Naringenin facilitates M2 macrophage polarization after myocardial ischemia-reperfusion by promoting nuclear translocation of transcription factor EB and inhibiting the NLRP3 inflammasome pathway.

Myocardial ischemia-reperfusion injury (MIRI) remains an unsolved puzzle in medical circles. Naringenin (NAR) is a flavonoid with cardioprotective potential. The purpose of this article was to discuss the protective mechanism of NAR in MIRI by regulating macrophage polarization. The MIRI mouse model was established and perfused with NAR before surgery. In the in vitro experiment, macrophages RAW264.7 were treated with lipopolysaccharide to induce M1 polarization after pretreatment with NAR. Rescue experiments were carried out to validate the functions of transcription factor EB (TFEB), the NLR pyrin domain containing 3 (NLRP3) inflammasome, and autophagy in macrophage polarization. NAR reduced histopathological injury and infarction of myocardial tissues in MIRI mice, inhibited M1 polarization and promoted M2 polarization of macrophages, diminished levels of pro-inflammatory factors, and augmented levels of anti-inflammatory factors. NAR facilitated TFEB nuclear translocation and inhibited the NLRP3 inflammasome pathway. Silencing TFEB or Nigericin partly nullified the effect of NAR on macrophage polarization. NAR increased autophagosome formation, autophagy flux, and autophagy level. Autophagy inhibitor 3-methyladenine partly invalidated the inhibition of NAR on the NLRP3 inflammasome pathway. In animal experiments, NAR protected MIRI mice through the TFEB-autophagy-NLRP3 inflammasome pathway. Collectively, NAR inhibited NLRP3 inflammasome activation and facilitated M2 macrophage polarization by stimulating TFEB nuclear translocation, thus protecting against MIRI.

miRNAs promote generation of porcine-induced pluripotent stem cells.

The pigs have similarities of organ size, immunology and physiology with humans. Porcine-induced pluripotent stem cells (piPSCs) have great potential application in regenerative medicine. Here, we established piPSCs induced from porcine fetal fibroblasts by the retroviral overexpression of Oct4, Sox2, Klf4, and c-Myc. The piPSCs not only express pluripotent markers but also have the capacity for differentiation in vivo and in vitro, including EB and teratoma formation. We supplemented microRNAs during the induction process because miR-302a, miR-302b, and miR-200c have been reported to be highly expressed in human and mouse embryonic stem cells and in iPSCs. In this study, we found that the overexpression of miR-302a, miR-302b, and miR-200c effectively improved the reprogramming efficiency and reduced the induction time for piPSCs in the OSKM and OSK induction systems. Due to the similar induction efficiency of 4F-induced piPSCs or of three factors combined with miR-302a, miR-302b, and miR-200c (3F-miRNA-induced piPSCs), we recommend the addition of miRNAs instead of c-Myc to reduce the tumorigenicity of piPSCs.

The generation of transgenic mice with fat1 and fad2 genes that have their own polyunsaturated fatty acid biosynthetic pathway.

BACKGROUND: Microorganisms and higher plants possess their own omega-3 and omega-6 polyunsaturated fatty acid (PUFAs) biosynthetic pathways. The n-6 fatty acid desaturase gene fad-2 codes for the n-6 desaturase enzyme that coverts oleic acid (OA 18:1 n-9) into linoleic acid (LA 18:2 n-6). The n-3 fatty acid desaturase gene fat-1 codes for the n-3 desaturase enzyme that converts n-6 PUFAs into n-3 PUFAs. Mammals lack n-3 and n-6 desaturase enzymes; therefore, they must obtain their omega-3 and omega-6 fatty acids by consuming plants or seafood. The beneficial effects of n-3 and n-6 PUFAs on human development and cardiovascular health have been well documented. METHODS: Here, we generated fat-1 and fad-2 transgenic mice by introducing mammal expression vectors containing the fat-1 and fad-2 genes via microinjection. RESULTS: Seven transgenic mice were obtained that expressed functional n-3 and n-6 desaturase enzymes. Analysis of the fatty acid contents of transgenic mouse livers revealed that n-6 and n-3 PUFA levels were greatly increased in the transgenic mice compared to wild-type mice. The use ratios of n-9 PUFAs (18:1 n-9) and n-6 PUFAs were both greater in the transgenic mice than in the wild-type controls. CONCLUSION: These transgenic mice were capable of producing their own omega-3 and omega-6 fatty acids. They have the same fatty acid metabolic pathways as higher plants and microbes.

