T. Chioato

Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury

BackgroundHepatocytes and stem cells transplantation may be an alternative to liver transplantation in acute or chronic liver disease. We aimed to evaluate the therapeutic potential of mesenchymal stem cells from human umbilical cord (UCMSCs), a readily available source of mesenchymal stem cells, in the CCl4-induced acute liver injury model.MethodsMesenchymal stem cells profile was analyzed by flow cytometry. In order to evaluate the capability of our UCMSCs to differentiate in hepatocytes, cells were seeded on three different supports, untreated plastic support, MatrigelTM and human liver acellular matrix. Cells were analyzed by immunocitochemistry for alpha-fetoprotein and albumin expression, qPCR for hepatocyte markers gene expression, Periodic Acid-Schiff staining for glycogen storage, ELISA for albumin detection and colorimetric assay for urea secretion.To assess the effects of undifferentiated UCMSCs in hepatic regeneration after an acute liver injury, we transplanted them via tail vein in mice injected intraperitoneally with a single dose of CCl4. Livers were analyzed by histological evaluation for damage quantification, immunostaining for Kupffer and stellate cells/liver myofibroblasts activation and for UCMSCs homing. Pro- and anti-inflammatory cytokines gene expression was evaluated by qPCR analysis and antioxidant enzyme activity was measured by catalase quantification.Data were analyzed by Mann–Whitney U-test, Kruskal-Wallis test and Cuzick’s test followed by Bonferroni correction for multiple comparisons.ResultsWe have standardized the isolation procedure to obtain a cell population with hepatogenic properties prior to in vivo transplantation. When subjected to hepatogenic differentiation on untreated plastic support, UCMSCs differentiated in hepatocyte-like cells as demonstrated by their morphology, progressive up-regulation of mature hepatocyte markers, glycogen storage, albumin and urea secretion. However, cells seeded on 3D-supports showed a minor or negligible differentiation capacity.UCMSCs-transplanted mice showed a more rapid damage resolution, as shown by histological analysis, with a lower inflammation level and an increased catalase activity compared to CCl4-treated mice.ConclusionsOur findings show that UCMSCs can be reliably isolated, have hepatogenic properties and following systemic administration are able to accelerate the resolution of an acute liver injury without any differentiation and manipulation. These features make UCMSCs strong candidates for future application in regenerative medicine for human acute liver disease.

Blood vessel-derived acellular matrix for vascular graft application.

To overcome the issues connected to the use of autologous vascular grafts and artificial materials for reconstruction of small diameter (<6 mm) blood vessels, this study aimed to develop acellular matrix- (AM-) based vascular grafts. Rat iliac arteries were decellularized by a detergent-enzymatic treatment, whereas endothelial cells (ECs) were obtained through enzymatic digestion of rat skin followed by immunomagnetic separation of CD31-positive cells. Sixteen female Lewis rats (8 weeks old) received only AM or previously in vitro reendothelialized AM as abdominal aorta interposition grafts (about 1 cm). The detergent-enzymatic treatment completely removed the cellular part of vessels and both MHC class I and class II antigens. One month after surgery, the luminal surface of implanted AMs was partially covered by ECs and several platelets adhered in the areas lacking cell coverage. Intimal hyperplasia, already detected after 1 month, increased at 3 months. On the contrary, all grafts composed by AM and ECs were completely covered at 1 month and their structure was similar to that of native vessels at 3 months. Taken together, our findings show that prostheses composed of AM preseeded with ECs could be a promising approach for the replacement of blood vessels.

