Francesca Timoneri

Global profiling of viral and cellular non-coding RNAs in Epstein-Barr virus-induced lymphoblastoid cell lines and released exosome cargos.

The human EBV-transformed lymphoblastoid cell line (LCL), obtained by infecting peripheral blood monocular cells with Epstein-Barr Virus, has been extensively used for human genetic, pharmacogenomic, and immunologic studies. Recently, the role of exosomes has also been indicated as crucial in the crosstalk between EBV and the host microenvironment. Because the role that the LCL and LCL exosomal cargo might play in maintaining persistent infection, and since little is known regarding the non-coding RNAs of LCL, the aim of our work was the comprehensive characterization of this class of RNA, cellular and viral miRNAs, and cellular lncRNAs, in LCL compared with PBMC derived from the same donors. In this study, we have demonstrated, for the first time, that all the viral miRNAs expressed by LCL are also packaged in the exosomes, and we found that two miRNAs, ebv-miR-BART3 and ebv-miR-BHRF1-1, are more abundant in the exosomes, suggesting a microvescicular viral microRNA transfer. In addition, lncRNA profiling revealed that LCLs were enriched in lncRNA H19 and H19 antisense, and released these through exosomes, suggesting a leading role in the regulation of the tumor microenvironment.

Characterization of Liver-Specific Functions of Human Fetal Hepatocytes in Culture.

This study was designed to assess liver-specific functions of human fetal liver cells proposed as a potential source for hepatocyte transplantation. Fetal liver cells were isolated from livers of different gestational ages (16-22 weeks), and the functions of cell preparations were evaluated by establishing primary cultures. We observed that 20- to 22-week-gestation fetal liver cell cultures contained a predominance of cells with hepatocytic traits that did not divide in vitro but were functionally competent. Fetal hepatocytes performed liver-specific functions at levels comparable to those of their adult counterpart. Moreover, exposure to dexamethasone in combination with oncostatin M promptly induced further maturation of the cells through the acquisition of additional functions (i.e., ability to store glycogen and uptake of indocyanine green). In some cases, particularly in cultures obtained from fetuses of earlier gestational ages (16-18 weeks gestation), cells with mature hepatocytic traits proved to be sporadic, and the primary cultures were mainly populated by clusters of proliferating cells. Consequently, the values of liver-specific functions detected in these cultures were low. We observed that a low cell density culture system rapidly prompted loss of the mature hepatocytic phenotype with downregulations of all the liver-specific functions. We found that human fetal liver cells can be cryopreserved without significant loss of viability and function and evaluated up to 1 year in storage in liquid nitrogen. They might, therefore, be suitable for cell banking and allow for the transplantation of large numbers of cells, thus improving clinical outcomes. Overall, our results indicate that fetal hepatocytes could be used as a cell source for hepatocyte transplantation. Fetal liver cells have been used so far to treat end-stage liver disease. Additional studies are needed to include these cells in cell-based therapies aimed to treat liver failure and inborn errors of metabolism.

Wharton’s jelly mesenchymal stromal cells immunomodulatory molecules: their journey from umbilical cord to differentiated cells

Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) have a unique ability to cross lineage borders. Their immunomodulatory and anti-inflammatory features, further render these cells promising for regenerative medicine applications. Few data are present in literature on the expression of immunomodulatory molecules in umbilical cord (UC) tissue and their maintenance in paired cultured WJ-MSCs, an important aspect in cellular therapy applications. In addition, few data exist on the maintenance of expression of immunomodulatory molecules in mature cell types differentiated from MSCs. Therefore we investigated, in vivo (in UC at full term) and in vitro (in either undifferentiated or differentiated WJ-MSCs), the expression of different markers and their maintenance alongside cell culture, ex vivo expansion and differentiation. IHC, ICC, RT-PCR and flow cytometry were used to detect expression of markers in both paired UC sections and WJ-MSCs. Differentiation was performed towards the standard mesenchymal lineages as well as hepatocyte-like cells. Paired ICC and IHC analyses showed that for most of the analyzed molecules the expression at the protein level is maintained in both UC tissue and WJ-MSCs. Structural molecules were expressed in both WJ and umbilical epithelium (UE), as well as in WJ-MSCs. We showed for the first time that UE and WJ were positive for both HLA-ABC and HLA-E, while HLA-DR was not detectable. The same data were confirmed on WJ-MSCs. Both B7-1 and B7-2 were absent in UC and WJ-MSCs, while we showed for the first time that B7-H3 was highly expressed in both WJ and WJMSCs. Differentiation experiments showed that immunomodulatory molecules were expressed upon application of complex differentiation protocols, in parallel to the acquisition of mature markers or functions. Some important conclusions may be drawn from the current experiments: i) WJMSCs mostly maintain the expression of molecules just present in their “niche”, under standard culture conditions; ii) the parallel expression of immunomodulatory molecules sheds new light on the ability of WJ-MSCs to modulate host immune responses; iii) the in vivo expression of molecules such as HLA-E and B7-H3 opens new questions on the role of WJ during pregnancy; iv) the expression of these molecules in differentiated cells provides key features for in vivo applications, in particular for hepatocyte-like cells.

