Lipidomics study of mesenchymal stromal cells derived from human placenta

The interest for lipid metabolism in the stem cell field has increased in the last few years (1,2,3). Membrane lipidomics embraces many aspects of cell metabolism and the role of lipids is now considered more than merely inert and structural in delimitating the extra- and intra-cellular compartments (4,5). Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. The aim of our work was to study membrane lipidomics of mesenchymal stromal cells derived from human placenta and correlate it to specific biological properties, by using chemically- defined tailored lipid supplements (Refeed®). In the experimental study, the cell membranes of freshly isolated mesenchymal stromal cells obtained from human fetal membranes (FM-MSCs) were characterized for fatty acid composition. Then, we investigated cell morphology, viability, proliferation, differentiation and immunomodulation after in-vitro exposure to Refeed® supplements. Control MSCs were cultured without lipid supplementation. Our results showed a significant reduction of membrane fluidity for in-vitro primary cells, with cell membrane fatty acid composition greatly differing from the in-vivo one. By tailoring lipid supplementation, the fatty acid composition and biophysical properties of in-vitro cell membranes resulted more similar to the in-vivo counterparts, with higher omega-6 fatty acid content and increased membrane fluidity. These modifications of membrane composition and properties had no effect on cell morphology and viability, whereas ameliorated cell proliferation rate, diffentiation ability and immunomodulatory properties. In particular, supplemented FMMSCs showed an increased expression of cell membrane molecules like Vascular Endothelial Growth Factor Receptors 1 (VEGFR-1 or Flt-1) and 2 (VEGFR-2 or KDR), that correlated with a more efficient response to angiogenic commitment. Moreover, regarding immunomodulation, supplemented FM-MSCs displayed an increased expression of the tolerogenic cell surface protein HLA-G, that positively influenced the in-vitro cell immunomodulatory ability. Finally, these data suggest that specific lipid supplementation have functional consequences on in-vitro MSC behavior and may influence cell-based therapeutic approaches

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

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

BACKGROUND: Term 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). RESULTS: The 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. CONCLUSION: The 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

Recent Patents and Advances on Tag-Less Microfluidic Stem Cell Sorting Methods: Applications for Perinatal Stem Cell Isolation

none9Interest in stem cell separation and purification from easily accessible clinical specimens is booming due to the increase of cell therapy applications. The recovery of pluripotent or multipotent stem cells in human sources different from the embryo requires the use of effective methods of cell sorting/enrichment. Among these sources, perinatal tissues retain cells with pivotal stem cell features such as high self-renewal ability, wide differentiation potential and immunomodulatory properties. In this perspective, methods exploiting cell biophysical differences in a less dependent process of identification based on specific markers are therefore promising. These methods allow cell isolation irrespective of the broad and diversified surface antigenic panel that usually limits the ability to easily distinguish cells as in the case of mesenchymal stromal/stem cell separation. In addition, the use of non- or minimally invasive tag-less techniques might be a way to preserve stem cell features of the selected product and reduce regulatory issues related to their use in regenerative applications. In this review, non-invasive cell sorting techniques based on microfluidic systems and relevant patents are described. In particular applications of emerging separation approach, Field-Flow Fractionation (FFF), for perinatal stem cell sorting are cited. Protocols and applications based on FFF-derived techniques are detailedmixedFrancesco Alviano;Barbara Roda;Martina Rossi;Maya Tanase;Kristel Martinelli;Cosetta Marchionni;Andrea Zattoni;Pierluigi Reschiglian;Laura BonsiFrancesco Alviano;Barbara Roda;Martina Rossi;Maya Tanase;Kristel Martinelli;Cosetta Marchionni;Andrea Zattoni;Pierluigi Reschiglian;Laura Bons

Restored in vivo-like membrane lipidomics positively influence in vitro features of cultured mesenchymal stromal/stem cells derived from human placenta

Background: The study of lipid metabolism in stem cell physiology has recently raised great interest. The role of lipids goes beyond the mere structural involvement in assembling extra- and intra-cellular compartments. Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. In the last years, it has been reported how in vitro cell culturing can modify membrane lipidomics. The aim of the present work was to study the membrane fatty acid profile of mesenchymal stromal cells (MSCs) derived from human fetal membranes (hFM-MSCs) and to correlate this to specific biological properties by using chemically defined tailored lipid supplements (Refeed\uc2\uae). Methods: Freshly isolated hFM-MSCs were characterized for their membrane fatty acid composition. hFM-MSCs were cultivated in vitro following a classical protocol and their membrane fatty acid profile at different passages was compared to the profile in vivo. A tailored Refeed\uc2\uae lipid supplement was developed with the aim of reducing the differences created by the in vitro cultivation and was tested on cultured hFM-MSCs. Cell morphology, viability, proliferation, angiogenic differentiation, and immunomodulatory properties after in vitro exposure to the tailored Refeed\uc2\uae lipid supplement were investigated. Results: A significant modification of hFM-MSC membrane fatty acid composition occurred during in vitro culture. Using a tailored lipid supplement, the fatty acid composition of cultured cells remained more similar to their in vivo counterparts, being characterized by a higher polyunsaturated and omega-6 fatty acid content. These changes in membrane composition had no effect on cell morphology and viability, but were linked with increased cell proliferation rate, angiogenic differentiation, and immunomodulatory properties. In particular, Refeed\uc2\uae-supplemented hFM-MSCs showed greater ability to express fully functional cell membrane molecules. Conclusions: Culturing hFM-MSCs alters their fatty acid composition. A tailored lipid supplement is able to improve in vitro hFM-MSC functional properties by recreating a membrane environment more similar to the physiological counterpart. This approach should be considered in cell therapy applications in order to maintain a higher cell quality during in vitro passaging and to influence the outcome of cell-based therapeutic approaches when cells are administered to patients

Characterization of Perinatal Stem Cell Spheroids for the Development of Cell Therapy Strategy

Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM

Characterization of Perinatal Stem Cell Spheroids for the Development of Cell Therapy Strategy

Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM

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