Characterizing neuroinflammation and identifying prenatal diagnostic markers for neural tube defects through integrated multi-omics analysis.

BACKGROUND: Neural Tube Defects (NTDs) are congenital malformations of the central nervous system resulting from the incomplete closure of the neural tube during early embryonic development. Neuroinflammation refers to the inflammatory response in the nervous system, typically resulting from damage to neural tissue. Immune-related processes have been identified in NTDs, however, the detailed relationship and underlying mechanisms between neuroinflammation and NTDs remain largely unclear. In this study, we utilized integrated multi-omics analysis to explore the role of neuroinflammation in NTDs and identify potential prenatal diagnostic markers using a murine model. METHODS: Nine public datasets from Gene Expression Omnibus (GEO) and ArrayExpress were mined using integrated multi-omics analysis to characterize the molecular landscape associated with neuroinflammation in NTDs. Special attention was given to the involvement of macrophages in neuroinflammation within amniotic fluid, as well as the dynamics of macrophage polarization and their interactions with neural cells at single-cell resolution. We also used qPCR assay to validate the key TFs and candidate prenatal diagnostic genes identified through the integrated analysis in a retinoic acid-induced NTDs mouse model. RESULTS: Our analysis indicated that neuroinflammation is a critical pathological feature of NTDs, regulated both transcriptionally and epigenetically within central nervous system tissues. Key alterations in gene expression and pathways highlighted the crucial role of STATs molecules in the JAK-STAT signaling pathway in regulating NTDs-associated neuroinflammation. Furthermore, single-cell resolution analysis revealed significant polarization of macrophages and their interaction with neural cells in amniotic fluid, underscoring their central role in mediating neuroinflammation associated with NTDs. Finally, we identified a set of six potential prenatal diagnostic genes, including FABP7, CRMP1, SCG3, SLC16A10, RNASE6 and RNASE1, which were subsequently validated in a murine NTDs model, indicating their promise as prospective markers for prenatal diagnosis of NTDs. CONCLUSIONS: Our study emphasizes the pivotal role of neuroinflammation in the progression of NTDs and underlines the potential of specific inflammatory and neural markers as novel prenatal diagnostic tools. These findings provide important clues for further understanding the underlying mechanisms between neuroinflammation and NTDs, and offer valuable insights for the future development of prenatal diagnostics.

PPARγ-dependent hepatic macrophage switching acts as a central hub for hUCMSC-mediated alleviation of decompensated liver cirrhosis in rats.

BACKGROUND: Decompensated liver cirrhosis (DLC), a terminal-stage complication of liver disease, is a major cause of morbidity and mortality in patients with hepatopathies. Human umbilical cord mesenchymal stem cell (hUCMSC) therapy has emerged as a novel treatment alternative for the treatment of DLC. However, optimized therapy protocols and the associated mechanisms are not entirely understood. METHODS: We constructed a DLC rat model consistent with the typical clinical characteristics combined use of PB and CCL4. Performing dynamic detection of liver morphology and function in rats for 11 weeks, various disease characteristics of DLC and the therapeutic effect of hUCMSCs on DLC in experimental rats were thoroughly investigated, according to ascites examination, histopathological, and related blood biochemical analyses. Flow cytometry analysis of rat liver, immunofluorescence, and RT-qPCR was performed to examine the changes in the liver immune microenvironment after hucMSCs treatment. We performed RNA-seq analysis of liver and primary macrophages and hUCMSCs co-culture system in vitro to explore possible signaling pathways. PPARγ antagonist, GW9662, and clodronate liposomes were used to inhibit PPAR activation and pre-exhaustion of macrophages in DLC rats' livers, respectively. RESULTS: We found that changing the two key issues, the frequency and initial phase of hUCMSCs infusion, can affect the efficacy of hUCMSCs, and the optimal hUCMSCs treatment schedule is once every week for three weeks at the early stage of DLC progression, providing the best therapeutic effect in reducing mortality and ascites, and improving liver function in DLC rats. hUCMSCs treatment skewed the macrophage phenotype from M1-type to M2-type by activating the PPARγ signaling pathway in the liver, which was approved by primary macrophages and hUCMSCs co-culture system in vitro. Both inhibition of PPARγ activation with GW9662 and pre-exhaustion of macrophages in DLC rats' liver abolished the regulation of hUCMSCs on macrophage polarization, thus attenuating the beneficial effect of hUCMSCs treatment in DLC rats. CONCLUSIONS: These data demonstrated that the optimal hUCMSCs treatment effectively inhibits the ascites formation, prolongs survival and significantly improves liver structure and function in DLC rats through the activation of the PPARγ signaling pathway within liver macrophages. Our study compared the efficacy of different hUCMSCs infusion regimens for DLC, providing new insights on cell-based therapies for regenerative medicine.