Wharton’s jelly mesenchymal stem cells differentiation into hepatocyte-like cells: functional characterization and expression of immunomodulatory molecules

Mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs) recently emerged as promising tools for cellular therapy due to their ability to differentiate into diverse cell types and their immunomodulatory features. Little is known on the expression of immunomodulatory molecules in mature cells differentiated from WJ-MSCs, therefore we aimed to characterize the extent of maintenance of the naive traits of these cells also in a highly specialized differentiated counterpart. WJ-MSCs were differentiated into hepatocyte-like cells (HLCs) with a four weeks protocol. RT-PCR, flow cytometry, IHC and ICC were performed to assess expression of key markers in both undifferentiated and differentiated cells. Hepatocyte specific assays such as PAS staining, CYP3A4 induction and activity, G6Pase activity, were performed to demonstrate the acquisition of traits of the mature hepatocyte phenotype. WJ-MSCs were successfully differentiated into HLCs using a multi-step protocol. These cells were able to store glycogen, incorporate specific live cells stains, perform enzymatic reactions and metabolic activities which are usually featured by mature hepatocytes. In addition, WJ-MSCs did express several immune-related molecules, for which an immunomodulatory role has been demonstrated both in vitro and in vivo. We demonstrated for the first time that key molecules as HLA-E and B7-H3 are expressed also by HLCs derived from WJ-MSCs. Our data showed for the first time that HLCs mostly maintain the expression of immune-modulating molecules, together with new markers and functional features of mature cells. In multiple pathologic conditions the intrinsic immunomodulatory ability of differentiated cells may help survive the interaction with the host immune system, even in the absence of a specific immunosuppressive therapy. This, together with the acquisition of key mature hepatocyte functions, may render WJ-MSCs promising in cell therapy applications for liver diseases.

Isolation and phenotypical characterization of mesenchymal stem cells from the Wharton's jelly of pre-term human umbilical cord

Extraembryonic tissues such as umbilical cord are considered a promising source of stem cells, potentially useful in therapy. Pre-term umbilical cords may be also a useful source of mesenchymal populations, given also that pre-term birth infants may develop diseases during childhood which may be reverted by a cell therapy approach. Pre-term cords can be also made available from therapeutic abortions, providing a further cell source to obtain high numbers of WJ-MSCs. Little is known about the phenotype and differentiative potential of these cells. Preterm UC were obtained following therapeutic abortions after mothers’ informed consent and processed within 12 hours from tissue collection. Characterization of cells was performed at P2 and P5, by both flow cytometry (FC) and ICC, to detect of classical MSC markers, immunomodulatory molecules, tissue-specific markers. The isolation protocol allowed to successfully derive WJ-MSCs which showed the typical morphology, and were routinely passaged up to passage 10. Multi-color flow cytometric analyses showed that isolated cells were positive for classical MSCs markers (CD29, CD44, CD73, CD90, CD105) and. negative (or weakly positive) for typical hematopoietic and endothelial markers (CD45, CD34, CD14, CD68, CD39 and CD31). In addition, we demonstrated the expression of tissue specific markers, both endodermal (CK18, CK19, alpha-fetoprotein and albumin) and neuro-ectodermal (nestin). This may indicate the potential of preterm WJ-MSC to undergo multiple differentiation pathways, as demonstrated for cells isolated from term UC. Preterm WJ-MSCs showed MHC class I expression (but not class II), suggesting hypoimmunogenic properties for these cells. Moreover, B7H3 expression should favor immune tolerance by the host following cellular transplantation. ICC allowed confirming part of the FC data and was used to assess the expression of further antigens. Present data demonstrate that preterm WJ-MSCs can be isolated and expanded, with high reproducibility. these cells do express classical MSC markers and immunomodulatory molecules, independently from the underlying pathology which led to abortion.