degeneration in ALS.


Author(s):  
Masamitsu Sone ◽  
Sou Nakamura ◽  
Sachiko Umeda ◽  
Harumi Ginya ◽  
Motohiko Oshima ◽  
...  
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