High-efficiency c-Myc-mediated induction of functional hepatoblasts from the human umbilical cord mesenchymal stem cells.

BACKGROUND: Direct reprogramming of human fibroblasts to hepatocyte-like cells was proposed to generate large-scale functional hepatocytes demanded by liver tissue engineering. However, the difficulty in obtaining large quantities of human fibroblasts greatly restricted the extensive implementation of this approach. Meanwhile, human umbilical cord mesenchymal stem cells (HUMSCs) are the preferred cell source for HLCs with the advantages of limited ethical concerns, easy accessibility, and propagation in vitro. However, no direct reprogramming protocol for converting HUMSCs to hepatoblast-like cells (HLCs) has been reported. METHODS: HLCs were successfully generated from HUMSCs by forced expression of FOXA3, HNF1A, and HNF4A (collectively as 3TFs) and c-Myc. In vitro and in vivo functional experiments were conducted to demonstrate the hepatic phenotype, characterization, and function of HUMSC-derived HLCs (HUMSC-iHeps). ChIP-seq and RNA-seq were integrated to reveal the potential molecular mechanisms underlying c-Myc-mediated reprogramming. RESULTS: We showed that c-Myc greatly improved the trans-differentiation efficiency for HLCs from HUMSCs, which remained highly efficient in reprogramming fibroblasts into HLCs, suggesting c-Myc could promote direct reprogramming and its potentially widespread applicability for generating large amounts of HLCs in vitro. Mice transplantation experiments further confirmed the therapeutic potential of HUMSC-iHeps by liver function restoration and survival prolongation. Besides, in vivo safety assessment demonstrated the low risk of the tumorigenic potential of HUMSC-iHeps. We found that c-Myc functioned predominantly at an early phase of reprogramming, and we further unraveled the regulatory network altered by c-Myc. CONCLUSIONS: c-Myc enhanced reprogramming efficiency of HLCs from HUMSCs. A large scale of functional HLCs generated more conveniently from HUMSCs could benefit biomedical studies and applications of liver diseases.

Nanomicelle protects the immune activation effects of Paclitaxel and sensitizes tumors to anti-PD-1 Immunotherapy.

