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The umbilical cord (UC) is an essential part of the placenta, contributing to foetal development by ensuring the blood flow between mother and foetus. The UC is formed within the first weeks of gestation by the enclosure of the vessels (one vein and two arteries) into a bulk of mucous connective tissue, named Wharton's jelly (WJ) and lined by the umbilical epithelium. Since their first identification, cells populating WJ were described as unusual fibroblasts (or myofibroblasts). Recent literature data further highlighted the functional interconnection between UC and the resident cells. The UC represents a reservoir of progenitor populations which are collectively grouped into MSCs (mesenchymal stem cells). Such cells have been sourced from each component of the cord, namely the sub-amnion layer, the WJ, the perivascular region, and the vessels. These cells mainly show adherence to the phenotype of adult MSCs (as bone marrow-derived ones) and can differentiate towards mature cell types belonging to all the three germ layers. In addition, cells from human UC are derived from an immunoprivileged organ, namely the placenta: in fact, its development and function depend on the elusion of the maternal immune response towards the semi-allogeneic embryo. This is reflected in the expression of immunomodulatory molecules by UC-derived MSCs. The present paper describes UC structural features and the cell types which can be derived, with a focus on their phenotype and the novel results which boosted the use of UC-derived cells for regenerative medicine applications.

Therapeutic options for end-stage organ failure are often limited to whole organ transplantation. The tolerance or rejection of the transplanted organ is driven by both early non-specific innate and specific adaptive responses. The use of mesenchymal stromal cells (MSCs) is considered a promising tool in regenerative medicine. Human umbilical cord (HUC) is an easily available source of MSCs, without relevant ethical issues. Moreover, Wharton's jelly-derived MSCs (WJ-MSCs), showed consistent immunomodulatory features that may be useful to promote immune tolerance in the host after transplantation. Few data are available on the phenotype of WJ-MSCs in situ. We investigated the expression of immune-related molecules, such as HLAs, IDO, CD276/B7-H3, and others, both in situ (HUC) and in in vitro-cultured WJ-MSCs. Morphological and biochemical techniques were used to define the expression of such molecules. In addition, we focused on the possible role of CD276/B7-H3 on T cells proliferation inhibition. We assessed CD276/B7-H3 expression by WJ-MSCs both in situ and alongside cell culture. WJ-MSCs were able to suppress T cell proliferation in mixed lymphocyte reaction (MLR). Moreover, we describe for the first time a specific role for CD276/B7-H3, since the immunomodulatory ability of WJ-MSCs was abolished upon anti-CD276/B7-H3 antibody addition to the MLR. These results further detail the immune regulation properties and tolerance induction exerted by human WJ-MSCs, in particular pointing to CD276/B7-H3 as one of the main involved factors. These data further suggest WJ-MSCs as potent tools to modulate local immune response in "support-type" regenerative medicine approaches.

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., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

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 Wharton's 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.

Mesenchymal (stromal) stem cells (MSC) are a broad class of stromal populations which are able to differentiate towards mature cell types, and do express molecules involved in immune modulation, tolerance induction and inflammation dampening. MSC can be virtually isolated from each adult organ, as well as from foetus-associated perinatal tissues. In particular, Wharton's jelly-derived MSC (WJ-MSC) bear all of these key properties, together with their ease of sourcing and lack of ethical issues. Cellular therapy is a key technique in regenerative medicine approaches, in particular for the treatment of diseases in which physiological processes of cellular repopulation are blocked by the underlying pathological conditions. Recent data enlightened the ability of administered cells to act also in a repopulation-independent fashion in target organs, since their peculiar immunomodulatory and anti-inflammatory features may favor organ self-repair by reactivating local progenitors by both cell-mediated or paracrine mechanisms. Translating classical regenerative medicine to "reparative medicine" or "support medicine" should represent a further therapeutic strategy independent from the differentiation capacity of MSC populations. Recent data further highlighted that WJ-MSC outperform BM-MSC (which are now being applied clinically) both in terms of immune modulation and lack of tumorigenesis (or tumor-promoting activities) in vivo. Starting from these premises, this paper analyzes the recent data on the biology of WJ-MSC, considering the role of both naive and differentiated cells in immune modulation. In particular, the role of tolerance promoting pathways via non-classical B7 costimulators or class Ib MHC molecules are examined. In addition, we also analyzed the interconnections with other mechanicistic pathways (as those driven by matrix degrading metalloproteinases) in immune modulation. Our observations strongly support the notion that WJ-MSC may constitute a new tool in regenerative and reparative medicine applications.