Giacomo Lanzoni

Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells with the ability to differentiate into endothelial cells in vitro

BackgroundTerm Amniotic membrane (AM) is a very attractive source of Mesenchymal Stem Cells (MSCs) due to the fact that this fetal tissue is usually discarded without ethical conflicts, leading to high efficiency in MSC recovery with no intrusive procedures. Here we confirmed that term AM, as previously reported in the literature, is an abundant source of hMSCs; in particular we further investigated the AM differentiation potential by assessing whether these cells may also be committed to the angiogenic fate. In agreement with the recommendation of the International Society for Cellular Therapy, the mesenchymal cells herein investigated were named Amniotic Membrane-human Mesenchymal Stromal Cells (AM-hMSC).ResultsThe recovery of hMSCs and their in vitro expansion potential were greater in amniotic membrane than in bone marrow stroma. At flow cytometry analysis AM-hMSCs showed an immunophenotypical profile, i.e., positive for CD105, CD73, CD29, CD44, CD166 and negative for CD14, CD34, CD45, consistent with that reported for bone marrow-derived MSCs. In addition, amniotic membrane-isolated cells underwent in vitro osteogenic (von Kossa stain), adipogenic (Oil Red-O stain), chondrogenic (collagen type II immunohistochemichal detection) and myogenic (RT-PCR MyoD and Myogenin expression as well as desmin immunohistochemical detection) differentiation. In angiogenic experiments, a spontaneous differentiation into endothelial cells was detected by in vitro matrigel assay and this behaviour has been enhanced through Vascular Endothelial Growth Factor (VEGF) induction. According to these findings, VEGF receptor 1 and 2 (FLT-1 and KDR) were basally expressed in AM-hMSCs and the expression of endothelial-specific markers like FLT-1 KDR, ICAM-1 increased after exposure to VEGF together with the occurrence of CD34 and von Willebrand Factor positive cells.ConclusionThe current study suggests that AM-hMSCs may emerge as a remarkable tool for the cell therapy of multiple diseased tissues. AM-hMSCs may potentially assist both bone and cartilage repair, nevertheless, due to their angiogenic potential, they may also pave the way for novel approaches in the development of tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.

Angiogenic Potential of Human Dental Pulp Stromal (STEM) Cells

Dental pulp is a heterogeneous microenviroment where unipotent progenitor and pluripotent mesenchymal stem cells cohabit. In this study we investigated whether human Dental Pulp Stromal (Stem) Cells (DP-SCs) committed to the angiogenic fate. DP-SCs showed the specific mesenchymal immunophenotypical profile positive for CD29, CD44, CD73, CD105, CD166 and negative for CD14, CD34, CD45, in accordance with that reported for bone marrow-derived SCs. The Oct-4 expression in DP-SCs, evaluated through RT-PCR analysis, increased in relation with the number of the passages in cell culture and decreased after angiogenic induction. In agreement with their multipotency, DP-SCs differentiated toward osteogenic and adipogenic commitments. In angiogenic experiments, differentiation of DP-SCs, through Vascular Endothelial Growth Factor (VEGF) induction, was evaluated by in vitro matrigel assay and by cytometric analysis. Accordingly, endothelial-specific markers like Flt-1 and KDR were basally expressed and they increased after exposure to VEGF together with the occurrence of ICAM-1 and von Willebrand Factor positive cells. In addition, VEGF-induced DP-SCs maintained endothelial cell-like features when cultured in a 3-D fibrin mesh, displaying focal organization into capillary-like structures. The DP-SC angiogenic potential may prove a remarkable tool for novel approaches to developing tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.

A decreased positivity for CD90 on human mesenchymal stromal cells (MSCs) is associated with a loss of immunosuppressive activity by MSCs

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.

Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics.

BACKGROUND AIMS The presence of ectopic tissues in the pathologic artery wall raises the issue of whether multipotent stem cells may reside in the vasculature itself. Recently mesenchymal stromal cells (MSC) have been isolated from different human vascular segments (VW MSC), belying the previous view that the vessel wall is a relatively quiescent tissue. METHODS Resident multipotent cells were recovered from fresh arterial segments (aortic arches, thoracic and femoral arteries) collected in a tissue-banking facility and used to establish an in situ and in vitro study of the stemness features and multipotency of these multidistrict MSC populations. RESULTS Notch-1+, Stro-1+, Sca-1+ and Oct-4+ cells were distributed along an arterial wall vasculogenic niche. Multidistrict VW MSC homogeneously expressed markers of stemness (Stro-1, Notch-1 and Oct-4) and MSC lineages (CD44, CD90, CD105, CD73, CD29 and CD166) whilst they were negative for hematopoietic and endothelial markers (CD34, CD45, CD31 and vWF). Each VW MSC population had characteristics of stem cells, i.e. a high efflux capability for Hoechst 33342 dye and the ability to form spheroids when grown in suspension and generate colonies when seeded at low density. Again, VW MSC cultured in induction media exhibited adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic differentiation, as documented by histochemical, immunohistochemical, molecular and electron microscopy analysis. CONCLUSIONS Overall, these findings may enlighten the physiopathologic mechanisms of vascular wall diseases as well as having potential implications for cellular, genetic and tissue engineering approaches to treating vascular pathologies when these are unresponsive to medical and surgical therapies.