Paclitaxel (PTX) has shown pleiotropic immunologic effects on the tumor microenvironment, and nanomicelle has emerged as a promising strategy for PTX delivery. However, the detailed mechanisms remain to be fully elucidated. Meanwhile, immunogenic cell death (ICD) is an effective approach to activate the immune system. This study investigated the ICD effect of PTX and how nanomicelle affected the immune-activation ability of PTX. Methods: The ICD effects of PTX were identified via the expression of ICD markers and cell vaccine experiment. Tumor size and overall survival in multiple animal models with treatment were monitored to evaluate the antitumor effects. The mechanisms of PTX-induced ICD and antitumor immunity were determined by detecting gene expression related to ER stress and analyzing immune cell profile in tumor after treatment. Results: We revealed the immune-regulation mechanism of PTX nanomicelle by inducing ICD, which can promote antigen presentation by dendritic cells (DCs) and activate antitumor immunity. Notably, nanomicelle encapsulation protected the ICD effects and immune activation, which were hampered by immune system impairment caused by chemotherapy. Compared with traditional formulations, a low dose of nanomicelle-encapsulated PTX (nano-PTX) treatment induced immune-dependent tumor control, which increased the infiltration and function of both T cells and DCs within tumors. However, this antitumor immunity was hampered by highly expressed PD-1 on tumor-infiltrating CD8+ T cells and upregulated PD-L1 on both immune cells and tumor cells after nano-PTX treatment. Combination therapy with a low dose of nano-PTX and PD-1 antibodies elicited CD8+ T cell-dependent antitumor immunity and remarkably improved the therapeutic efficacy. Conclusions: Our results provide systemic insights into the immune-regulation ability of PTX to induce ICD, which acts as an inducer of endogenous vaccines through ICD effects, and also provides an experimental basis for clinical combination therapy with nano-PTX and PD-1 antibodies.

TFAP2C facilitates somatic cell reprogramming by inhibiting c-Myc-dependent apoptosis and promoting mesenchymal-to-epithelial transition.

Transcription factors are known to mediate the conversion of somatic cells to induced pluripotent stem cells (iPSCs). Transcription factor TFAP2C plays important roles in the regulation of embryonic development and carcinogenesis; however, the roles of Tfap2c in regulating somatic cell reprogramming are not well understood. Here we demonstrate Tfap2c is induced during the generation of iPSCs from mouse fibroblasts and acts as a facilitator for iPSCs formation. Mechanistically, the c-Myc-dependent apoptosis, which is a roadblock to reprogramming, can be significantly mitigated by Tfap2c overexpression. Meanwhile, Tfap2c can greatly promote mesenchymal-to-epithelial transition (MET) at initiation stage of OSKM-induced reprogramming. Further analysis of gene expression and targets of Tfap2c during reprogramming by RNA-sequencing (RNA-seq) and ChIP-qPCR indicates that TFAP2C can promote epithelial gene expression by binding to their promoters directly. Finally, knockdown of E-cadherin (Cdh1), an important downstream target of TFAP2C and a critical regulator of MET antagonizes Tfap2c-mediated reprogramming. Taken together, we conclude that Tfap2c serves as a strong activator for somatic cell reprogramming through promoting the MET and inhibiting c-Myc-dependent apoptosis.

Modulating the Tumor Microenvironment via Oncolytic Viruses and CSF-1R Inhibition Synergistically Enhances Anti-PD-1 Immunotherapy.

Immunotherapy based on the immune checkpoint blockade has emerged as the most promising approach for cancer therapy. However, the proportion of colorectal cancer patients who benefit from immunotherapy is small due to the immunosuppressive tumor microenvironment. Hence, combination immunotherapy is an ideal strategy to overcome this limitation. In this study, we developed a novel combination of CSF-1R (colony-stimulating factor 1 receptor) inhibitor (PLX3397), oncolytic viruses, and anti-PD-1 antibody. Our results demonstrated that the triple treatment synergistically conferred significant tumor control and prolonged the survival of mouse models of colon cancer. Approximately 43% and 82% of mice bearing the CT26 and MC38 tumor, respectively, survived long term following the triple treatment. This combination therapy reprogrammed the immunosuppressive tumor microenvironment toward a CD8+ T cell-biased anti-tumor immunity by increasing T cell infiltration in the tumor and augmenting anti-tumor CD8+ T cell function. Our results provide a robust strategy for clinical combination therapy.

Human Adipose-Derived Mesenchymal Stem Cell-Secreted CXCL1 and CXCL8 Facilitate Breast Tumor Growth By Promoting Angiogenesis.

