From nucleus pulposus mesenchymal stem cells towards neural differentiation: an interesting prospect

Regenerative medicine arouses great interest for the treatment of many neurological diseases. Since nucleus pulposus of the invertebral discs is a postembryonic vestige of the notochord, it has been hypothesized that mesenchymal stem cells (MSCs) isolated from nucleus pulposus (NP-MSCs) can more easily differentiate into neurons. In this study, MSCs from nucleus pulposus were successfully isolated and characterized. Then, neural differentiation was induced by using a medium consisted of DMEM/F12 supplemented with B27 and the growth factors FGF and EGF for 10 days. Immunocytochemistry, molecular studies, SEM and TEM microscopy analyses were performed. NP-MSCs exhibited the typical features of MSCs, revealing spindle-shape morphology, specific immunophenotype attributable to MSCs and the ability to differentiate in osteogenic and chondrogenic lineages. After neurodifferentiation induction, compared to NP-MSCs in only DMEM/F12, proliferation rate decreased and cells changed morphology acquiring an increased number of the so-called neural-like extensions. Neural progenitor marker NESTIN and mature neuronal marker ENOLASE-2 were up-regulated, while GFAP was not detected. Moreover, cells after differentiation were small rounded and fusiform, with tendency to organize in clumps; they had elongated extrusions containing oriented cytoskeletal elements, classifiable as microtubules and intermediate filaments, as visualized by SEM and TEM microscopy. Dense vesicles similar to lipid droplet were also observed. NP-MSCs in differentiation medium were able to form neurospheres. In conclusion, even if more analysis have to be done and the way to treat neurodegenerative disease with regenerative medicine is still long, NP-MSCs represent a promising resource

The inflamed microenvironment: role on MSCs immunobiology and cancer

Inflammation and cancer are an inseparable binomial. The majority of cancers are triggered by somatic mutations and environmental factors with a common element: inflammation. Inflammation creates a microenvironment in which neoplastic cells can profit from the trophic factors secreted by inflammatory cells, useful to interfere with the anti-tumor response. Among the others, mesenchymal stem cells (MSCs) participate to microenvironment creation by a strong paracrine effect. The linkage between MSCs and inflammation is bidirectional: the inflamed microenvironment affects the complex MSCs immunobiology, but also MSCs can sustain inflammation. Here, we tried to clarify the influence of inflammation on the immunobiology of MSCs and deepen the paracrine effect of MSCs on tumor growth. MSCs were isolated from periprosthetic capsule caused by breast implant, affected by inflammation (I-MSCs). The contralateral part of the same patient, not inflamed, was used as control (C-MSCs). A panel of selected cytokines were analyzed by Real-Time PCR and ELISA. The cytokines expression was different in I-MSCs compared to C-MSCs, revealing that inflammation affects MSCs immunobiology. Then, C- and I-MSCs were indirectly co-cultured with MCF7 cells from breast adenocarcinoma. New analyses on proliferation rate and cytokines expression were performed. C- and I-MSCs gave almost the same results. The over-secretion of all the cytokines referred to the Th1 pathway and the decrease of those belonging to the Th2 pathway revealed the absence of a switch from Th1 to Th2 important to induce a chronic inflammation. The levels of TGF-β and G-CSF linked to the skill to damage the antigen-presenting cell function were decreased. In conclusion, even if MCF-7 proliferation increased after co-culture with I-MSCs, MSCs-derived paracrine effect does not sustain breast adenocarcinoma. These results absolve the breast implants from the insult to enhance adenocarcinoma onset

Crosstalk between Mesenchymal Stem Cells and tumor cells: the role of inflammation

