Plumbagin, a Natural Compound with Several Biological Effects and Anti-Inflammatory Properties.

Phytochemicals from various medicinal plants are well known for their antioxidant properties and anti-cancer effects. Many of these bioactive compounds or natural products have demonstrated effects against inflammation, while some showed a role that is only approximately described as anti-inflammatory. In particular, naphthoquinones are naturally-occurring compounds with different pharmacological activities and allow easy scaffold modification for drug design approaches. Among this class of compounds, Plumbagin, a plant-derived product, has shown interesting counteracting effects in many inflammation models. However, scientific knowledge about the beneficial effect of Plumbagin should be comprehensively reported before candidating this natural molecule into a future drug against specific human diseases. In this review, the most relevant mechanisms in which Plumbagin plays a role in the process of inflammation were summarized. Other relevant bioactive effects were reviewed to provide a complete and compact scenario of Plumbagin's potential therapeutic significance.

Non-Permissive Parvovirus B19 Infection: A Reservoir and Questionable Safety Concern in Mesenchymal Stem Cells.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells with differentiation, immunoregulatory and regenerative properties. Because of these features, they represent an attractive tool for regenerative medicine and cell-based therapy. However, MSCs may act as a reservoir of persistent viruses increasing the risk of failure of MSCs-based therapies and of viral transmission, especially in immunocompromised patients. Parvovirus B19V (B19V) is a common human pathogen that infects bone marrow erythroid progenitor cells, leading to transient or persistent anemia. Characteristics of B19V include the ability to cross the placenta, infecting the fetus, and to persist in several tissues. We thus isolated MSCs from bone marrow (BM-MSCs) and fetal membrane (FM-MSCs) to investigate their permissiveness to B19V infection. The results suggest that both BM- and FM- MSCs can be infected by B19V and, while not able to support viral replication, allow persistence over time in the infected cultures. Future studies are needed to understand the potential role of MSCs in B19V transmission and the conditions that can favor a potential reactivation of the virus.

The Medium Obtained from the Culture of Hodgkin Lymphoma Cells Affects the Biophysical Characteristics of a Fibroblast Cell Model.

The neoplastic Hodgkin-Reed-Sternberg (HRS) cells in Hodgkin lymphoma (HL) represent only 1-10% of cells and are surrounded by an inflammatory microenvironment. The HL cytokine network is a key point for the proliferation of HRS cells and for the maintenance of an advantageous microenvironment for HRS survival. In the tumor microenvironment (TME), the fibroblasts are involved in crosstalk with HRS cells. The aim of this work was to study the effect of lymphoma cell conditioned medium on a fibroblast cell population and evaluate modifications of cell morphology and proliferation. Hodgkin lymphoma-derived medium was used to obtain a population of "conditioned" fibroblasts (WS-1 COND). Differences in biophysical parameters were detected by the innovative device Celector®. Fibroblast-HL cells interactions were reproduced in 3D co-culture spheroids. WS-1 COND showed a different cellular morphology with an enlarged cytoplasm and enhanced metabolism. Area and diameter cell values obtained by Celector® measurement were increased. Co-culture spheroids created with WS-1 COND showed a tighter aggregation than those with non-conditioned WS-1. The presence of soluble factors derived from HRS cells in the conditioned medium was adequate for the proliferation of fibroblasts and conditioned fibroblasts in a 3D HL model allowed to develop a representative model of the in vivo TME.

Perinatal Stem Cell Therapy to Treat Type 1 Diabetes Mellitus: A Never-Say-Die Story of Differentiation and Immunomodulation.

Human term placenta and other postpartum-derived biological tissues are promising sources of perinatal cells with unique stem cell properties. Among the massive current research on stem cells, one medical focus on easily available stem cells is to exploit them in the design of immunotherapy protocols, in particular for the treatment of chronic non-curable human diseases. Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells and perinatal cells can be harnessed both to generate insulin-producing cells for beta cell replenishment and to regulate autoimmune mechanisms via immunomodulation capacity. In this study, the strong points of cells derived from amniotic epithelial cells and from umbilical cord matrix are outlined and their potential for supporting cell therapy development. From a basic research and expert stem cell point of view, the aim of this review is to summarize information regarding the regenerative medicine field, as well as describe the state of the art on possible cell therapy approaches for diabetes.

Effective Label-Free Sorting of Multipotent Mesenchymal Stem Cells from Clinical Bone Marrow Samples.