Autologous adipose tissue or adipose tissue with additive adipose-derived mesenchymal stem cells (ADSCs) is used in the breast reconstruction of breast cancer patients who undergo mastectomy. ADSCs play an important role in the angiogenesis and adipogenesis, which make it much better than other materials. However, ADSCs may promote residual tumor cells to proliferate or metastasize, and the mechanism is still not fully understood. In this study, we demonstrated that human ADSCs (hADSCs) could facilitate tumor cells growth after co-injection with MCF7 and ZR-75-30 breast cancer cells (BCCs) by promoting angiogenesis, but hADSCs showed limited effect on the growth of MDA-MB-231 BCCs. Intriguingly, compared with ZR-75-30 tumor cells, MCF7 tumor cells were more potentially promoted by hADSCs in the aspects of angiogenesis and proliferation. Consistent with this, cytokine and angiogenesis array analyses showed that after co-injection with hADSCs, the CXCL1 and CXCL8 concentration were significantly increased in MCF7 tumor, but only moderately increased in ZR-75-30 tumor and did not increase in MDA-MB-231 tumor. Furthermore, we found that CXCL1/8 were mainly derived from hADSCs and could increase the migration and tube formation of human umbilical vein endothelial cells (HUVECs) by signaling via their receptors CXCR1 and CXCR2. A CXCR1/2-specific antagonist (SCH527123) attenuated the angiogenesis and tumor growth in vivo. Our findings suggest that CXCL1/8 secreted by hADSCs could promote breast cancer angiogenesis and therefore provide better understanding of safety concerns regarding the clinical application of hADSCs and suggestion in further novel therapeutic options. Stem Cells 2017;35:2060-2070.

Rapid generation of functional hepatocyte-like cells from human adipose-derived stem cells.

BACKGROUND: Liver disease is a major cause of death worldwide. Orthotropic liver transplantation (OLT) represents the only effective treatment for patients with liver failure, but the increasing demand for organs is unfortunately so great that its application is limited. Hepatocyte transplantation is a promising alternative to OLT for the treatment of some liver-based metabolic disorders or acute liver failure. Unfortunately, the lack of donor livers also makes it difficult to obtain enough viable hepatocytes for hepatocyte-based therapies. Currently, a fundamental solution to this key problem is still lacking. Here we show a novel non-transgenic protocol that facilitates the rapid generation of functional induced hepatocytes (iHeps) from human adipose-derived stem cells (hADSCs), providing a source of available cells for autologous hepatocytes to treat liver disease. METHODS: We used collagenase digestion to isolate hADSCs. The surface marker was detected by flow cytometry. The multipotential differentiation potency was detected by induction into adipocytes, osteocytes, and chondrocytes. Passage 3-7 hADSCs were induced into iHeps using an induction culture system composed of small molecule compounds and cell factors. RESULTS: Primary cultured hADSCs presented a fusiform or polygon appearance that became fibroblast-like after passage 3. More than 95 % of the cells expressed the mesenchymal cell markers CD29, CD44, CD166, CD105, and CD90. hADSCs possessed multipotential differentiation towards adipocytes, osteocytes, and chondrocytes. We rapidly induced hADSCs into iHeps within 10 days in vitro; the cellular morphology changed from fusiform to close-connected cubiform, which was similar to hepatocytes. After induction, most of the iHeps co-expressed albumin and alpha-1 antitrypsin; they also expressed mature hepatocyte special genes and achieved the basic functions of hepatocyte. Moreover, iHep transplantation could improve the liver function of acute liver-injured NPG mice and prolong life. CONCLUSIONS: We isolated highly purified hADSCs and rapidly induced them into functional hepatocyte-like cells within 10 days. These results provide a source of available cells for autologous hepatocytes to treat liver disease.

Efficient inhibition of intraperitoneal ovarian cancer growth in nude mice by liposomal delivery of short hairpin RNA against STAT3.