Mesenhymal Stem Cells (MSCs) are self-renewal multipotent cells that can be isolated from different adult tissues. There is a growing interest in the role exerted by MSCs in cancer progression. MSCs exhibit a marked tropism for tumors and par- ticipate to the creation of the stroma and related inflammation, which has a critical role in carcinogenesis, progression and metastasis. Nevertheless, while many studies showed that MSCs promote tumor progression and metastasis, others reported that MSCs suppress tumor growth. These contradictory results may be due to the origin of the MSCs, their degree of differentiation, the tumor model and other factors that are not yet elucidated. Aim of this work was to establish the role of the paracrine effect exerted by MSCs isolated from inflamed (I-MSCs) and control (C-MSCs) tis- sues towards human MCF7 and KI-JK cell lines, respectively derived from a breast cancer and an anaplastic large T cell lymphoma (ALCL). After stemness characteriza- tion, MSCs were indirectly co-cultured with MCF7 or KI-JK for 7 days; subsequently the proliferation rate and the expression of specific genes were tested. Genes were selected according to their role in inflammation and cancer previously reported in literature and explicate their action by different mechanisms: chemokines with pro- (CXCL2, CXCL9) or anti-angiogenic effect (CXCL10); chemokines (CCL2, CXCL12, CXCL5) for the recruitment of myeloid-derived suppressor cells (MDSCs); interleu- kins distinctive for chronic (IL2, IL4) and acute (IL8, IL16) inflammation; cytokines belonging to the Th2 subset (CCL22, IL13, IL22, CCL17, CCL18); cytokines (IL6, IL10 and TGFβ1) involved in the manipulation of the antigen-presenting cells function. Our data confirm a role of MSCs in cancer; an increase of pro-angiogenic chemokines as well as of interleukin related to acute phase of inflammation, a general switch of the T cell response from the Th1 cell subset to the Th2 subset and the induction of MDSCs were observed. Surprisingly, these effects have been mainly found in both cancer cell lines after co-culture with C-MSCs; it may mean that I-MSCs, suffering of chronic inflammation, are less responsive than C-MSCs to new stress/stimuli. Fur- ther experiments will be necessary to better address the role of MSCs and inflamma- tion on cancer progression; nevertheless, this study highlight as MSCs are not simply guardian but active actors in cancer fate

Mesoderm stem cells and inflammation: role in the Pathogenesis and potential therapy of selected Gynecological Deseases and primary Myopathies

Le cellule staminali mesenchimali (MSCs) sono un tipo specifico di cellule staminali adulte con un elevato potenziale proliferativo e differenziativo verso cellule specializzate di derivazione mesodermica. Le MSCs svolgono anche una funzione paracrina attraverso il rilascio di molteplici fattori di crescita, chemochine e citochine. Le MSCs si comportanto da sentinelle che percepiscono il microambiente e agiscono di conseguenza, passando da un fenotipo pro-infiammatorio ad uno immunosoppressivo in base ai segnali che ricevono. Nel seguente lavoro sono valutati l’esistenza e il ruolo delle MSCs nella patogenesi e nella potenziale terapia di selezionate patologie ginecologiche con una componente infiammatoria come il leiomioma uterino, la neoplasia intraepiteliale cervicale (CIN) e in miopatie primarie, quali la Distrofia Muscolare di Duchenne (DMD). Nel primo studio, sono state identificate le cellule progenitrici nel leiomioma e nel miometrio sano ed è stata investigata la correlazione tra tali cellule e l’infiammazione nell’insorgenza del leiomioma. I dati suggeriscono che una overespressione di citochine relative all’infiammazione cronica nei progenitori del leiomioma potrebbe favorire un microambiente adeguato per l’insorgenza di questa patologia. Nel secondo studio, le MSCs sono state isolate da cervici di pazienti giovani (yC-MSCs) e pazienti vecchie (oC-MSCs) e i risultati mostrano come la loro immunobiologia sia condizionata dall’età dei donatori, influenzando anche il tasso di regressione della CIN. Inoltre, nel crosstalk con le cellule HeLa, yC-MSCs svolgono maggiormente un ruolo anti-tumorale sostenendo un’infiammazione acuta. L’obiettivo del terzo studio è stato quello di trovare una corretta strategia per aumentare la produzione di distrofina nella DMD mediante terapia genica. Pertanto, i mioblasti isolati da donatori di DMD sono stati trasdotti con la proteina fluorescente verde (GFP) e un vettore lentivirale esprimente l’snRNA per indurre il salto dell’esone; i dati indicano che i mioblasti trasdotti erano abili a differenziare in senso miogenico esprimendo la distrofina funzionale.Mesenchymal stem or stromal cells (MSCs) are a specific type of adult stem cells with an extensive proliferation and differentiation potential towards specialized cells developing from the mesoderm. MSCs are also characterized by paracrine function through the release of multiple growth factors, chemokines and cytokines. MSCs play as sentinel that feel the microenvironment and act consequently, switching from a pro-inflammatory phenotype to an immunosuppressive phenotype according to the signals they receive. In the present work the existence and the role of MSCs in the pathogenesis and potential therapy of selected gynecological diseases with an inflammatory component as uterine leiomyoma, cervical intraepithelial neoplasia (CIN), and in primary myopathies, as Duchenne Muscular Dystrophy (DMD) were evaluated. In the first study, progenitor cells were identified both in leiomyomas and normal myometrium, and the correlation between these cells and inflammation in leiomyoma onset has been investigated. The data suggest that the upregulation of cytokines related to chronic inflammation in leiomyoma progenitors could favour a microenvironment suitable for the onset of this pathology. In the second study, MSCs from cervix of young (yC-MSCs) and old patients (oC-MSCs) were isolated and results show as their immunobiology is affected by the age of donors, influencing in turn the regression rate of CIN. In addition, in the crosstalk with HeLa cells, yC-MSCs play an anti-tumoral role sustaining an acute inflammatory environment. The goal of the third study was to find a correct strategy to enhance the production of dystrophin protein in DMD through gene therapy. Therefore, myoblasts isolated from DMD donor were transduced with green fluorescent protein (GFP) and a lentiviral vector expressing the snRNA to induce exon skipping; data indicate that transduced myoblasts were able to perform myogenic differentiation expressing a functional dystrophin protein

