Francesco Alviano

Concise review: Isolation and characterization of cells from human term placenta: Outcome of the First International Workshop on Placenta Derived Stem Cells

Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy‐based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23–24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications.

Toward cell therapy using placenta-derived cells: disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table

Among the many cell types that may prove useful to regenerative medicine, mounting evidence suggests that human term placenta-derived cells will join the list of significant contributors. In making new cell therapy-based strategies a clinical reality, it is fundamental that no a priori claims are made regarding which cell source is preferable for a particular therapeutic application. Rather, ongoing comparisons of the potentiality and characteristics of cells from different sources should be made to promote constant improvement in cell therapies, and such comparisons will likely show that individually tailored cells can address disease-specific clinical needs. The principle underlying such an approach is resistance to the notion that comprehensive characterization of any cell type has been achieved, neither in terms of phenotype nor risks-to-benefits ratio. Tailoring cell therapy approaches to specific conditions also requires an understanding of basic disease mechanisms and close collaboration between translational researchers and clinicians, to identify current needs and shortcomings in existing treatments. To this end, the international workshop entitled Placenta-derived stem cells for treatment of inflammatory diseases: moving toward clinical application was held in Brescia, Italy, in March 2009, and aimed to harness an understanding of basic inflammatory mechanisms inherent in human diseases with updated findings regarding biological and therapeutic properties of human placenta-derived cells, with particular emphasis on their potential for treating inflammatory diseases. Finally, steps required to allow their future clinical application according to regulatory aspects including good manufacturing practice (GMP) were also considered. In September 2009, the International Placenta Stem Cell Society (IPLASS) was founded to help strengthen the research network in this field.

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 AIMSnThe 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.nnnMETHODSnResident 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.nnnRESULTSnNotch-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.nnnCONCLUSIONSnOverall, 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.

BACKGROUNDnIt 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.nnnMETHODSnWe 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.nnnRESULTSnThere 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.nnnDISCUSSIONnOverall 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 AIMSnBone 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.nnnMETHODSnIntestinal 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 Novel Stem Cell Tag-Less Sorting Method

Growing interest in stem cell research has led to the development of a number of new methods for isolation. The lack of homogeneity in stem cell preparation blurs standardization, which however is recommended for successful applications. Among stem cells, mesenchymal stem cells (MSCs) are promising candidates for cell therapy applications. This paper presents a fractionation protocol based on a tag-less, flow-assisted method of purifying, distinguishing and sorting MSCs. The protocol entails a suspension of cells in a transport fluid being injected into a ribbon-like capillary device by continuous flow. In a relatively short time (about 30xa0min) sorted cells are collected. The protocol has been applied to the improvement of MSC isolation, with a specific view to reducing cell manipulation operations, keeping instrumental simplicity and increasing analytical information for cell characterization. Applications such as MSC purification from epithelial contaminants, MSC characterization from various human sources and sorting of MSC subpopulations with high differentiation potential are described. The low cost, full biocompatibility and scale-up potential of the protocol presented could make the procedure attractive for stem cell selection.

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.

Thrombocytopenia with absent radii (TAR) syndrome: from hemopoietic progenitor to mesenchymal stromal cell disease?

Thrombocytopenia with absent radii (TAR) syndrome is a rare autosomal recessive disease characterized by hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. Its expression includes skeletal, hematologic, and cardiac system abnormalities. According to some authors, the association of disparate skeletal and hematologic abnormalities is related to simultaneous development of the heart, radii, and megakaryocytes at 6 to 8 weeks gestation. Thrombocytopenia that generally presents at birth or during the neonatal period can also occur subsequently. Data as to the physiopathology of TAR syndrome are scanty because of the low frequency of the disease and frequent unavailability of samples for bone marrow. The few studies on colony formation suggest that thrombocytopenia could be due to a decreased response to thrombopoietin that affects both proliferation and differentiation. The genetic basis of this syndrome remains unclear because c-mpl gene mutations are not a likely cause of thrombocytopenia and they are also frequent in the normal population. This is also the case for the mutations to the multifunctional growth factor transforming growth factor (TGF)-beta2 gene as described in our laboratory. Finally, the deletion on chromosome 1q21.1 described by Klopocki and colleagues is not considered sufficient to determine the TAR syndrome phenotype. We have reported that bone marrow adherent stromal cells from patients with TAR syndrome do not express CD105 antigen (expressed in normal mesenchymal cells), part of the receptor complex for TGF-beta1 and TGF-beta3. Thus, the hypothesis that the clinical phenotype of TAR could derive from damage to a common osteo/chondrogenic and hemopoietic progenitor warrants further study.