AIM: Signal transducer and activator of transcription 3 (STAT3) plays an important role in the tumor formation, prognosis and chemoresistance of ovarian cancer. Our goal was to investigate the effect of silencing STAT3 on ovarian cancer cell apoptosis, proliferation, angiogenesis and expression of key targets in vitro and in vivo. METHODS: The ovarian cancer cell lines A2780CP and A2780s were used. STAT3 was knocked down by the plasmid-based short hairpin RNA (shRNA) expression system. In vitro, a colony formation assay and Hoechst staining were used to examine cell proliferation and apoptosis. The expression level of STAT3 and apoptosis-related proteins were determined by Western blot. The A2780CP intraperitoneal model was used to evaluate the effect of shSTAT3 on tumor growth in mice. Proliferation, apoptosis, and angiogenesis in tumor tissues were measured by proliferating cell nuclear antigen, TUNEL and CD31 immunostaining, respectively. RESULTS: Treatment with shSTAT3 resulted in apoptosis and inhibition of cell proliferation in vitro. Western blot analysis demonstrated that shSTAT3 induced the expression of cleaved caspase-3 and reduced the expression of survivin, Bcl-2 and vascular endothelial growth factor. In vivo, the tumor weight was reduced to 13.46% of 5% glucose by shSTAT3/lipoplexes (P < 0.01), accompanied with apoptosis induction (P < 0.01), proliferation inhibition (P < 0.01) and angiogenesis inhibition (P < 0.01). CONCLUSIONS: We find that treatment with shSTAT3 inhibits tumor growth in vitro and in vivo by inducing apoptosis and inhibiting cell proliferation. This work should provide the scientific foundation for future investigation of shSTAT3 as a strategy for ovarian cancer gene therapy and the combination of gene therapy with chemotherapy.

Vector-based miR-15a/16-1 plasmid inhibits colon cancer growth in vivo.

miR-15 (microRNA 15) and miR-16 are frequently deleted or down-regulated in many cancer cell lines and various tumour tissues, suggesting that miR-15a/16-1 plays important roles in tumour progression and might be a method for cancer treatment. We have developed a vector-based plasmid to explore the anti-tumour efficacy of miR-15a/16-1 in colon cancer in vivo. It is proposed that miR-15a and miR-16-1 target cyclin B1 (CCNB1), which associates with several tumorigenic features such as survival and proliferation. The levels of miR-15a and miR-16-1 in colon cancer cells were inversely correlated with CCNB1 expression, and there was consensus between miR-15a/16-1 and CCNB1 mRNA sequences by analysing homology. Vector-based miR-15a/16-1 expression plasmid was constructed and transfected into HCT 116 and SW620 colon cancer cells in vitro. The effects produced on cell viability and angiogenesis were analysed using flow cytometric analysis, colony formation analysis and tube formation analysis. CCNB1 expression down-regulation was checked by Western blotting. Systemic delivery of miR-15a/16-1 plasmids encapsulated in cationic liposome led to a significant inhibition of subcutaneous tumour growth and angiogenesis in tumour tissues, whereas no effects were observed with liposome carrying the non-specific plasmid. In summary, miR-15a/16-1 has been applied in colon cancer treatment in vivo, and resulted in effective colon tumour xenografts growth arrest and angiogenesis decrease. These findings suggest that systemic delivery of vector-based miR-15a/16-1 expression plasmid can be an approach to colon cancer therapy.

Suppression of epidermal growth factor receptor (EGFR) expression by small hairpin RNA inhibits the growth of human nonsmall cell lung cancers bearing wild-type and mutant EGFR.

In the present study, we have used plasmid-based RNA interference (RNAi) strategy to downregulate the expression of epidermal growth factor receptor (EGFR) in EGFR wild-type (H292) and mutant (H1975) lung tumor models. The targeted knockdown of EGFR by small hairpin RNA not only inhibited growth of H292 xenograft but also inhibited H1975 lung cancer cell and xenograft, which bore L858R/T790M EGFR and was resistant to EGFR tyrosine kinase inhibitors. These data demonstrated that small hairpin RNA was an effective therapy against mutant EGFR-expressing cancer cells and thus considered to be a promising strategy in the treatment of lung cancers.

