Biology subjects

Sakurai, H.

Publications and source records attributed to Sakurai, H..

3 recordsLinked to original sources

bioRxiv2022

Hit-and-run silencing of endogenous DUX4 by targeting DNA hypomethylation on D4Z4 repeats in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD), a progressive skeletal muscle disorder, is epigenetically characterized by DNA hypomethylation of the D4Z4 repeats in the 4q35 region, which enables aberrant DUX4 expression. Sustainable DUX4 suppression is thus a promising therapeutic strategy by which to prevent disease progression, but most of the supposed methods to achieve this depend on the expression of a mediator biochemical entity that would potentially narrow the quality of life of individuals with FSHD in the clinical context. In this study, we report that by applying hit-and-run silencing with dCas9-mediated epigenetic editing targeting DNA hypomethylation on D4Z4 repeats, we could achieve the suppression of endogenous DUX4 in our FSHD patient-derived iPSC model. Notably, DNA methylation was significantly upregulated in FSHD cells and suppression effects were observed for at least two weeks after intervention, which was not the case with transient treatments of typical dCas9-KRAB alone. Off-target analysis showed that despite the potential genome-wide risk for DNA methylation, the impact on the transcriptome was limited. We propose that hit-and-run silencing could be a promising option to prevent disease progression with minimum intervention for individuals with FSHD, motivating further study for clinical development. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/487997v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@19e85dforg.highwire.dtl.DTLVardef@672798org.highwire.dtl.DTLVardef@58717dorg.highwire.dtl.DTLVardef@83044b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2021

Skeletal muscle releases extracellular vesicles with distinct protein and miRNA signatures that accumulate and function within the muscle microenvironment

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since the skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish SkM-EVs from others, whether the skeletal muscle releases EVs in vivo and how much the skeletal muscle-derived EVs (SkM-EVs) account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings reveal that the skeletal muscle releases exosome-like small EVs with distinct protein and miRNA profiles in vivo and that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.

physiology↗

bioRxiv2021

Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patients pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) can potentially recapitulate disease pathology more accurately than existing disease models when differentiated into affected cell types. Thus, successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection procedures, and clonal variations must be overcome. Moreover, their functionality to exhibit muscular contraction has never been reported. Here, we demonstrated that bulk MYOD1- hiPSCs established with puromycin selection, but not with G418 selection, showed high differentiation efficiency, generating more than 80% Myogenin (MyoG)+ and Myosin heavy chain (MHC)+ muscle cells within seven days. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties compared with those of clonally established MYOD1- hiPSCs, suggesting that the bulk method may minimize the effects of clonal variations. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical pulse stimulation, indicating their functionality. Together, the findings indicate that our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and facilitates the generation of muscular disease models. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/468571v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@12c091eorg.highwire.dtl.DTLVardef@ad1ccorg.highwire.dtl.DTLVardef@53ba90org.highwire.dtl.DTLVardef@fd65f5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