Mesenchymal stem cells (MSC) make up less than 1% of the bone marrow (BM). Several methods are used for their isolation such as gradient separation or centrifugation, but these methodologies are not direct and, thus, plastic adherence outgrowth or magnetic/fluorescent-activated sorting is required. To overcome this limitation, we investigated the use of a new separative technology to isolate MSCs from BM; it label-free separates cells based solely on their physical characteristics, preserving their native physical properties, and allows real-time visualization of cells. BM obtained from patients operated for osteochondral defects was directly concentrated in the operatory room and then analyzed using the new technology. Based on cell live-imaging and the sample profile, it was possible to highlight three fractions (F1, F2, F3), and the collected cells were evaluated in terms of their morphology, phenotype, CFU-F, and differentiation potential. Multipotent MSCs were found in F1: higher CFU-F activity and differentiation potential towards mesenchymal lineages compared to the other fractions. In addition, the technology depletes dead cells, removing unwanted red blood cells and non-progenitor stromal cells from the biological sample. This new technology provides an effective method to separate MSCs from fresh BM, maintaining their native characteristics and avoiding cell manipulation. This allows selective cell identification with a potential impact on regenerative medicine approaches in the orthopedic field and clinical applications.

Unravelling Heterogeneity of Amplified Human Amniotic Fluid Stem Cells Sub-Populations.

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector® (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector® profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

A Reliable Flow-Based Method for the Accurate Measure of Mass Density, Size and Weight of Live 3D Tumor Spheroids.

Gathering precise information on mass density, size and weight of cells or cell aggregates, is crucial for applications in many biomedical fields with a specific focus on cancer research. Although few technical solutions have been presented for single-cell analysis, literature does not cover this aspect for 3D models such as spheroids. Since the research interest on such samples is notably rising, here we describe a flow-apparatus, and the associated physical method and operative protocol for the accurate measurements of mass density, size and weight. The technique is based on the detection of the terminal velocity of a free-falling sample into a specifically conceived analysis flow-channel. Moreover, in order to demonstrate the accuracy and precision of the presented flow-device, analyses were initially carried out on standardized polystyrene beads. Finally, to display the application of the proposed system for biological samples, mass density, size and weight of live SW620 tumor spheroids were analyzed. The combined measurements of such parameters can represent a step toward a deeper understanding of 3D culture models.

Characterization of the Tissue and Stromal Cell Components of Micro-Superficial Enhanced Fluid Fat Injection (Micro-SEFFI) for Facial Aging Treatment.

BACKGROUND: New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for regenerative purposes, the cellular content of the sample should be considered. OBJECTIVES: To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples. METHODS: Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector,® an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays. RESULTS: Microscopic and quality analyses of micro-SEFFI samples by Celector® confirmed the high fluidity and sample cellular composition in terms of red blood cell contamination, the presence of cell aggregates, and extracellular matrix fragments. ASCs were isolated from adipose tissue harvested using SEFFI and micro-SEFFI systems. These cells were demonstrated to have a good proliferation rate and differentiation potential towards mesenchymal lineages. CONCLUSIONS: Despite the small sizes and low cellularity observed in micro-SEFFI-derived tissue, we were able to isolate stem cells. This result partially explains the regenerative potential of autologous micro-SEFFI tissue grafts. In addition, using this novel Celector® technology, tissues used for aging treatment were characterized analytically, and the adipose tissue composition was evaluated with no need for extra sample processing.

Early Developmental Zebrafish Embryo Extract to Modulate Senescence in Multisource Human Mesenchymal Stem Cells.

Stem cells undergo senescence both in vivo, contributing to the progressive decline in self-healing mechanisms, and in vitro during prolonged expansion. Here, we show that an early developmental zebrafish embryo extract (ZF1) could act as a modulator of senescence in human mesenchymal stem cells (hMSCs) isolated from both adult tissues, including adipose tissue (hASCs), bone marrow (hBM-MSCs), dental pulp (hDP-MSCs), and a perinatal tissue such as the Wharton's Jelly (hWJ-MSCs). In all the investigated hMSCs, ZF1 decreased senescence-associated ß-galactosidase (SA ß-gal) activity and enhanced the transcription of TERT, encoding the catalytic telomerase core. In addition, it was associated, only in hASCs, with a transcriptional induction of BMI1, a pleiotropic repressor of senescence. In hBM-MSCs, hDP-MSCs, and hWJ-MSCs, TERT over-expression was concomitant with a down-regulation of two repressors of TERT, TP53 (p53), and CDKN1A (p21). Furthermore, ZF1 increased the natural ability of hASCs to perform adipogenesis. These results indicate the chance of using ZF1 to modulate stem cell senescence in a source-related manner, to be potentially used as a tool to affect stem cell senescence in vitro. In addition, its anti-senescence action could also set the basis for future in vivo approaches promoting tissue rejuvenation bypassing stem cell transplantation.

