Project description:we conducted temporal bulk RNAseq on 60-day-old mature hiPS-CMs infected with either LacZ (control) or CDK1, CDK4, CCNB, CCND (4F) adenovirus for 24, 48 or 72 h.
Project description:We conducted temporal single cell RNAseq on 60-day-old mature hiPS-CMs infected with either LacZ (control) or CDK1, CDK4, CCNB, CCND (4F) adenovirus for 24, 48 or 72 h.
Project description:The regenerative capacity of the heart after myocardial infarction (MI) is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 and Cdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in 15-20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after MI. Here, we aim to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Temporal bulk RNAseq of mature hiPS-CMs treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in cardiomyocytes after 48 h post-infection with 4F, which was associated with sarcomere disassembly and metabolic reprogramming.
Project description:Purpose: To induce hiPS-CMs maturation by using engineering technics, including culturing on the anisotropic pattern, has been widely explored. However, the underlying mechanisms of the benefits driven by the aligned topographic stimuli are still pending. To obtain insights into the underlying molecular pathways/signaling involved in the facilitation of hiPS-CMs maturation driven by specific topographic stimuli, we performed RNA-seq for the hiPS-CMs samples after culturing on the different patterns Methods: hiPS-CMs cultured on the flat bottom 24-well plate (MS-80240; Sumilon)/random nanofiber substrate (NanoECM, 2401; Funakoshi)/aligned nanofiber substrate (NanoAligned, 2402; Funakoshi) for one week. Then the cells were harvested as Flat, Random, Align group samples. Total RNA was extracted using the RNeasy Plus mini kit (740990.250; Takara), the concentration of RNA was measured by using NanoDrop (2000/2000c Spectrophotometers, Thermo Fisher). The Illumina package bcl2fastq software was used for base-calling. The raw reads were mapped to the human reference genome sequences (GRCh38) using TopHat ver. 2.1.1 in combination with Bowtie2 ver. 2.3.4.1 Results: The principal component analysis(PCA) analysis revealed that the hiPS-CMs from flat and random patterns showed the most variance. And the differentially expressed genes (DEGs) were detected with theDESeq2 package, showed that the flat group samples occupied most of the enrichment gene expression. The enriched DEGs termed by Gene Ontology(GO) Biological Process revealed that the upregulated genes in flat group are mainly related to the regulation of cell movement, including extracellular matrix organization, cell adhesion, and cell migration. The well-known cardiac maturation markers such as MYH7 and TNNI3 showed significantly upregulated in align group, as well as cardiac structural(MLC2, TNNT2, GJA1), and calcium handling relevant genes( CASQ2, CAMK2B, CAV3) also showed a higher expression than that of in flat group. The most up-regulated gene sets related pathways in align group include cardiac development and heart morphogenesis, negative regulation of binding, and microtubule-based process. In contrast, the relative down-regulated gene enriched pathways in align group are mostly involved in KRT gene family, which also plays a role in cell movement Conclusions: hiPS-CMs by culturing on the aligned pattern for a short-term have facilitated maturation of hiPS-CMs. The up-regulated gene sets in align group related to regulating cell cycle pathways. The genome-wide expression analysis provided insights into the underlying molecular pathways/signaling involved in the specific topographic stimuli induced maturation of hiPS-CMs
Project description:We carried out bulk RNA sequencing of normal adult mouse CMs (Control-CMs) and adult mouse CMs with ectopic overexpression of Yamanaka factors (Oct4, Sox2, Klf4, Myc, OSKM-CMs), based on a hypothesis that OSKM would induce at least partial CM dedifferentiation in vivo. More than 2,000 genes were differentially expressed between Control- and OSKM-CMs, which were enriched for gene ontology (GO) terms related to multiple key aspects of CM dedifferentiation. This study provides a genome-wide transcriptional profile of dedifferentiating CMs induced by OSKM.
Project description:Expression data from hiPS lines after commitment towards mesenchymal lineage (hiPS-MSC) and expression data of mesenchymal lines (MSC), used as positive control of commitment. Following this, hiPS-MSC and MSC are seeded on scaffold to differentiate in a ligamentous (middle) and osseous (edges) part (post 21 days 3D differentiation) We used microarrays to validate efficient commitment of hiPS to generate hiPS-MSC and do whole transcriptome comparison of hiPS-MSC vs isolated MSC (positive control) and then compare the enhanced biological functions after differentiation of these cells on a scaffold