Abstract Background: Acute liver failure (ALF) is a complicated condition that is characterized by global hepatocyte death and often requires immediate liver transplantation. However, this therapy is limited by shortage of donor organs. Mesenchymal stem cells (MSCs) and hepatocytes are two attractive sources of cell-based therapies to treat ALF. The combined transplantation of hepatocytes and MSCs is considered to be more effective for the treatment of ALF than single-cell transplantation. We have previously demonstrated that HNF4α-overexpressing human umbilical cord MSCs (HNF4α-UMSCs) promoted the expression of hepatic-specific genes. In addition, microencapsulation allows exchange of nutrients, forming a protective barrier to the transplanted cells . Moreover, encapsulation of hepatocytes improves the viability and synthetic ability of hepatocytes and circumvents immune rejection. This study aimed to investigate the therapeutic effect of microencapsulation of hepatocytes and HNF4α-UMSCs in ALF mice. Methods: Human hepatocytes and UMSCs were obtained from liver and umbilical cord separately, followed by co-encapsulation and transplantation into mice by intraperitoneal injection. LPS/D-gal was used to induce ALF by intraperitoneal injection 24 h after transplantation. In addition, Raw 264.7cells (a macrophage cell line) were used to elucidate the effect of HNF4α-UMSCs-hepatocyte microcapsules on polarization of macrophages. The protein chip of conditioned mediums (CMs) of UMSCs and HNF4α-UMSCs were used to define the important paracrine factors and investigate the possible mechanism of HNF4α-UMSCs for the treatment of ALF in mice. Results: HNF4α-UMSCs can enhance the function of primary hepatocytes in alginate–poly-L-lysine–alginate (APA) microcapsules. The co-encapsulation of both HNF4α-UMSCs and hepatocytes achieved better therapeutic response in ALF mice by promoting M2 macrophage polarization and inflammatory resolution effect mainly modulated by the paracrine factor HB-EGF from HNF4α-UMSCs. Conclusions: The present study confirms that the co-encapsulation of HNF4α-UMSC and hepatocytes could exert an efficient effect on ALF mainly by HB-EGF and provides a novel therapeutic strategy for the treatment of ALF.
Abstract Background: Advances in research on stem cell therapy provide new feasible solutions for acute liver failure (ALF) treatment. Recent studies have demonstrated that the expression of hepatocyte nuclear factor 4α (HNF4α) reset within the damaged hepatocytes can restore normal physiological function. This study aimed to determine the role of human umbilical cord mesenchymal stem cells (HuMSCs) overexpressing HNF4α in ALF treatment.Method: We isolated and cultured HuMSCs in vitro, reversed it by lent virus expression HNF4α (hereinafter referred to as HuMSC-HNF4α). HuMSC-HNF4α was intraperitoneally administrated into the mice immediately after exposed to D-galactosamine / lipopolysaccharide (D-galn / LPS). To investigate their effects in ALF, we performed liver histological and serumbiochemical analysis. Macrophages differentiation and cytokines secreted by HuMSCs were evaluated to elucidate its mechanisms.Results: We found HuMSC-HNF4α has more obvious therapeutic effects on ALF than the negative control virus transfected the HuMSCs (HuMSC-CON). In vitro, HuMSC-HNF4α promotes the polarization of liver macrophages (Kupffer cells) to M2 phenotype, inhibits the inflammatory response of macrophages and reduces the levels of inflammatory factors such as TNF-α, IL-1β to reduce liver damage.Conclusion: Our research confirmed that the therapeutic effect of HuMSC-HNF4α on ALF is not the same as the previous passive support but an active intervention on excessive inflammation in the body. This provides new ideas for research and clinical practice in the future.
To establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for therapeutic usage in hepatocyte transplantation and bioartificial liver support systems for the treatment of acute and chronic liver diseases,and for experimental usage as an in vitro model of the liver.Adult hepatocytes from 20 human donors undergoing partial hepatectomy were isolated using a two-step extracoporeal collagenase perfusion technique.Seven preincubation time points (2h,6h,12h,24h,36h,48h and 72h) were selected for optimization.After pre-incubation at 4 degrees C for 12-24h in HepatoZYME-SFM (the optimal condition),hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules,transferred to a complete medium containing 10% dimethyl sulphoxide and immediately placed into an isopropanol progressive freezing container for overnight freezing at -80 degrees C followed by immersion in liquid nitrogen the next day.During the post-thawing culture period,the cells were tested for albumin secretion,urea synthesis,cell cycling,transcription and protein synthesis (measuring mRNA and protein levels),and the morphological structure and pathology,for comparison with the features from before microencapsulated cryopreservation (PMC).The viability and plating efficiency of the hepatocytes isolated using the two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0%,respectively.The pre-incubation times of 12h and 24h (viability:61.4+/-4.8% and 62.0+/-5.6%; plating efficiency:3.2+/-5.8% and 62.6+/-3.6%,respectively) showed significantly higher albumin secretion than all other time points tested (F =40.3,all P less than 0.05).Compared with the immediate cryopreservation (immediately frozen control) hepatocytes,the PMC hepatocytes showed significantly better transcription and protein synthesis and higher albumin secretion and urea levels.The PMC group did not show a significantly different level of albumin production from the directly cultured hepatocytes (culture day 2:ll9.2ng/ml vs.131.36ng/ml,P =0.051; day 3:110ng/ml vs.120.4ng/ml,P=0.063; day 4:98.2ng/ml vs.109.8ng/ml,P more than 0.05).However,over culturing days 2,3 and 4,comparison of the PMC hepatocytes to the immediate cryopreservation hepatoeytes showed the former to have significantly higher secretion of albumin (119.2ng/ml vs.101.2ng/ml,110.0ng/ml vs.87.6ng/ml and 98.2ng/ml vs.73.8ng/ml; all P less than 0.05) and urea level (7.83 mug/ml vs.6.79 mug/ml,6.83 mug/ml vs.5.89 mug/ml and 5.85 mug/ml vs.4.83 mug/ml; all P less than 0.05).The post-thawed PMC hepatoeytes showed preservation of the morphological structure,while the immediate cryopreservation hepatocytes did not.The two-step extracorporeal collagenase perfusion technique after partial hepatectomy is a novel,simple,and reliable method for hepatocyte isolation.Pre-incubation at 4 degrees C for 12-24h before the microencapsulation cryopreservation allows for efficient recovery of functional and morphological integrity after thawing and provides viable hepatoeytes that may be useful for clinical applications in pharmacotoxicology,bioartificial liver therapy and cell therapy in humans.
