Simona Corrao

Isolation and characterization of Oct-4 + /HLA-G + mesenchymal stem cells from human umbilical cord matrix: differentiation potential and detection of new markers

The presence of multipotent cells in several adult and embryo-related tissues opened new paths for their use in regenerative medicine. Extraembryonic tissues such as umbilical cord are considered a promising source of stem cells, potentially useful in therapy. The characterization of cells from the umbilical cord matrix (Wharton’s Jelly) and amniotic membrane revealed the presence of a population of mesenchymal-like cells, sharing a set of core-markers expressed by “mesenchymal stem cells”. Several reports enlightened the differentiation capabilities of these cells, even if at times the lack of an extensive characterization of surface markers and immune co-stimulators expression revealed hidden pitfalls when in vivo transplantation was performed. The present work describes a novel isolation protocol for obtaining mesenchymal stem cells from the umbilical cord matrix. These cells are clonogenic, retain long telomeres, can undergo several population doublings in vitro, and can be differentiated in mature mesenchymal tissues as bone and adipose. We describe for the first time that these cells, besides expressing all of the core-markers for mesenchymal stem cells, feature also the expression, at both protein and mRNA level, of tolerogenic molecules and markers of all the three main lineages, potentially important for both their differentiative potential as well as immunological features.

New emerging potentials for human Wharton's jelly mesenchymal stem cells: immunological features and hepatocyte-like differentiative capacity.

In recent years, human mesenchymal stem cells (MSC) have been extensively studied. Their key characteristics of long-term self-renewal and a capacity to differentiate into diverse mature tissues favor their use in regenerative medicine applications. Stem cells can be found in embryonic and extraembryonic tissues as well as in adult organs. Several reports indicate that cells of Whartons jelly (WJ), the main component of umbilical cord extracellular matrix, are multipotent stem cells, expressing markers of bone marrow mesenchymal stem cells (BM-MSC), and giving rise to different cellular types of both connective and nervous tissues. Whartons jelly mesenchymal stem cells (WJ-MSC) express markers previously characterized in embryonic stem cells (ESC), such as Nanog and Oct3/4A. WJ-MSC further emerge as promising hypoimmunogenic cells, due to the expression of molecules able to modulate NK cells and expand regulatory T-cell populations. Moreover, it is now accepted that the differentiative capacities of such cells span all the mesoderm-derived tissues, extending to neuroectodermal as well as endodermal lineages. In this review, we compare very recent data on the potential of WJ-MSC to undergo hepatocyte-like differentiation with the results obtained from other adult MSC populations. Data in the literature strongly suggest that WJ-MSC can differentiate into diverse cell types, showing a unique ability to cross lineage borders. This, together with their in vitro proliferative potential and their immunoregulatory features, renders these cells extremely promising for regenerative medicine applications in different pathological settings.

Human Hsp10 and Early Pregnancy Factor (EPF) and their relationship and involvement in cancer and immunity: Current knowledge and perspectives

This article is about Hsp10 and its intracellular and extracellular forms focusing on the relationship of the latter with Early Pregnancy Factor and on their roles in cancer and immunity. Cellular physiology and survival are finely regulated and depend on the correct functioning of the entire set of proteins. Misfolded or unfolded proteins can cause deleterious effects and even cell death. The chaperonins Hsp10 and Hsp60 act together inside the mitochondria to assist protein folding. Recent studies demonstrated that these proteins have other roles inside and outside the cell, either together or independently of each other. For example, Hsp10 was found increased in the cytosol of different tumors (although in other tumors it was found decreased). Moreover, Hsp10 localizes extracellularly during pregnancy and is often indicated as Early Pregnancy Factor (EPF), which is released during the first stages of gestation and is involved in the establishment of pregnancy. Various reports show that extracellular Hsp10 and EPF modulate certain aspects of the immune response with anti-inflammatory effects in patients with autoimmune conditions improving clinically after treatment with recombinant Hsp10. Moreover, Hsp10 and EPF are involved in embryonic development, acting as a growth factor, and in cell proliferation/differentiation mechanisms. Therefore, it becomes evident that Hsp10 is not only a co-chaperonin, but an active player in its own right in various cellular functions. In this article, we present an overview of various aspects of Hsp10 and EPF as they participate in physiological and pathological processes such as the antitumor response and autoimmune diseases.

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.

Hsp60 and heme oxygenase-1 (Hsp32) in acute myocardial infarction.