Effects of TET1 knockdown on gene expression and DNA methylation in porcine induced pluripotent stem cells.

TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression of POU5F1 (OCT4), SOX2, KLF4, and MYC (C-MYC). siRNAs targeting to TET1 were used to transiently knockdown the expression of TET1 in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation of TET1. The effects of TET1 knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown of TET1 resulted in the downregulated expression of pluripotency-related genes, such as LEFTY2, KLF2, and SOX2, and the upregulated expression of differentiation-related genes including PITX2, HAND1, GATA6, and LEF1. However, POU5F1, MYC, KLF4, and NANOG were actually not downregulated. Further analysis showed that the methylation levels of the promoters for POU5F1 and MYC increased significantly after TET1 downregulation, whereas there were no obvious changes in the promoters of SOX2, KLF4, and NANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest that TET1 may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation.

The effects of the WT1 gene on apoptosis and development-related gene expression in porcine kidney fibroblasts and swine testis cells.

The Wilm's tumor 1 gene (WT1) encodes zinc finger proteins that function as tumor suppressors, and play important roles in the development of the genito-urinary system and other organs. Its precise function in the development of porcine tissues and organs, which is an attractive transplantation resource for certain human diseases, is still unclear. Here, we sought to define the role of WT1 in porcine kidney and testis tissues using porcine kidney fibroblasts (PKFs) and swine testis (ST) cells as in vitro models, both of which express WT1. The recombinant plasmids pLV3-WT1 shRNA and pIRES2 -WT1-EGFP were constructed to respectively down- and up-regulate the WT1 gene in porcine cells. The role of WT1 in cell proliferation was investigated by RT-PCR, immunocytochemical staining, apoptosis analysis, and Western blot. The pLV3-WT1 shRNA dramatically reduced WT1 expression at both the transcription and protein levels. The down-regulation of WT1 directly led to early cell apoptosis, and changes in Sf1, Sox9, and Gdnf gene expression in PKFs and ST cells. In contrast, up-regulation of WT1 gave no obvious phenotype in ST cells. Our results demonstrate that WT1 is essential for the survival of PKFs and ST cells because it regulates apoptosis- and development-related genes in the cells; however, no obvious effect was observed when WT1 was over-expressed.

Sanwei sandalwood decoction improves function of the gut microbiota in heart failure.

Objective: To investigate the effects of Sanwei sandalwood decoction on improving function of the intestinal flora in doxorubicin-induced heart failure in rats. Materials and methods: Thirty Sprague-Dawley rats were screened and randomly assigned into a blank group, a model group, and a Sanwei sandalwood decoction group (treatment group). The rat model of heart failure was prepared and established in the latter two groups. After successful model establishment, the treatment group received Sanwei sandalwood decoction by continuous gavage at 2 g/kg, once daily for 4 weeks. The other groups were given an equivalent volume of saline. After the final dose, fecal samples were collected from each group and analyzed by macrogenomics and nontargeted metabolomics to characterize the intestinal flora and associated metabolites. Results: The composition of gut microbiota was significantly different between the three groups. There were 778,808 common genes between the blank and model groups, while 49,315 genes were lost and 521,008 were gained in the model group relative to the blank group. At the phylum level, all groups of rat fecal samples were dominated by Firmicutes, Bacteroidota, Actinobacteria, and Proteobacteria. At the genus level, the microbial community composition in all experimental groups of rat fecal samples was dominated by Lactobacillus, Bifidobacterium, Limosilactobacillus, Allobaculum, Prevotella, and Ligilactobacillus spp. Interestingly, cluster analysis was performed on the top 30 KEGG ontology (KO) terms displaying significant differences in relative abundance in the rat fecal microbiome among experimental groups. The relative frequency of posttranslational modification, coenzyme transport and metabolism, cell wall, membrane, and envelope biogenesis in the eggNOG and CAZy databases. In the nontargeted metabolomics, the group principal component analysis revealed that the groups were well distinguished from one another. The different metabolites were screened with VIP >1, and the KEGG different metabolite classification and enrichment analysis revealed that there includes 15 metabolites pathway, including loxoprofen, conifery-l-acetate, trichilin A, and others. The arachidonic acid pathway also accounted for a significant portion of the KEGG pathway classification analysis. Conclusion: Sanwei sandalwood decoction positively affects the intestinal microbial environment of rats with heart failure, improving the gut dysbiosis that is caused by the condition. This treatment intervention inhibits the growth of pathogenic bacteria and promotes the growth of beneficial species.