Optimized protocol for immunostaining of experimental GFP-expressing and human hearts

Morphological and histochemical analysis of the heart is fundamental for the understanding of cardiac physiology and pathology. The accurate detection of different myocardial cell populations, as well as the high-resolution imaging of protein expression and distribution, within the diverse intracellular compartments, is essential for basic research on disease mechanisms and for the translatability of the results to human pathophysiology. While enormous progress has been made on the imaging hardware and methods and on biotechnological tools [e.g., use of green fluorescent protein (GFP), viral-mediated gene transduction] to investigate heart cell structure and function, most of the protocols to prepare heart tissue samples for analysis have remained almost identical for decades. We here provide a detailed description of a novel protocol of heart processing, tailored to the simultaneous detection of tissue morphology, immunofluorescence markers and native emission of fluorescent proteins (i.e., GFP). We compared a variety of procedures of fixation, antigen unmasking and tissue permeabilization, to identify the best combination for preservation of myocardial morphology and native GFP fluorescence, while simultaneously allowing detection of antibody staining toward sarcomeric, membrane, cytosolic and nuclear markers. Furthermore, with minimal variations, we implemented such protocol for the study of human heart samples, including those already fixed and stored with conventional procedures, in tissue archives or bio-banks. In conclusion, a procedure is here presented for the laboratory investigation of the heart, in both rodents and humans, which accrues from the same tissue section information that would normally require the time-consuming and tissue-wasting observation of multiple serial sections.

Preliminary investigation of blood vessel-derived acellular matrix for vascular graft application

Although autologous vascular grafts and artificial materials have been used for reconstruction of small diameter (<5mm) blood vessels, the poor availability of vessels and the occurrence of intimal hyperplasia and progressive atherosclerotic degeneration represent shortcomings of these vascular prostheses. Therefore, this preliminary study aimed to develop acellular matrix (AM)-based vascular grafts. Rat thoracic aortas were decellularized by means of a detergent-enzymatic treatment [1], whereas endothelial cells (ECs) were obtained through enzymatic digestion of rat skin followed by immunomagnetic separation of CD31-positive cells. Twenty male Lewis rats (8 week old) received either only AM and previously in vitro reendothelized AM as abdominal aorta Interposition grafts (about 2 cm). After 1 (n=10) and 3 (n=10) months from surgery, grafts were explanted and morphologically examined by scanning electron microscopy and Movat staining. The detergent enzymatic treatment completely removed the cellular part of vessels and both MHC class I and class II antigens. After 1 month from surgery, the luminal surface of implanted AMs was partially covered by ECs and several platelets adhered in the areas lacking cell coverage. Intimal hyperplasia, already detected after 1 month, increased at 3 months. On the contrary, all the grafts composed by AM and ECs were completely covered at 1 month and their structure was similar to that of native vessels at 3 months. Taken together, our findings show that prostheses composed of AM pre-seeded with ECs could be a promising approach for the replacement of blood vessels.

1070 IMMUNE RESPONSE AND GENDER DISPARITY INFLUENCE LIVER REGENERATION AFTER ACUTE HEPATIC INJURY

and therefore regulate development and regression of liver fibrosis in sortilin −/− mice. Methods: Expression of neurotrophin receptors and ligands was measured by qRT-PCR in quiescent, activated and fully activated rat HSC, then the effect of NGF, proNGF and BDNF on proliferation, activation and apoptosis of HSC was assessed in 7d and 14d cultures. Thioacetamide-induced fibrosis was performed in wild type (WT) and sortilin −/− mice. Results: Fully activated rat HSC express increased mRNA levels for NGF, BDNF, trkB, p75NTR and sortilin compared to quiescent and activated HSC, while trkA is unchanged. Interestingly, neurotrophins strongly induce mRNA expression of a-smooth muscle actin (a-SMA), collagen I and TGF-b1 in activated HSC, but not in fully activated HSC. On the other hand, fully activated HSC display a dramatic increase in proinflammatory cytokines TNFa, SDF-1 and MCP-1 after neurotrophin treatment. Surprisingly, we observed that NGF and proNGF induce a modest statistically significant, and BDNF a strong mitogenic effect both in activated and fully activated HSC, but no apoptosis. In vivo results with sortilin −/− mice demonstrate that these mice have increased fibrosis, accompanied by enhanced aSMA and collagen I compared to WT mice, in a model of thioacetamide-induced fibrosis. Conclusions: The neurotrophin system plays different roles during HSC activation stages. Whereas, at earlier stages of HSC activation, neurotrophins increase activation, at later stages of activation, they induce a proinflammatory phenotype. Sortilin may participate in the response of HSC to neurotrophins, as sortilin −/− mice have increased liver fibrosis.