Taking advantage of the potential of mesenchymal stromal cells in liver regeneration:Cells and extracellular vesicles as therapeutic strategies

Cell-based therapies for acute and chronic liver diseases are under continuous progress. Mesenchymal stem/stromal cells(MSCs) are multipotent cells able to migrate selectively to damaged tissue and contribute to its healing and regeneration. The MSC pro-regenerative effect occurs due to their immunomodulatory capacity and their ability to produce factors that promote cell protection and survival. Likewise,it has been observed that part of their paracrine effect is mediated by MSC-derived extracellular vesicles(EVs). EVs contain proteins,lipids and nucleic acids(DNA,m RNA,mi RNA,lnc RNA) from the cell of origin,allowing for intercellular communication. Recently,different studies have demonstrated that MSC-derived EVs could reproduce,at least in part,the biological effects obtained by MSCbased therapies. Moreover,due to EVs' stability for long periods of time and easy isolation methods they have become a therapeutic option to MSCs treatments. This review summarizes the latest results achieved in clinical trials using MSCs as cell therapy for liver regeneration,the role of EVs in liver physiopathology and the potential of MSC-derived EVs as intercellular mediators and therapeutic tools in liver diseases.

Role of neuroinflammation in neurodegeneration development

Studies in neurodegenerative diseases,including Alzheimer’s disease,Parkinson’s disease and Amyotrophic lateral sclerosis,Huntington’s disease,and so on,have suggested that inflammation is not only a result of neurodegeneration but also a crucial player in this process.Protein aggregates which are very common pathological phenomenon in neurodegeneration can induce neuroinflammation which further aggravates protein aggregation and neurodegeneration.Actually,inflammation even happens earlier than protein aggregation.Neuroinflammation induced by genetic variations in CNS cells or by peripheral immune cells may induce protein deposition in some susceptible population.Numerous signaling pathways and a range of CNS cells have been suggested to be involved in the pathogenesis of neurodegeneration,although they are still far from being completely understood.Due to the limited success of traditional treatment methods,blocking or enhancing inflammatory signaling pathways involved in neurodegeneration are considered to be promising strategies for the therapy of neurodegenerative diseases,and many of them have got exciting results in animal models or clinical trials.Some of them,although very few,have been approved by FDA for clinical usage.Here we comprehensively review the factors affecting neuroinflammation and the major inflammatory signaling pathways involved in the pathogenicity of neurodegenerative diseases,including Alzheimer’s disease,Parkinson’s disease,and Amyotrophic lateral sclerosis.We also summarize the current strategies,both in animal models and in the clinic,for the treatment of neurodegenerative diseases.

Advances of adeno-associated virus applied in gene therapy to hemophilia from bench work to the clinical use

Hemophilia A and B are diseases caused by a single gene deficiency and are thus suitable for gene therapy.In recent clinical research,adeno-associated virus(AAV)was employed by several teams in the treatment of hemophilia A and B,and the outcomes were encouraging.In this review,we summarized the most recent research on the mechanism and application of AAV in the treatment of hemophilia,trying to analyze the advantages of AAV gene therapy and the main challenges in its clinical use.We also summarized the clinical trials involving hemophilia,especially those employing AAV gene therapy to treat hemophilia A and B,some of which have already been completed and some that are still ongoing.From the reports of the completed clinical trials,we tried to determine the correlations among AAV dose,AAV serotype,immune response,and gene expression time.Finally,taking into account the most recent studies investigating AAV capsid modification,transgene optimization,and AAV chaperones,we summarized the direction of basic research and clinical applications of AAV in the future.

DNA end binding activity and Ku70/80 heterodimer expression in human colorectal tumor

AIM: To determine the DNA binding activity and protein levels of the Ku70/80 heterodimer, the functional mediator of the NHEJ activity, in human colorectal carcinogenesis.METHODS: The Ku70/80 DNA-binding activity was determined by electrophoretic mobility shift assays in 20 colon adenoma and 15 colorectal cancer samples as well as matched normal colonic tissues. Nuclear and cytoplasmic protein expression was determined by immunohistochemistry and Western blot analysis.RESULTS: A statistically significant difference was found in both adenomas and carcinomas as compared to matched normal colonic mucosa (P＜0.00). However,changes in binding activity were not homogenous with approximately 50% of the tumors showing a clear increase in the binding activity, 30% displaying a modest increase and 15% showing a decrease of the activity.Tumors, with increased DNA-binding activity, also showed a statistically significant increase in Ku70 and Ku86nuclear expression, as determined by Western blot and immunohistochemical analyses (P＜0.001). Cytoplasmic protein expression was found in pathological samples,but not in normal tissues either from tumor patients or from healthy subjects.CONCLUSION: Overall, our DNA-binding activity and protein level are consistent with a substantial activation of the NHEJ pathway in colorectal tumors. Since the NHEJ is an error prone mechanism, its abnormal activation can result in chromosomal instability and ultimately lead to tumorigenesis.

