Tiziana Corsello

Human Wharton's jelly mesenchymal stem cells maintain the expression of key immunomodulatory molecules when subjected to osteogenic, adipogenic and chondrogenic differentiation in vitro: new perspectives for cellular therapy.

Rheumatoid arthritis and osteoarthritis are the main diseases that imply an inflammatory process at the joints involving the articular cartilage. Recently, mesenchymal stem cells (MSCs) derived from perinatal tissues were considered good candidates for cellular therapy of musculoskeletal and orthopaedic diseases, since they can differentiate into multiple cell types and are an easily accessible cellular source. Therefore, several protocols exist on the differentiation of mesenchymal stem cells of different origins into osteoblasts and chondrocytes. Another key feature of MSCs is their capacity to modulate the immune system responses in vitro and in vivo. This may have critical outcomes in diseases of the musculoskeletal system where an inflammatory or autoimmune process is at the basis of the main disease. In the present paper, after isolation of MSCs from Whartons Jelly (WJ-MSCs), we performed the three standard differentiation protocols. The acquisition of the differentiated phenotype was demonstrated by the specific histological stains. As the main objective of this work, we determined the expression of immunomodulatory molecules (by immunohistochemistry and qualitative RT-PCR), both in undifferentiated cells and after differentiation. We demonstrated for the first time that immune-related molecules (as B7-H3/CD276 and HLA-E) which have been characterized in undifferentiated MSCs, are also expressed by the differentiated progeny. This strongly suggests that also after the acquisition of a mature phenotype, WJ-MSCs-derived cells may maintain their immune privilege. This evidence, which deserves much work to be confirmed in vivo and in other MSCs populations, may provide a formal proof of the good results globally achieved with WJMSCs as cellular therapy vehicle.

Isolation and characterization of CD276+/HLA-E+ human subendocardial mesenchymal stem cells from chronic heart failure patients: analysis of differentiative potential and immunomodulatory markers expression.

Mesenchymal stem cells (MSCs) are virtually present in all postnatal organs as well as in perinatal tissues. MSCs can be differentiated toward several mature cytotypes and interestingly hold potentially relevant immunomodulatory features. Myocardial infarction results in severe tissue damage, cardiomyocyte loss, and eventually heart failure. Cellular cardiomyoplasty represents a promising approach for myocardial repair. Clinical trials using MSCs are underway for a number of heart diseases, even if their outcomes are hampered by low long-term improvements and the possible presence of complications related to cellular therapy administration. Therefore, elucidating the presence and role of MSCs that reside in the post-infarct human heart should provide essential alternatives for therapy. In the current article we show a novel method to reproducibly isolate and culture MSCs from the subendocardial zone of human left ventricle from patients undergoing heart transplant for post-infarct chronic heart failure (HSE-MSCs, human subendocardial mesenchymal stem cells). By using both immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR), we demonstrated that these cells do express key MSCs markers and do express heart-specific transcription factors in their undifferentiated state, while lacking strictly cardiomyocyte-specific proteins. Moreover, these cells do express immunomodulatory molecules that should disclose their further potential in immune modulation processes in the post-infarct microenvironment. Another novel datum of potentially relevant interest is the expression of cardiac myosin heavy chain at nucclear level in HSE-MSCs. Standard MSCs trilineage differentiation experiments were also performed. The present paper adds new data on the basic biological features of heart-resident MSCs that populate the organ following myocardial infarction. The use of heart-derived MSCs to promote in-organ repair or as a cellular source for cardiomyoplasty is a fascinating and challenging task, which deserves further research efforts.

Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin

Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein.