CEA cell adhesion molecule 5 enriches functional human hematopoietic stem cells capable of long-term multi-lineage engraftment.

Hematopoietic stem cell (HSC) surface markers improve the understanding of cell identity and function. Here, we report that human HSCs can be distinguished by their expression of the CEA Cell Adhesion Molecule 5 (CEACAM5, CD66e), which serves as a marker and a regulator of HSC function. CD66e+ cells exhibited a 5.5-fold enrichment for functional long term HSCs compared to CD66e- cells. CD66e+CD34+CD90+CD45RA- cells displayed robust multi-lineage repopulation and serial reconstitution ability in immunodeficient mice compared to CD66e-CD34+CD90+CD45RA-cells. CD66e expression also identified almost all repopulating HSCs within the CD34+CD90+CD45RA- population. Together, these results indicated that CEACAM5 is a marker that enriches functional human hematopoietic stem cells capable of long-term multi-lineage engraftment.

A DL-4- and TNFα-based culture system to generate high numbers of nonmodified or genetically modified immunotherapeutic human T-lymphoid progenitors.

Several obstacles to the production, expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy. In the context of HSCT, delayed naïve T-cell recovery contributes to poor outcomes. A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors (HTLPs), allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus. However, it is challenging to produce HTLPs in the high numbers required to meet clinical needs. Here, we found that adding tumor necrosis factor alpha (TNFα) to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival. This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy (including CAR T-cell therapy).

Ex vivo generated human T-lymphoid progenitors as a tool to accelerate immune reconstitution after partially HLA compatible hematopoietic stem cell transplantation or after gene therapy.

Prolonged T-cell immunodeficiency following HLA- incompatible hematopoietic stem cell transplantation (HSCT) represents a major obstacle hampering the more widespread use of this approach. Strategies to fasten T-cell reconstitution in this setting are highly warranted as opportunistic infections and an increased risk of relapse account for high rates of morbidity and mortality especially during early month following this type of HSCT. We have implemented a feeder free cell system based on the use of the notch ligand DL4 and cytokines allowing for the in vitro differentiation of human T-Lymphoid Progenitor cells (HTLPs) from various sources of CD34+ hematopoietic stem and precursor cells (HSPCs). Co- transplantion of human T-lymphoid progenitors (HTLPs) and non- manipulated HSPCs into immunodeficient mice successfully accelerated the reconstitution of a polyclonal T-cell repertoire. This review summarizes preclinical data on the use of T-cell progenitors for treatment of post- transplantation immunodeficiency and gives insights into the development of GMP based protocols for potential clinical applications including gene therapy approaches. Future clinical trials implementing this protocol will aim at the acceleration of immune reconstitution in different clinical settings such as SCID and leukemia patients undergoing allogeneic transplantation. Apart from pure cell-therapy approaches, the combination of DL-4 culture with gene transduction protocols will open new perspectives in terms of gene therapy applications for primary immunodeficiencies.

Baboon envelope LVs efficiently transduced human adult, fetal, and progenitor T cells and corrected SCID-X1 T-cell deficiency.

T cells represent a valuable tool for treating cancers and infectious and inherited diseases; however, they are mainly short-lived in vivo. T-cell therapies would strongly benefit from gene transfer into long-lived persisting naive T cells or T-cell progenitors. Here we demonstrate that baboon envelope glycoprotein pseudotyped lentiviral vectors (BaEV-LVs) far outperformed other LV pseudotypes for transduction of naive adult and fetal interleukin-7-stimulated T cells. Remarkably, BaEV-LVs efficiently transduced thymocytes and T-cell progenitors generated by culture of CD34+ cells on Delta-like ligand 4 (Dll4). Upon NOD/SCIDγC-/- engraftment, high transduction levels (80%-90%) were maintained in all T-cell subpopulations. Moreover, T-cell lineage reconstitution was accelerated in NOD/SCIDγC-/- recipients after T-cell progenitor injection compared with hematopoietic stem cell transplantation. Furthermore, γC-encoding BaEV-LVs very efficiently transduced Dll4-generated T-cell precursors from a patient with X-linked severe combined immunodeficiency (SCID-X1), which fully rescued T-cell development in vitro. These results indicate that BaEV-LVs are valuable tools for the genetic modification of naive T cells, which are important targets for gene therapy. Moreover, they allowed for the generation of gene-corrected T-cell progenitors that rescued SCID-X1 T-cell development in vitro. Ultimately, the coinjection of LV-corrected T-cell progenitors and hematopoietic stem cells might accelerate T-cell reconstitution in immunodeficient patients.