The Cytomegalovirus Enhancer Induces an Immediate Response to the Myosin Light Chain 2v Promoter during P19CL6 Cell Differentiation

The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhancer and a 250 bp MLC-2v promoter in front of the GFP gene to further evaluate the role of the CMV enhancer. This plasmid (pCBVenh/MLC-2vpro/EGFP) was stably introduced into P19CL6 cells, and the transfectant differentiated into cardiomyocytes with DMSO. Upon DMSO addition, GFP was immediately transcribed (within 2 days) and the amount of the transcript increased with cultivation. Concomitantly, GFP fluorescence was detected in the cells under a microscope. However, native MLC-2v was transcribed later on day 4. This expression time course is different from that of GFP. Clearly the CMV enhancer responded immediately to DMSO. Since GATA DNA-binding proteins play crucial roles in the initiation of cardiomyocyte differentiation, such a response could be ascribed to the presence of multiple GATA motifs in the enhancer sequence but not in the native MLC-2v promoter. Thus the CMV enhancer may be not only useful for gene therapy and monitoring cell differentiation but also the study of the role of GATA transcription factors expressed in P19CL6 cells.

Efficacy and safety of human umbilical cord-derived mesenchymal stem cells in the treatment of refractory immune thrombocytopenia:a prospective,single arm,phase I trial

Patients with refractory immune thrombocytopenia(ITP)frequently encounter substantial bleeding risks and demonstrate limited responsiveness to existing therapies.Umbilical cord-derived mesenchymal stem cells(UC-MSCs)present a promising alternative,capitalizing on their low immunogenicity and potent immunomodulatory effects for treating diverse autoimmune disorders.This prospective phase I trial enrolled eighteen eligible patients to explore the safety and efficacy of UC-MSCs in treating refractory ITP.The research design included administering UC-MSCs at escalating doses of 0.5×10^(6)cells/kg,1.0×10^(6)cells/kg,and 2.0×10^(6)cells/kg weekly for four consecutive weeks across three cohorts during the dose-escalation phase,followed by a dose of 2.0×10^(6)cells/kg weekly for the dose-expansion phase.Adverse events,platelet counts,and changes in peripheral blood immunity were monitored and recorded throughout the administration and follow-up period.Ultimately,12(with an addition of three patients in the 2.0×10^(6)cells/kg group due to dose-limiting toxicity)and six patients were enrolled in the dose-escalation and dose-expansion phase,respectively.Thirteen patients(13/18,72.2%)experienced one or more treatment emergent adverse events.Serious adverse events occurred in four patients(4/18,22.2%),including gastrointestinal hemorrhage(2/4),profuse menstruation(1/4),and acute myocardial infarction(1/4).The response rates were 41.7%in the dose-escalation phase(5/12,two received 1.0×10^(6)cells/kg per week,and three received 2.0×10^(6)cells/kg per week)and 50.0%(3/6)in the dose-expansion phase.The overall response rate was 44.4%(8/18)among all enrolled patients.To sum up,UC-MSCs are effective and well tolerated in treating refractory ITP(ClinicalTrials.gov ID:NCT04014166).

HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs

Genome-edited human induced pluripotent stem cells(iPSCs)hold great promise for therapeutic applications.However,low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockout and homology-directed repair(HDR)-edited iPSC lines,particularly for silent genes.This is partially due to chromatin compaction,inevitably limiting Cas9 access to the target DNA.Among the six HDAC inhibitors we examined,vorinostat,or suberoylanilide hydroxamic acid(SAHA),led to the highest HDR efficiency at both open and closed loci,with acceptable toxicity.HDAC inhibitors equally increased non-homologous end joining(NHEJ)editing efficiencies(~50%)at both open and closed loci,due to the considerable HDAC inhibitor-mediated increase in Cas9 and sgRNA expression.However,we observed more substantial HDR efficiency improvement at closed loci relative to open chromatin(2.8 vs.1.7-fold change).These studies provide a new strategy for HDRediting of silent genes in iPSCs.