STAT3 inhibition suppresses hepatic stellate cell fibrogenesis: HJC0123, a potential therapeutic agent for liver fibrosis

Hepatic Stellate Cells (HSCs) are the major source of the excessive extracellular matrix (ECM) production that replaces liver parenchyma with fibrous tissue during liver fibrosis. The signal transducer and activator of transcription 3 (STAT3) promotes HCSs survival, proliferation, and activation contributing to fibrogenesis. We have previously used a fragment-based drug design approach and have discovered a novel STAT3 inhibitor, HJC0123. Here, we explored the biological effects of HJC0123 on the fibrogenic properties of HSCs. HJC0123 treatment resulted in the inhibition of HSCs proliferation at submicromolar concentrations. HJC0123 reduced the phosphorylation, nuclear translocation, and transcriptional activity of STAT3. It decreased the expression of STAT3-regulated proteins, induced cell cycle arrest, promoted apoptosis and downregulated SOCS3. HJC0123 treatment inhibited HSCs activation and downregulated ECM protein fibronectin and type I collagen expression. In addition, HJC0123 increased IL-6 production and decreased TGF-β induced Smad2/3 phosphorylation. These results demonstrate that HJC0123 represents a novel STAT3 inhibitor that suppresses the fibrogenic properties of HSCs, suggesting its therapeutic potential in liver fibrosis.

Transformation of primary human hepatocytes in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Currently, there is limited knowledge of neoplastic transformation of hepatocytes in HCC. In clinical practice, the high rate of HCC local recurrence suggests the presence of different hepatocyte populations within the liver and particularly in the tumor proximity. The present study investigated primary human hepatocyte cultures obtained from liver specimens of patients affected by cirrhosis and HCC, their proliferation and transformation. Liver samples were obtained from seven HCC cirrhotic patients and from three patients with normal liver (NL). Immediately after surgery, cell outgrowth and primary cultures were obtained from the HCC lesion, the cirrhotic tissue proximal (CP, 1-3 cm) and distal (CD, >5 cm) to the margin of the neoplastic lesion, or from NL. Cells were kept in culture for 16 weeks. Morphologic analyses were performed and proliferation rate of the different cell populations compared over time. Glypican-3, Heppar1, Arginase1 and CD-44 positivity were tested. The degree of invasiveness of cells acquiring neoplastic characteristics was studied with a transwell migration assay. We observed that HCC cells maintained their morphology and unmodified neoplastic characteristics when cultured. Cells isolated from CP, showed a progressive morphologic transformation in HCC-like cells accompanied by modification of markers expression with signs of invasiveness. Absence of HCC contamination in the CP isolates was confirmed. In CD samples some of these characteristics were present and at significantly lower levels. With the present study, we are the first to have identified and describe the existence of human hepatocytes near the cancerous lesion that can transform in HCC in vitro.

Respiratory Syncytial Virus Infection Changes Cargo Composition of Exosome Released from Airway Epithelial Cells

Exosomes are microvesicles known to carry biologically active molecules, including RNA, DNA and proteins. Viral infections can induce profound changes in exosome composition, and exosomes have been implicated in viral transmission and pathogenesis. No information is current available regarding exosome composition and function during infection with Respiratory Syncytial Virus (RSV), the most important cause of lower respiratory tract infections in children. In this study, we characterized exosomes released from RSV-infected lung carcinoma-derived A549 cells. RNA deep sequencing revealed that RSV exosomes contain a diverse range of RNA species like messenger and ribosomal RNA fragments, as well as small noncoding RNAs, in a proportion different from exosomes isolated from mock-infected cells. We observed that both RNA and protein signatures of RSV were present in exosomes, however, they were not able to establish productive infection in uninfected cells. Exosomes isolated from RSV-infected cells were able to activate innate immune response by inducing cytokine and chemokine release from human monocytes and airway epithelial cells. These data suggest that exosomes may play an important role in pathogenesis or protection against disease, therefore understating their role in RSV infection may open new avenues for target identification and development of novel therapeutics.

Role of Hydrogen Sulfide in NRF2- and Sirtuin-Dependent Maintenance of Cellular Redox Balance

Hydrogen sulfide (H2S) has arisen as a critical gasotransmitter signaling molecule modulating cellular biological events related to health and diseases in heart, brain, liver, vascular systems and immune response. Three enzymes mediate the endogenous production of H2S: cystathione β-synthase (CBS), cystathione γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). CBS and CSE localizations are organ-specific. 3-MST is a mitochondrial and cytosolic enzyme. The generation of H2S is firmly regulated by these enzymes under normal physiological conditions. Recent studies have highlighted the role of H2S in cellular redox homeostasis, as it displays significant antioxidant properties. H2S exerts antioxidant effects through several mechanisms, such as quenching reactive oxygen species (ROS) and reactive nitrogen species (RNS), by modulating cellular levels of glutathione (GSH) and thioredoxin (Trx-1) or increasing expression of antioxidant enzymes (AOE), by activating the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2). H2S also influences the activity of the histone deacetylase protein family of sirtuins, which plays an important role in inhibiting oxidative stress in cardiomyocytes and during the aging process by modulating AOE gene expression. This review focuses on the role of H2S in NRF2 and sirtuin signaling pathways as they are related to cellular redox homeostasis.

