Histological Study on in vitro Co-cultivation of the Myocardium Tissue and Cells with Mouse Embryonic Fibroblasts

The histological observation was experimentally conducted on in vitro cultured mouse embryonic myocardium cells and myocardiumoid cell mass. The mouse embryo tissue were cultured and regular pulsatile myocardiumoid tissue could be found. During in vitro culture, the myofilament bundles in the cell were gradually increasing and strongly connectted each other with embryonic age and there were loose muscle fibers initially and intercalated discs were close to each other. The lose myofilament bundles were developed in muscle fibers with age and the distance between intercalated discs was enlarged. There were myofilamentoid structure in inactive cells and filament peripherily.

Mitofusion 2 Overexpression Decreased Proliferation of Human Embryonic Lung Fibroblasts in Acute Respiratory Distress Syndrome through Inhibiting RAS-RAF-1-ERK1/2 Pathway

Acute respiratory distress syndrome(ARDS)is one of the most fatal diseases worldwide.Pulmonary fibrosis occurs early in ARDS,and its severity plays a crucial role in ARDS mortality rate.Some studies suggested that fibroproliferation is an essential mechanism in ARDS.Mitofusion2(Mfn2)overexpression plays a role in inhibiting cell proliferation.However,the role and potential mechanism of Mfn2 on the proliferation of fibroblasts is still unknown.In this study,we aimed at exploring the effect of Mfn2 on the human embryonic lung fibroblasts(HELF)and discussed its related mechanism.The HELF were treated with the Mfn2 overexpressing lentivirus(adv-Mfn2).The cell cycle was detected by flow cytometry.MTT,PCR and Western blotting were used to investigate the effect of Mfn2 on the proliferation of the HELF,collagen expression,the RAS-RAF-1-ERK1/2 pathway and the expression of cycle-related proteins(p21,p27,Rb,Raf-1,p-Raf-1,Erk1/2 and p-Erk1/2).The co-immunoprecipitation assay was used to explore the interaction between Mfn2 and Ras.The results showed that the overexpression of Mfn2 inhibited the proliferation of the HELF and induced the cell cycle arrest at the G0/G1 phase.Meanwhile,Mfn2 also inhibited the expression of collagen I,p-Erk and p-Raf-1.In addition,an interaction between Mfn2 and Ras existed in the HELF.This study suggests that the overexpression of Mfn2 can decrease the proliferation of HELF in ARDS,which was associated with the inhibition of the RAS-RAF-1-ERK1/2 pathway.The results may offer a potential therapeutic intervention for patients with ARDS.

Fibroblasts upregulate expression of adhesion molecules and promote lymphocyte retention in 3D fibroin/gelatin scaffolds

Bioengineered scaffolds are crucial components in artificial tissue construction.In general,these scaffolds provide inert three-dimensional(3D)surfaces supporting cell growth.However,some scaffolds can affect the phenotype of cultured cells,especially,adherent stromal cells,such as fibroblasts.Here we report on unique properties of 3D fibroin/gelatin materials,which may rapidly induce expression of adhesion molecules,such as ICAM-1 and VCAM-1,in cultured primary murine embryonic fibroblasts(MEFs).In contrast,two-dimensional(2D)fibroin/gelatin films did not show significant effects on gene expression profiles in fibroblasts as compared to 3D culture conditions.Interestingly,TNF expression was induced in MEFs cultured in 3D fibroin/gelatin scaffolds,while genetic or pharmacological TNF ablation resulted in diminished ICAM-1 and VCAM-1 expression by these cells.Using selective MAPK inhibitors,we uncovered critical contribution of JNK to 3D-induced upregulation of these adhesion molecules.Moreover,we observed ICAM-1/VCAM-1-dependent adhesion of lymphocytes to fibroblasts cultured in 3D fibroin/gelatin scaffolds,but not on 2D fibroin/gelatin films,suggesting functional reprogramming in stromal cells,when exposed to 3D environment.Finally,we observed significant infiltration of lymphocytes into 3D fibroin/gelatin,but not into collagen scaffolds in vivo upon subcapsular kidney implantation in mice.Together our data highlight the important features of fibroin/gelatin scaffolds,when they are produced as 3D sponges rather than 2D films,which should be considered when using these materials for tissue engineering.

An optimized prime editing system for efficient modification of the pig genome

Prime editing(PE)is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-tobase conversions.However,its low efficiency hampers the application in creating novel breeds and biomedical models,especially in pigs and other important farm animals.Here,we demonstrate that the pig genome is editable using the PE system,but the editing efficiency was quite low as expected.Therefore,we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts(PEFs).First,we modified the peg RNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine,which significantly enhanced PE efficiency by improving the expression of peg RNA and targeted cleavage.Then,we targeted SAMHD1,a deoxynucleoside triphosphate triphosphohydrolase(d NTPase)that impedes the reverse transcription process in retroviruses,and found that treatment with its inhibitor,cephalosporin C zinc salt(CPC),increased PE efficiency up to 29-fold(4-fold on average),presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase(M-MLV RT)in the PE system.Moreover,PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors(HDACis).Among the four HDACis tested,panobinostat was the most efficient,with an efficiency up to 122-fold(7-fold on average),partly due to the considerable HDACi-mediated increase in transgene expression.In addition,the synergistic use of the three strategies further enhanced PE efficiency in PEFs.Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.