miR-17, miR-21, and miR-143 Enhance Adipogenic Differentiation from Porcine Bone Marrow-Derived Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (BMSCs) have multilineage differentiation abilities toward adipocytes and osteoblasts. Recently, numerous studies have focused on the roles of microRNAs (miRNAs) in the process of adipogenic differentiation of human and mouse cells. However, the role of miRNAs in adipogenic differentiation process of porcine BMSCs (pBMSCs) remains unclear. In this study, pBMSCs were induced to differentiate into adipocytes using a chemical approach, and the roles of miR-17, miR-21, and miR-143 in this process were investigated. Our results showed that pBMSCs could be chemically induced to differentiate into adipocytes and that the expression of miR-17, miR-21, and miR-143 increased during differentiation. Then, overexpression of mimics of miR-17, miR-21, and miR-143 increased the number of oil red O-positive cells of adipocyte differentiation. The expression levels of CCAAT/enhancer-binding protein alpha (C/EBPα) mRNA showed increases of 1.8-, 1.5-, and 1.2-fold in the groups expressing mimics of miR-21, miR-17, and miR-143, respectively, at day 20. These results demonstrate that miR-17, miR-21, and miR-143 are involved in and promote the adipogenic differentiation of pBMSCs. This study provides an experimental basis for establishing a stable and efficient adipogenic differentiation model for applications in cell therapy and tissue engineering.

[The effect of astragalus injection on the expression of endoplasmic reticulum stress chaperonin GRP78 and GRP94 mRNA in adriamycin-injured cardiomyocytes with calumenin silencing by shRNA].

OBJECTIVE: To investigate the effect of astragalus injection on the endoplasmic reticulum stress in adriamycin(ADR)-injured cardiomyocytes with calumenin silencing by shRNA. METHODS: Firstly, the stable lentiviral calumenin shRNAvector was constructed. Secondly, in vitro cultured neonatal rat cardiac myocytes were randomly divided into control group、normal group (3 mg/L ADR), lentivirus infection group (lentivirus infection+3 mg/L ADR), Astragalus group 1 (3 mg/L ADR+Astragalus), Astragalus group 2 (lentivirus infection+3 mg/L ADR+Astragalus). The mRNA epression level of calumenin expression and reticulum stress chaperone in GRP78, GRP94 of each group was monitored by real-time PCR. RESULTS: ①Compared with that of the control group, the calumenin mRNA expression in the normal group was reduced(P<0.05), yet its mRNA level in lentivirus the infection group and the Astragalus group 2 was further reduced(P<0.01). Compared with that of the normal group, the mRNA contents of calumenin in the Astragalus group 1 was increased(P<0.05). The expression of calumenin in Astragalus group 2 was increased comparing with lentivirus infection group (P<0.01). ②Compared with that in the control group, the expression of reticulum stress chaperone in GRP78, GRP 94 in lentivirus infection group and normal group was significantly increased (P<0.01); Compared with that in the normal group, the expression of reticulum stress chaperone in GRP78, GRP94 in Astragalus group 1 was reduced(P<0.01); Compared with that in the lentivirus infection group, the expression of reticulum stress chaperone in GRP78, GRP94 in the Astragalus group 2 was obviously decreased(P<0.01). CONCLUSIONS: ①Calumenin can alleviate endoplasmic reticulum stress induced by ADR-injured myocardial cells. ②Astragalus injection can restrain the endoplasmic reticulum stress induced by adriamycin, which may be achieved by the calumenin protein.

Sequence-specific inhibition of microRNA via CRISPR/CRISPRi system.

Here, we report a convenient and efficient miRNA inhibition strategy employing the CRISPR system. Using specifically designed gRNAs, miRNA gene has been cut at a single site by Cas9, resulting in knockdown of the miRNA in murine cells. Using a modified CRISPR interference system (CRISPRi), inactive Cas9 can reversibly prevent the expression of both monocistronic miRNAs and polycistronic miRNA clusters. Furthermore, CRISPR/CRISPRi is also capable of suppressing genes in porcine cells.