Heat shock proteins (Hsps) are produced in response to various stressors, including ischemia-reperfusion, and they can exit cells and reach the blood. In this pilot study, we determined serum levels of Hsp60 and heme-oxygenase-1 (HO-1; also named Hsp32) in subjects with acute myocardial infarction (AMI) to assess their clinical significance and potential prognostic value. We also performed a bioinformatics analysis of the 2 molecules in search of structural clues on the mechanism of their release from cells. We studied 40 patients consecutively admitted for AMI (male:female patient ratio=20:20, mean age: 64 ± 13 years) and 40 matched controls. A blood sample was drawn for biochemical analyses within 24 h of symptoms onset, and Hsp60 and HO-1 concentrations were determined by enzyme-linked immunosorbent assay (ELISA). All patients were followed up for 6 months to register adverse post-AMI cardiovascular events. A multivariate analysis demonstrated that elevated Hsp60 (P=0.0361), creatine phosphokinase-muscle brain (CK-MB) (P=0.0446), and troponin (P=0.0490) were predictive of post-AMI adverse events. In contrast, increased HO-1 showed a significant association with less severity of coronary artery diseases (P=0.0223). These findings suggest that Hsp60 and HO-1 play distinct roles in the pathogenesis of AMI and subsequent AMI-related pathology. The possibility that these proteins differ in their roles and mechanisms of action in AMI and post-AMI pathology was supported also by the bioinformatics estimates of probability of their localization in various subcellular compartments. The results clear the way for subsequent investigation on the pathogenetic role and clinical significance of Hsp60 and HO-1 in AMI.

Perinatal and Wharton's Jelly-Derived Mesenchymal Stem Cells in Cartilage Regenerative Medicine and Tissue Engineering Strategies

Stem cells can be found in embryonic and extraembryonic tissues as well as in adult organs. In particular, research in the last few years has delineated the key features of perinatal stem cells derived from fetus-associated tissues. These cells show multiple differentiation potential, can be easily expanded ex vivo, and raise no ethical concerns as regards their use. Several reports indicate that cells isolated from Whartons jelly (WJ), the main component of umbilical cord extracellular matrix, are multipotent stem cells that express markers shared by other mesenchymal stem cells (MSC) and give rise to different mature cell types belonging to all three germ layers. Moreover, WJ-MSC display promising hypoimmunogenic and immunomodulatory properties, since they express molecules able to modulate NK cells and expand regulatory T-cell populations. In this review, we focus on the use of perinatal stem cells for regenerative medicine aimed at cartilage repair and regeneration. Cartilage is a specialized connective tissue which has poor regeneration and self-repair capacity in vivo. Traumatic injury or autoimmune processes are among the main causes of cartilage damage and degeneration, for which new hope comes from tissue engineering using stem cells which have undergone chondrocyte-like differentiation. We analyze the in vitro and in vivo data on the use of perinatal stem cells, in particular WJ-MSC, for cartilage regenerative medicine. The high variability of cell sources, the use of different types of scaffolds and matrixes, and the administration of several combinations of growth factors clearly point out the need for further research to optimize this cellular therapy approach and translate the results obtained from bench to clinic.

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.

New frontiers in regenerative medicine in cardiology: the potential of Wharton's jelly mesenchymal stem cells.

Cardiomyopathies are still the first cause of death in the world. The identification of resident stem cells, comprising those derived from sub-endocardial stroma, suggests the possible self regeneration of the heart under autocrine/paracrine modulation in the cardiac microenvironment. Nevertheless, because of the limited in vivo regeneration potential of damaged cardiac tissue, the use of drugs and ultimately cardiac transplantation remain the common treatments of heart diseases and defects. The differentiative potential of embryonic and mesenchymal stem cells (MSCs) derived from different tissues (such as bone marrow and adipose tissue) was extensively explored in cell therapy for regenerative medicine. Many groups have been focused, in recent years, on isolation, characterization, and differentiation potential of MSCs derived from perinatal (or extraembryonic) tissues, mainly the placenta and the human umbilical cord. In this review, we summarized recent works about the stemness of Whartons jelly stromal cells and their potential in cardiac regeneration with favourable use in cell therapy and regenerative medicine. The peculiar features of these cells, as the expression of cardiac-specific transcription factors and immunomodulatory molecules suggest that human umbilical cord may be considered as a reliable alternative source of MSC useful for advanced therapy in cardiac regenerative medicine.

Hsp10: anatomic distribution, functions, and involvement in human disease

There is growing evidence that molecular chaperones/heat shock proteins are involved in the pathogenesis of a number of human diseases, known as chaperonopathies. A better molecular understanding of the pathogenetic mechanisms is essential for addressing new strategies in diagnostics, therapeutics and clinical management of chaperonopathies, including those in which Hsp10 is involved. This chaperonin has been studied for a long time as a member of the mitochondrial protein-folding machine. However, although in normal cells Hsp10 is mainly localized in the mitochondrial matrix, it has also been found during and after stress in other subcellular compartments, such as cytosol, vesicles and secretory granules, alone or in combination with other proteins. In these extramitochondrial locales, Hsp10 plays an active role in cell signalling. For example, cancer cells often show altered levels of Hsp10, compared to normal cells. Hsp10 may also be found in the extracellular space and in the bloodstream, with a possible immunomodulatory activity. This minireview focuses on some studies to date on the involvement of Hsp10 in human disease pathogenesis.

CD1a down-regulation in primary invasive ductal breast carcinoma may predict regional lymph node invasion and patient outcome.

Aims:  CD1a is a molecule belonging to the highly conserved group of CD1 proteins. Its expression in dendritic cells is related to the presentation of tumour‐derived glycolipid antigens to T cells and, consequently, the development of a successful antitumour response. The aim was to investigate the presence of CD1a+ cells in both primary tumours and lymph nodes (LN) of a series of 35 invasive ductal carcinomas by both immunohistochemistry and reverse transcription‐polymerase chain reaction.