Exploring the roles of MSCs in infections: focus on bacterial diseases.

Despite human healthcare advances, some microorganisms continuously react evolving new survival strategies, choosing between a commensal fitness and a pathogenic attitude. Many opportunistic microbes are becoming an increasing cause of clinically evident infections while several renowned infectious diseases sustain a considerable number of deaths. Besides the primary and extensively investigated role of immune cells, other cell types are involved in the microbe-host interaction during infection. Interestingly, mesenchymal stem cells (MSCs), the current leading players in cell therapy approaches, have been suggested to contribute to tackling pathogens and modulating the host immune response. In this context, this review critically explores MSCs' role in E. coli, S. aureus, and polymicrobial infections. Summarizing from various studies, in vitro and in vivo results support the mechanistic involvement of MSCs and their derivatives in fighting infection and in contributing to microbial spreading. Our work outlines the double face of MSCs during infection, disease, and sepsis, highlighting potential pitfalls in MSC-based therapy due to the MSCs' susceptibility to pathogens' weapons. We also identify potential targets to improve infection treatments, and propose the potential applications of MSCs for vaccine research.

Restoring In Vivo-Like Membrane Lipidomics Promotes Exosome Trophic Behavior from Human Placental Mesenchymal Stromal/Stem Cells.

Human mesenchymal stem cells (hMSCs) are an effective tool in regenerative medicine notably for their intrinsic plentiful paracrine activity rather than differentiating properties. The hMSC secretome includes a wide spectrum of regulatory and trophic factors, encompassing several naked molecules as well as different kinds of extracellular vesicles (EVs). Among EVs, exosomes represent an intriguing population, able to shuttle proteins, transcription factors, and genetic materials, with a relevant role in cell-to-cell communication, modulating biological responses in recipient cells. In this context, the extracellular milieu can greatly impact the paracrine activity of stem cells, modifying their metabolism, and the dynamics of vesicle secretion. In the present study, we investigated the effects elicited on exosome patterning by tailored, ad hoc formulated lipid supplementation (Refeed®) in MSCs derived from human fetal membranes (hFM-MSCs). Wound healing experiments revealed that stem cell exposure to exosomes obtained from Refeed®-supplemented hFM-MSCs increased their migratory capability, although the amount of exosomes released after Refeed® supplementation was lower than that yielded from non-supplemented cells. We found that such a decrease was mainly due to a different rate of exosomal exocytosis rather than to an effect of the lipid supplement on the endocytic pathway. Endoplasmic reticulum homeostasis was modified by supplementation, through the upregulation of PKR-like ER kinase (PERK) and inositol-requiring enzyme 1α (IRE1α). Increased expression of these proteins did not lead to stress-induced, unfolded protein response (UPR)-mediated apoptosis, nor did it affect phosphorylation of p38 kinase, suggesting that PERK and IRE1α overexpression was due to augmented metabolic activities mediated by optimization of a cellular feeding network afforded through lipid supplementation. In summary, these results demonstrate how tailored lipid supplementation can successfully modify the paracrine features in hFM-MSCs, impacting both intracellular vesicle trafficking and secreted exosome number and function.

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®). 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® 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® 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®-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.

Systemic delivery of HER2-retargeted oncolytic-HSV by mesenchymal stromal cells protects from lung and brain metastases.

Fully retargeted oncolytic herpes simplex viruses (o-HSVs) gain cancer-specificity from redirection of tropism to cancer-specific receptors, and are non-attenuated. To overcome the hurdles of systemic delivery, and enable oncolytic viruses (o-viruses) to reach metastatic sites, carrier cells are being exploited. Mesenchymal stromal cells (MSCs) were never tested as carriers of retargeted o-viruses, given their scarse-null expression of the cancer-specific receptors. We report that MSCs from different sources can be forcedly infected with a HER2-retargeted oncolytic HSV. Progeny virus spread from MSCs to cancer cells in vitro and in vivo. We evaluated the organ distribution and therapeutic efficacy in two murine models of metastatic cancers, following a single i.v. injection of infected MSCs. As expected, the highest concentration of carrier-cells and of viral genomes was in the lungs. Viral genomes persisted throughout the body for at least two days. The growth of ovarian cancer lung metastases in nude mice was strongly inhibited, and the majority of treated mice appeared metastasis-free. The treatment significantly inhibited also breast cancer metastases to the brain in NSG mice, and reduced by more than one-half the metastatic burden in the brain.

In vitro differentiation of human amniotic epithelial cells into insulin-producing 3D spheroids.