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.

Nuclear localization and new isoforms detection give new insights on Hsp10 functions in normal and cigarette smoke-stressed lung cells

Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular dis- tribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) is a potent stressor for the respiratory system, but its effects on the expression, function, and cellular locali- zation of mitochondrial chaperonins are still largely unknown. We studied in vivo (airways biopsies) the localization of Hsp10 and Hsp60 in patients (smokers and non-smokers) affected by mild-moderate COPD, and charac- terized the effects of non-lethal doses of CS extract (CSE) on the expression of these molecules in two human cell lines: lung fibroblasts (HFL-1) and bronchial epithelial cells (16HBE). We applied various in vitro methods: IHC, subcellular fractionation analyses (SFA), western blotting (WB), ICC, transmission electron microscopy (TEM) immunogold, chromati protein extracts (CPE), as well as 2D-gel based proteomics analyses. Bioinformatics was used to gather structural in silico data. IHC showed that Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers. ICC, SFA, and WB showed that 16HBE and HFL-1 cells featured nuclear Hsp10, before and after CSE exposure; TEM immunogold further confirmed this observation. Proteomics data showed that CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts. Bioinformatics analyses indicated that Hsp10 can localize in extramitochondrial sites, such as the nucleus, even if Hsp10 lacks known DNA-binding motifs or nuclear import signals. Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein as suggested by CPE/gel retardation experiments. Data reported here indicate that in human cells of the respiratory mucosa there are at least three different intracellular locales for Hsp10: mitochondrial, nuclear, and cyto- solic. Further experiments are en route for the definition of the mechanisms underlying the transfer of Hsp10 to the nucleus and other cellular/extracellular compartments. This work was supported by grants from University of Palermo (FFR 2012) to GLR.

Wharton’s Jelly Mesenchymal Stem Cells for the Treatment of Type 1 Diabetes

Type 1 diabetes is an autoimmune disease caused by the destruction of endocrine pancreas β cells by T lymphocytes, for which genetic and environmental risk factors have been proposed. Patients require daily infusions of recombinant insulin to overcome the reduced production by their own cells, but there is an increasing demand for a permanent and efficient supplementation which could better modulate the need for the hormone during the normal activities. For this reason, transplant-based therapeutic models have been proposed such as whole organ transplantation and Langerhans islets transplantation. These techniques are limited by many factors such as the lack of donors, the risks linked to the surgical practice, and the rejection reactions of the transplanted organ. Further opportunities may come from the research on stem cells. Multiple studies showed that multiple populations of stem cells may be differentiated toward pancreatic endocrine-like ones, which may express insulin in vitro and in vivo and often respond to glucose challenge. Another developing field in stem cells research is that on the immunomodulatory ability of some stem populations, in particular those defined as “perinatal,” derived from fetus-associated tissues usually discarded at birth. Wharton’s jelly mesenchymal stem cells (WJ-MSCs), which derive from the mature mucous tissue constituting the bulk of the umbilical cord, can also differentiate toward beta cells. Moreover, these cells feature important immunomodulatory activities, which seem to be maintained also in differentiated populations, which should render these cells even more promising for cell therapy applications in type 1 diabetes. This chapter analyzes the literature regarding the features and potential of WJ-MSCs for the therapy of type 1 diabetes, in the light of multiple possible therapeutic approaches, in which cells should be used both undifferentiated and differentiated, and in cotransplantation with islets. We propose that WJ-MSCs transplantation may be useful both to regenerate β cells and also prevent the autoimmune destruction of remnant and neogenetic β cells in patients.