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.

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.

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

BACKGROUND: Hepatocytes 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. METHODS: Mesenchymal 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Hybrid cardiomyocytes derived by cell fusion in heterotopic cardiac xenografts.

Cardiomyocytes expressing host markers, such as the Y chromosome in sex-mismatched transplants, have been described in human allografts, suggesting that circulating cells can contribute to cardiac regeneration. It has not been established, however, whether host-derived cardiomyocytes result from transdifferentiation of stem cells or cell fusion. To address this issue, we used heterotopic heart xenografts and looked for markers of donor and recipient cells. Golden Syrian hamsters or transgenic mice expressing nuclear beta-galactosidase under the control of the cardiac troponin I promoter served as organ donors, while GFP+ transgenic rats were used as recipients. GFP+ cells, including abundant CD-45+ inflammatory cells and rare undifferentiated cells expressing early cardiac markers (GATA-4 or MEF2C), were found in xenografts harvested two weeks after surgery. In addition, rare GFP+ mature cardiomyocytes were found in 7 of 8 hamster xenografts and 6 of 6 mouse xenografts. The proportion of these cells was very low (0.0001% to 0.0344% in hamster xenografts) but similar to the one observed in control rat heart allografts. Without exception, all GFP+ cardiomyocytes also expressed donor markers, i.e., hamster membrane antigens or lacZ, so they must derive from cell fusion, not transdifferentiation.