A functional role for soluble HLA-G antigens in immune modulation mediated by mesenchymal stromal cells.

BACKGROUND It has been suggested that soluble factors produced by bone marrow (BM) mesenchymal stromal cells (MSC) play a fundamental role in mediating immune modulation. HLA-G antigens (Ag) are major histocompatibility complex (MHC) class Ib molecules characterized by a limited polymorphism and a splicing mechanism that regulates the production of membrane-bound and soluble isoforms. Interleukin-10 (IL-10) cytokine is one of the main up-modulators of soluble HLA-G Ag (sHLA-G) production by CD14+ peripheral blood monocyte cells and increased IL-10 levels are reported to be associated with MSC immune modulation. METHODS We investigated, by specific enzyme-linked immunosorbent assay (ELISA), the possible role of sHLA-G molecules in the inhibition of the peripheral blood mononuclear cell (PBMC) response to phytohemagglutinin (PHA) mediated by MSC from different sources. RESULTS There was a significant correlation between the presence of increased levels of sHLA-G and IL-10 in the MSC/PBMC/PHA culture supernatants and lymphoproliferative inhibition. Neutralizing experiments performed with monoclonal Ab directed against HLA-G and IL-10 molecules confirmed the inhibitory ability of sHLA-G Ag. Furthermore, exogenous IL-10 induced sHLA-G molecule secretion by MSC alone in a polymorphic way, while a longitudinal analysis confirmed the loss of MSC inhibitory functions in relation to in vitro MSC aging. DISCUSSION Overall the results obtained suggest a functional role for sHLA-G molecules in inhibiting the PBMC response mediated by MSC. Moreover, the ability of IL-10 to induce sHLA-G Ag production by MSC alone could be proposed as a marker of MSC functional ability.

Isolation of stem cell populations with trophic and immunoregulatory functions from human intestinal tissues: potential for cell therapy in inflammatory bowel disease

BACKGROUND AIMS 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. METHODS 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.

A tag-less method of sorting stem cells from clinical specimens and separating mesenchymal from epithelial progenitor cells†

The interest in stem cell (SC) isolation from easily accessible clinical specimens is booming. The lack of homogeneity in pluri/multipotent SC preparation blurs standardization, which however is recommended for successful applications. Multipotent mesenchymal SCs (MSCs) in fact express a broad panel of surface antigens, which limit the possibility of sorting homogeneous preparations by using an immunotag‐based method.

A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential

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-(3)H)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.

Gravitational field-flow fractionation of human hemopoietic stem cells.

New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.

Susceptibility of human placenta derived mesenchymal stromal/stem cells to human herpesviruses infection.

Fetal membranes (FM) derived mesenchymal stromal/stem cells (MSCs) are higher in number, expansion and differentiation abilities compared with those obtained from adult tissues, including bone marrow. Upon systemic administration, ex vivo expanded FM-MSCs preferentially home to damaged tissues promoting regenerative processes through their unique biological properties. These characteristics together with their immune-privileged nature and immune suppressive activity, a low infection rate and young age of placenta compared to other sources of SCs make FM-MSCs an attractive target for cell-based therapy and a valuable tool in regenerative medicine, currently being evaluated in clinical trials. In the present study we investigated the permissivity of FM-MSCs to all members of the human Herpesviridae family, an issue which is relevant to their purification, propagation, conservation and therapeutic use, as well as to their potential role in the vertical transmission of viral agents to the fetus and to their potential viral vector-mediated genetic modification. We present here evidence that FM-MSCs are fully permissive to infection with Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Varicella zoster virus (VZV), and Human Cytomegalovirus (HCMV), but not with Epstein-Barr virus (EBV), Human Herpesvirus-6, 7 and 8 (HHV-6, 7, 8) although these viruses are capable of entering FM-MSCs and transient, limited viral gene expression occurs. Our findings therefore strongly suggest that FM-MSCs should be screened for the presence of herpesviruses before xenotransplantation. In addition, they suggest that herpesviruses may be indicated as viral vectors for gene expression in MSCs both in gene therapy applications and in the selective induction of differentiation.