Primary hepatocellular carcinoma progresses from liver fibrosis and cirrhosis to eventually result in liver failure and distant metastasis.Surgical resection is the preferred method of treatment for liver cancer while interventional treatment and liver transplantation are the choices to treat end-stage liver cancer.Unfortunately,partial hepatectomy and interventional treatment are not ideal due to the resulting consequence of hepatocyte dysfunction.Extensive clinical application of liver transplants is limited by the lack of available donors and high costs.Over the past decade,researches on bone marrow mesenchymal stem cells (BMSCs)have made remarkable achievements in the medical field.In this review,we summarize the recent progress of BMSCs in the treatment of liver diseases.
Key words:
Bone marrow mesenchymal stem cells; Liver cancer; Liver regeneration; Tumor metastasis
Pharmacologic perturbation projects, such as Connectivity Map (CMap) and Library of Integrated Network-based Cellular Signatures (LINCS), have produced many perturbed expression data, providing enormous opportunities for computational therapeutic discovery. However, there is no consensus on which methodologies and parameters are the most optimal to conduct such analysis. Aiming to fill this gap, new benchmarking standards were developed to quantitatively evaluate drug retrieval performance. Investigations of potential factors influencing drug retrieval were conducted based on these standards. As a result, we determined an optimal approach for LINCS data-based therapeutic discovery. With this approach, homoharringtonine (HHT) was identified to be a candidate agent with potential therapeutic and preventive effects on liver cancer. The antitumor and antifibrotic activity of HHT was validated experimentally using subcutaneous xenograft tumor model and carbon tetrachloride (CCL 4 )-induced liver fibrosis model, demonstrating the reliability of the prediction results. In summary, our findings will not only impact the future applications of LINCS data but also offer new opportunities for therapeutic intervention of liver cancer.
We performed multi-region sampling and sequencing on 14 patients with HCC, collecting a total of 75 tumor samples with spatial information and molecular data. In addition, 21 matched adjacent liver samples were also collected. All samples were subjected to RNA sequencing (RNA-seq). RNA sequencing was performed on Illumina NovaSeq 6000 platform with 40M pair-end 150bp reads per sample. Whole-exome sequencing (WES) was performed on 36 samples from patients T10, T13 and T18 (n = 26) as well as adjacent non-tumor tissues (n = 10). Paired end, 150bp read-length sequencing was then performed on Illumina NovaSeq 6000 platform with a mean sequencing coverage of 100X. Raw sequencing data has been deposited at the National Omics Data Encyclopedia (NODE) under the accession code OEP002956 ( http://www.biosino.org/node/project/detail/OEP002956).
Mesenchymal stem cells (MSCs) hold promise as cellular vehicles for the delivery of therapeutic gene products because they can be isolated, expanded, and genetically modified in vitro and possess tumor-oriented homing capacity in vivo. (1) Hepatocyte nuclear factor 4α (HNF4α) is a dominant transcriptional regulator of hepatocyte differentiation and hepatocellular carcinogenesis (HCC). (2,3) We have previously demonstrated that overexpression of HNF4α activates various hepatic-specific genes and enhances MSC differentiation. (4) However, the extent that overexpression of HNF4α in MSCs influences HCC progression has yet to be examined. Here we sought to investigate what effect MSCs overexpressing HNF4α (MSC-HNF4α) have on human hepatoma cells in vitro and in vivo. Conditioned medium collected from in vitro MSC-HNF4α cultures significantly inhibited hepatoma cell growth and metastasis compared with controls. Additionally, nude mice administered MSC-HNF4α exhibited significantly smaller tumors compared with controls in vivo. Immunoblot analysis of HCC cells treated with MSC-HNF4α displayed downregulated β-catenin, cyclinD1, c-Myc, MMP2 and MMP9. Taken together, our results demonstrate that MSC-HNF4α inhibits HCC progression by reducing hepatoma cell growth and metastasis through downregulation of the Wnt/β-catenin signaling pathway.
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