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Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSC) are currently under evaluation in phase III clinical trials for inflammatory bowel disease and other intestinal disease manifestations. The therapeutic efficacy of these treatments may derive from a combination of the differentiation, trophic and immunomodulatory abilities of the transplanted cells. We investigated intestinal tissues as sources of MSC: such cells may support tissue-specific functions and hold advantages for engraftment and contribution in the gastrointestinal environment.Intestinal specimens were collected, and the mucosa and submucosa mechanically separated and enzymatically digested. Mesenchymal stromal populations were isolated, expanded and characterized under conditions commonly used for MSC. The differentiation potential, trophic effect and immunomodulatory ability were investigated. Results We successfully isolated and extensively expanded populations showing the typical MSC profile: CD29+, CD44+, CD73+, CD105+ and CD166+, and CD14(-), CD34(-) and CD45(-). Intestinal mucosal (IM) MSC were also CD117+, while submucosal cultures (ISM MSC) showed CD34+ subsets. The cells differentiated toward osteogenic, adipogenic and angiogenic commitments. Intestinal-derived MSC were able to induce differentiation and organization of intestinal epithelial cells (Caco-2) in three-dimensional collagen cultures. Immunomodulatory activity was evidenced in co-cultures with normal heterologous phytohemagglutinin-stimulated peripheral blood mononuclear cells. Conclusions Multipotent MSC can be isolated from intestinal mucosal and submucosal tissues. IM MSC and ISM MSC are able to perform trophic and immunomodulatory functions. These findings could open a pathway for novel approaches to intestinal disease treatment.

Human umbilical cord blood units (UCBs) are an alternative source in allogeneic-stem-cell transplantation. Human leukocyte antigen (HLA)-G is a tolerogenic molecule with a possible implication in UCB immunoregulatory effect. HLA-G expression was observed in UCB myeloid and plasmacytoid dendritic cells; in contrast, CD34(+) cells did not produce this molecule. CD34(+) cells are primitive hematopoietic progenitor cells that are present in UCB and are necessary for long-term engraftment via production of immunoregulatory molecules and a hematopoietic progeny that supports cellular recovery. The role of these cells in UCB transplantation needs further evaluation of HLA-G expression in CD34(+) cells and their hematopoietic progeny. We confirmed the absence of HLA-G expression in CD34(+) cells, whereas CD34(+)-derived progeny secreted HLA-G molecules and expressed HLA-G mRNA in in vitro cultures. Furthermore, soluble HLA (sHLA)-G molecules purified from the culture supernatants of CD34(+)-derived progeny were able to suppress lymphoproliferative response in an HLA-G dose-dependent manner. Overall these results identify CD34(+)-derived hematopoietic progeny as producers of HLA-G molecules and support a role of this antigen as an immuno-modulatory factor in UCB.

Background aims. The beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC). Methods. Thirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-3H) thymidine. Results. The results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502). Conclusions. We propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.

Biologic and clinical interest in human mesenchymal stromal cells (hMSC) has risen over the last years, mainly due to their immunosuppressive properties. In this study, we investigated the basis of immunomodulant possible variability using hMSC from different sources (amniotic membrane, chorion, and bone marrow from either healthy subjects or patients with hematological malignancies, HM) and having discordant positivity for several immunological markers. The CD90+ hMSC reduced lymphoproliferative response in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) via sHLA-G and IL-10 up-modulation. On the contrary, hMSC showing a significantly lower expression for CD90 antigen, elicited a lymphoproliferative allogeneic response in PHA/PBMCs without any increase in soluble HLA-G and IL-10 levels. These data seems to suggest that CD90 molecule may be considered a novel predictive marker for hMSC inhibitory ability, and might cooperate with HLA-G molecule in regulating suppressive versus stimulatory properties of hMSC. These results may have clinical implication in either transplantation or in regenerative medicine fields.

In a limited study, comprising only ten patients, we have previously reported that allogeneic irradiated RCC-cell-line cells, engineered to produce IL-2 (ACHN-IL-2), admixed with autologous metastatic formalin-treated tumour cells were used to vaccinate MRCC patients in progression of disease and also receiving IL-2 immunotherapy. The cells, admixed to autologous TC, were administered subcutaneously. We now report an extended study on thirty patients and one hundred thirty-one controls. Patients received 4-20 injections (mean 10 +/- 4), containing an average of 92 x 10(6) +/- 45 x 10(6) ACHN-IL-2 transfected cells (a minimum of 25 x 10(6), and a maximum of 200 x 10(6)). Autologous TC, admixed to allogeneic, were also administered by 4-16 s.c. injections (mean 7 +/- 3), i.e. a total of 12 x 10(6)-160 x 10(6) cells. Vaccination was administered during 73-1451 (307 +/- 316) days, and the follow-up continued for 1122 +/- 1240 days (106-5137). Throughout this period, the patients continued receiving the previously set immunotherapy treatment. No adverse side effects related to the treatment were noticed. One complete and four partial tumour responses were observed, as well as nine cases of stable disease. Thirteen patients died in the treated group (43%) and 63 (44%) in the control group. Responding patients resumed progression in 4-11 months and died 18 and 36 months after beginning the vaccine therapy. The Gehan Wilcoxon's test showed a significantly (P0.01) better survival in the vaccinated patients compared to that of the controls. Thus, we confirm, in an increased number of patients and an extensive follow-up, that our vaccination protocol is safe, devoid of adverse side effects, and promising.