Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stromal Cells as an Efficient Nanocarrier to Deliver siRNA or Drug to Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Treatment of PDAC remains a major challenge. This study aims to evaluate, in vitro, the use of human umbilical cord mesenchymal stromal cell (UC-MSC)-derived EVs to specifically target pancreatic cancer cells. EVs were isolated from the FBS-free supernatants of the cultured UC-MSCs by ultracentrifugation and characterized by several methods. EVs were loaded with scramble or KRASG12D-targeting siRNA by electroporation. The effects of control and loaded EVs on different cell types were evaluated by assessing cell proliferation, viability, apoptosis and migration. Later, the ability of EVs to function as a drug delivery system for doxorubicin (DOXO), a chemotherapeutic drug, was also evaluated. Loaded EVs exhibited different kinetic rates of uptake by three cell lines, namely, BxPC-3 cells (pancreatic cancer cell line expressing KRASwt), LS180 cells (colorectal cell line expressing KRASG12D) and PANC-1 cells (pancreatic cell line expressing KRASG12D). A significant decrease in the relative expression of the KRASG12D gene after incubation with KRAS siRNA EVs was observed by real-time PCR. KRASG12D siRNA EVs significantly reduced the proliferation, viability and migration of the KRASG12D cell lines compared to scramble siRNA EVs. An endogenous EV production method was applied to obtain DOXO-loaded EVs. Briefly, UC-MSCs were treated with DOXO. After 24 h, UC-MSCs released DOXO-loaded EVs. DOXO-loaded EVs were rapidly taken up by PANC-1 cells and induced apoptotic cell death more efficiently than free DOXO. In conclusion, the use of UC-MSC-derived EVs as a drug delivery system for siRNAs or drugs could be a promising approach for the targeted treatment of PDAC.info:eu-repo/semantics/publishe

A molecular score by quantitative PCR as a new prognostic tool at diagnosis for chronic lymphocytic leukemia patients.

Several markers have been proposed to predict the outcome of chronic lymphocytic leukemia (CLL) patients. However, discordances exist between the current prognostic factors, indicating that none of these factors are totally perfect.Evaluation StudiesJournal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

In Vivo Production of Mesenchymal Stromal Cells After Injection of Autologous Platelet-Rich Plasma Activated by Recombinant Human Soluble Tissue Factor in the Bone Marrow of Healthy Volunteers.

Autologous mesenchymal stromal cell (MSC)-based therapies offer one of the most promising and safe methods for regeneration or reconstruction of tissues and organs. Routine procedures to obtain adequate amount of autologous stem cells need their expansion through culture, with risks of contamination and cell differentiation, leading to the loss of cell ability for therapies. We suggest the use of human bone marrow (BM) as a physiological bioreactor to produce autologous MSC by injection of autologous platelet-rich plasma activated by recombinant human soluble tissue factor (rhsTF) in iliac crest. A trial on 13 healthy volunteers showed the feasibility and harmlessness of the procedure. The phenotype and cellularity of BM cells were not modified, on day 3 after injection. Endothelial progenitor cells (EPC) were mobilized to the bloodstream, without stimulation of hematopoietic stem cells (HSC). MSC level in BM increased with a specific commitment to preosteoblastic cell population both in vivo and in vitro. This self-stimulation system of BM seems thus to be a promising feasible process 3 days before clinical cell therapy applications.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe

Inflammation and Toll-like receptor ligation differentially affect the osteogenic potential of human mesenchymal stromal cells depending on their tissue origin.

Mesenchymal stromal cells (MSCs) can be isolated not only from bone marrow (BM) but also from other tissues, including adipose tissue (AT) and umbilical cord Wharton's Jelly (WJ). Thanks to their ability to differentiate into various cell types, MSC are considered attractive candidates for cell-based regenerative therapy. In degenerative clinical settings, inflammation or infection is often involved. In the present work, we hypothesized that an inflammatory environment and/or Toll-like receptor (TLR) ligation could affect the MSC differentiation potential. MSC were isolated from BM, AT, and WJ. Inflammation was mimicked by a cytokine cocktail, and TLR activation was induced through TLR3 and TLR4 ligation. Osteogenesis was chosen as a model for differentiation. Osteogenic parameters were evaluated by measuring Ca2+ deposits and alkaline phosphatase (ALP) activity at day 7, 14, and 21 of the culture in an osteogenic medium. Our results show that WJ-MSC exhibit a much lower osteogenic potential than the other two MSC types. However, inflammation was able to strongly increase the osteogenic differentiation of WJ-MSC as calcification, and ALP activity appeared as early as day 7. However, this latter enzymatic activity remained much lower than that disclosed by BM-MSC. TLR3 or TLR4 triggering increased the osteogenesis in AT- and, to lesser extent, in BM-MSC. In conclusion, WJ-MSC constitutively disclose a lower osteogenic potential as compared with BM and AT-MSC, which is not affected by TLR triggering but is strongly increased by inflammation, then reaching the level of BM-MSC. These observations suggest that WJ-MSC could constitute an alternative of BM-MSC for bone regenerative applications, as WJ is an easy access source of large amounts of MSC that can effectively differentiate into osteoblasts in an inflammatory setting.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Isolation and characterization of human mesenchymal stromal cells subpopulations: comparison of bone marrow and adipose tissue.

Preparations of Mesenchymal Stromal Cells (MSC) are generally obtained from unfractionated tissue cells resulting in heterogeneous cell mixtures. Several markers were proposed to enrich these cells but the majority of these markers are defined for Bone Marrow (BM). Moreover, the surface markers of freshly isolated MSC also differ from those of cultured MSC in addition of a phenotypic variation depending on the MSC source. For tissue engineering applications, it is crucial to start with a well-defined cell population. In this study, we performed immunomagnetic selections with 5 single surface markers to isolate MSC subpopulations from BM and Adipose Tissue (AT): CD271, SUSD2, MSCA-1, CD44 and CD34. We determined the phenotype, the clonogenicity, the proliferation, the differentiation capacity and the immunoregulatory profile of the sub-populations obtained in comparison with unselected cells. We showed that native BM-MSC can be enriched from the positive fractions of MSCA-1, SUSD2 and CD271 selections. In contrast, we observed that SUSD2 and MSCA-1 were unable to identify MSC from AT meaning they are not expressed in situ. Only the CD34+ selection successfully isolated MSC from AT. Interestingly, we observed that CD271 selection can define AT cell subsets with particular abilities but only in lipoaspiration samples, not in abdominoplasties samples. Importantly, we found a population of clearly CD34+ fresh BM-MSC displaying different properties. A single marker-based selection for MSC enrichment should be more advantageous for cell therapy and would enable the standardization of efficient and safe therapeutic intervention through the use of a well identified and homogeneous cell population.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Mesenchymal stromal cells from the foreskin: Tissue isolation, cell characterization and immunobiological properties.

Because of their self-renewal capacity, multilineage potential and immunomodulatory properties, MSCs are an attractive tool for cell-based immunotherapy strategies. Foreskin, considered as a biological waste material, has been shown to be a reservoir of therapeutic cells.SCOPUS: ar.jinfo:eu-repo/semantics/publishe