From 2646 to 15: differentially regulated miRNAs between progenitors from normal myometrium and leiomyoma

Uterine leiomyomas (fibroids) are smooth muscle neoplasms of the myometrial layer of the uterus and the most common benign tumors in women. Although their etiology is still unclear, progenitor cells (PCs) seem to be implicated

Mesenchymal Stem Cells from Cervix and Age: New Insights into CIN Regression Rate

Cervical intraepithelial neoplasia (CIN) is a precancerous lesion of the uterine cervix that can regress or progress to cervical cancer; interestingly, it has been noted that young women generally seem to have higher rates of spontaneous regression and remission, suggesting a correlation between the patient’s age and regression/progression rates of CIN. Even if the underlying mechanisms are still unclear, inflammation seems to play a pivotal role in CIN fate and inflammatory processes are often driven by mesenchymal stem cells (MSCs). This study was aimed at evaluating if age affects the behavior of MSCs from the cervix (C-MSCs) that in turn may modulate inflammation and, finally, regression rate. Fourteen samples of the human cervix were recovered from two groups of patients, “young” (mean age 28 ± 2) and “old” (mean age 45 ± 3), during treatment using the loop electrosurgical excision procedure (LEEP) technique. Progenitor cells were isolated, deeply characterized, and divided into young (yC-MSCs) and old cervixes (oC-MSCs); the senescence, expression/secretion of selected cytokines related to inflammation, and the effects of indirect cocultures with HeLa cells were analyzed. Our results show that isolated cells satisfy the fixed criteria for stemness and display age-related properties; yC-MSCs express a higher level of cytokines related to acute inflammation than oC-MSCs. Finally, in the crosstalk with HeLa cells, MSCs derived from the cervixes of young patients play a stronger antitumoral role than oC-MSCs. In conclusion, the immunobiology of MSCs derived from the cervix is affected by the age of donors and this can correlate with the regression rate of CIN by influencing their paracrine effect. In addition, MSCs from a young cervix drives an antitumoral effect by sustaining an acute inflammatory environment

New miRNAs network in human mesenchymal stem cells derived from skin and amniotic fluid.

Mesenchymal stem cells (MSCs), isolated from different adult sources, have great appeal for therapeutic applications due to their simple isolation, extensive expansion potential, and high differentiative potential.In our previous studies we isolated MSCs form amniotic fluid (AF-MSCs) and skin (S-MSCs) and characterized them according to their phenotype, pluripotency, and mRNA/microRNAs (miRNAs) profiling using Card A from Life Technologies.Here, we enlarge the profiling of AF-MCSs and S-MSCs to the more recently discovered miRNAs (Card B by Life Technologies) to identify the miRNAs putative target genes and the relative signaling pathways. Card B, in fact, contains miRNAs whose role and target are not yet elucidated.The expression of the analyzed miRNAs is changing between S-MSCs and AF-MSCs, indicating that these two types of MSCs show differences potentially related to their source. Interestingly, the pathways targeted by the miRNAS deriving from Card B are the same found during the analysis of miRNAs from Card A.This result confirms the key role played by WNT and TGF-β pathways in stem cell fate, underlining as other miRNAs partially ignored up to now deserve to be reconsidered. In addition, this analysis allows including Adherens junction pathways among the mechanisms finely regulated in stem cell behavior