Intravenous liposomal delivery of the short hairpin RNAs against Plk1 controls the growth of established human hepatocellular carcinoma.

Polo-like kinase 1 (Plk1) is a key cell cycle regulator that is frequently overexpressed in human hepatocellular carcinomas. Blockade of the Plk1 pathway has been reported to be capable of inducing anti-tumor effect. Here, plasmids containing U6 promoter-driven shRNAs against human Plk1 were constructed and transfected in human hepatocellular carcinoma cell line HepG2. ShRNA targeting Plk1 almost completely reduced Plk1 expression in HepG2 hepatocellular carcinoma cells, as confirmed by RT-PCR and Western blot. As a consequence, HepG2 cells exhibited reduced proliferation and enhanced apoptosis in vitro. Most importantly, Treatment with Plk shRNA-DOTAP:Chol complex significantly suppressed the growth of HepG2 xenografts, accompanied with phenotypic changes in tumor cells, including proliferation inhibition and apoptosis induction. Our study suggested that shRNA-mediated silencing of Plk1 might be a novel therapeutic approach against human hepatocellular carcinoma by inhibiting tumor cells proliferation and inducing apoptosis.

RNA interference-mediated silencing of focal adhesion kinase inhibits growth of human colon carcinoma xenograft in nude mice.

Focal adhesion kinase (FAK), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human colon cancer. In the present study, we utilized the short hairpin RNA (shRNA) to knock down the expression of FAK in SW480 human colon cancer cells in vitro. Furthermore, nude mice bearing human colon carcinoma SW480 were established and treated with plasmids encoding FAK shRNA encapsulated in DOTAP: Chol cationic liposome through tail vein injection. Tumor growth and potential side effect were observed during the treatment. Assessments of angiogenesis, cell proliferation, and apoptosis were performed by using immunohistochemistry against CD31, proliferating cell nuclear antigen (PCNA) and TUNEL assays, respectively. The results indicated that DOTAP: Chol could efficiently deliver the therapeutic plasmids systemically to tumor xenografts, resulting in suppression of tumor growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumor volume by approximately 86% compared with control groups (p < 0.01), accompanied with angiogenesis inhibition (p < 0.05), tumor cell proliferation suppression (p < 0.05) and apoptosis induction (p < 0.05). Taken together, our data demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human colon carcinoma.

Prokaryotic expression, purification and characterization of a novel pro-apoptosis protein hPNAS-4.

Recently, hPNAS-4 [human PNAS-4; also known as C1orf121 (Genbank accession number NP-057160)] was reported to be a novel pro-apoptotic protein in mammalian cells. However, at present there is little information about hPNAS-4 and its molecular mechanisms. In our present studies, the hPNAS-4 gene was first cloned into the pGEX-6p-1 vector with a GST (glutathione transferase) tag to express in soluble form in Escherichia coli induced with 0.5 mM IPTG (isopropyl beta-D-thiogalactoside) at 25 degrees C, and the recombinant hPNAS-4 was purified to near homogeneity with 96% purity by affinity chromatography and anion-exchange chromatography. The purified hPNAS-4 protein was further identified by liquid chromatography-ESI (electrospray ionization)-MS analysis and used to raise polyclonal antibodies that could specifically recognize the hPNAS-4 protein. In addition, hPNAS-4 is localized to the cytoplasm and the perinuclear compartment. Furthermore, the overexpression of hPNAS-4 in human A549 lung cancer cells resulted in decrease of cell viability via apoptosis. This study represented an important step to investigate the characterization for the new pro-apoptosis protein hPNAS-4, which should aid the discovery of new drug targets for the development of effective therapeutic approaches to cancer in the future.