Regenerative medicine and stem cell therapy may represent the solution for the treatment of non-curable human diseases such as type 1 diabetes. In this context of growing demand for functional and safe stem cells, human amniotic epithelial cells (hAECs) from term placenta have attracted increasing interest for their wide availability, stem cell properties, and differentiation plasticity, which make them a promising tool for stem cell-based therapeutic applications. We initially assayed the stemness characteristics of hAECs in serum-free conditions. Subsequently we developed a culture procedure on extracellular matrix for the formation of three-dimensional (3D) spheroids. Finally, we tested the immunomodulation and differentiation potential of hAEC spheroids: the presence of pancreatic endocrine hormones was revealed with transmission electron microscopy and immunofluorescence analyses; the release of C-peptide in hyperglycemic conditions was assayed with ELISA. The serum-free culture conditions we applied proved to maintain the basic stemness characteristics of hAECs. We also demonstrated that 3D spheroids formed by hAECs in extracellular matrix can be induced to differentiate into insulin-producing cells. Finally, we proved that control and induced cells equally inhibit the proliferation of activated mononuclear cells. The results of this study highlight the properties of amnion derived epithelial cells as promising and abundant source for cell-based therapies. In particular we are the first group to show the in vitro pancreatic induction of hAECs cultured on extracellular matrix in a 3D fashion. We accordingly propose the outcomes of this study as a novel contribution to the development of future cell replacement therapies involving placenta-derived cells.

Neutrophil gelatinase-associated lipocalin increases HLA-G(+)/FoxP3(+) T-regulatory cell population in an in vitro model of PBMC.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is emerging as a mediator of various biological and pathological states. However, the specific biological role of this molecule remains unclear, as it serves as a biomarker for many conditions. The high sensitivity of NGAL as a biomarker coupled with relatively low specificity may hide important biological roles. Data point toward an acute compensatory, protective role for NGAL in response to adverse cellular stresses, including inflammatory and oxidative stress. The aim of this study was to understand whether NGAL modulates the T-cell response through regulation of the human leukocyte antigen G (HLA-G) complex, which is a mediator of tolerance. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood mononuclear cells (PBMCs) were obtained from eight healthy donors and isolated by centrifugation on a Ficoll gradient. All donors gave informed consent. PBMCs were treated with four different concentrations of NGAL (40-320 ng/ml) in an iron-loaded or iron-free form. Changes in cell phenotype were analyzed by flow cytometry. NGAL stimulated expression of HLA-G on CD4+ T cells in a dose- and iron-dependent manner. Iron deficiency prevented NGAL-mediated effects, such that HLA-G expression was unaltered. Furthermore, NGAL treatment affected stimulation of regulatory T cells and in vitro expansion of CD4(+) CD25(+) FoxP3(+) cells. An NGAL neutralizing antibody limited HLA-G expression and significantly decreased the percentage of CD4(+) CD25(+) FoxP3(+) cells. CONCLUSIONS/SIGNIFICANCE: We provide in vitro evidence that NGAL is involved in cellular immunity. The potential role of NGAL as an immunomodulatory molecule is based on its ability to induce immune tolerance by upregulating HLA-G expression and expansion of T-regulatory cells in healthy donors. Future studies should further evaluate the role of NGAL in immunology and immunomodulation and its possible relationship to immunosuppressive therapy efficacy, tolerance induction in transplant patients, and other immunological disorders.

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.

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.

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.

BACKGROUND: 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. METHODS: We present a tag-less, flow-assisted method to purify, distinguish, and sort pluri/multipotent SCs obtained from clinical specimens, based on differences in the biophysical properties that cells acquire when in suspension under fluidic conditions. A suspension of cells in a transport fluid is injected into a ribbon-like capillary device by continuous flow. In a relatively short time (about 30 min), sorted cells are collected. RESULTS: We obtained baseline separation between MSCs and epithelial cells, which are important contaminants of isolated MSCs. The extent of separation is evaluated by flow cytometry through detection of a specific epithelial antigen. MSCs from various human sources also prove to have different, characteristic, highly-reproducible fractionation profiles. Finally, we evaluated the dissimilar differentiation potential among cell fractions obtained from sorting a single MSC source. After differentiation induction, a fraction displayed a differentiation yield close to 100%, whereas unfractionated cells contained only 40% of responding cells. CONCLUSIONS: The results demonstrate that the method presented is able to obtain selected and well-characterized living MSCs with an increased differentiation yield. Its reduced cost, full biocompatibility, and scale-up potential could make this method an effective procedure for stem cell selection.

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 30 min) 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.