Mesenchymal Stem/stromal Cells Cultures Research Articles

Persistent Contamination By Bone and Bone Marrow-Derived Macrophages Obscures Functional Assessment of Tissue-Dependent Heterogeneity in Mesenchymal Stromal Cells

Mesenchymal stem/stromal cells (MSCs) in the bone marrow microenvironment (BMME) serve as an essential support for the hematopoietic stem cell pool and are enriched for skeletal stem cells capable of regenerating bone, cartilage and adipose tissues. Purification of murine MSCs is essential to explore the functions of MSCs not only in skeletal generation but also in the regulation of the BMME. However, conventional protocols have not sufficed to purify murine MSCs, limiting the functional assessment of MSCs ex vivo. Careful evaluation of macrophage contribution in conventional ex vivo cultures of MSCs obtained from either bone marrow (BM-MSCs) or bone associated cells (BAC-MSCs) revealed abundant and persistent contamination with macrophages (BM mean 53.35%, BAC: mean 43.52% at passage3). Therefore, we developed a novel culture system by targeting three immunological markers; F4/80, Ly6C, and CD45. The functional and transcriptional analysis with this isolation procedure ensured clear dissection of macrophages and MSCs. MSCs were isolated from bone marrow (BM-MSCs) and bone (bone associated cells: BAC-MSCs).BM-MSCs display more proliferative progenitor like characteristics while the BAC-MSCs display dormant stem cell like characteristics. Previously Sox9 was reported to be a marker of murine undifferentiated mesenchymal precursors that possess tri-lineage differentiation capacity and are equivalent to CXCL12-expressing stromal cells from adult mice. Interestingly, Sox9 was significantly expressed in BACs (mean 4.75% in MSC (Lin-/CD45-/CD31-/Sca-1+/CD51+)) but not in BM cells (mean 0.733% in MSC (Lin-/CD45-/CD31-/Sca-1+/CD51+)) in vivo by flow cytometric analysis. In our ex vivo MSC cultures, we confirmed that BM-MSCs are enriched for Sox9 expressing cells, similar to that of BAC-MSCs indicating that we successfully purified a very rare multipotent stromal cell population from both BM and BACs. Comparing MSCs from culture that included macrophages to those that successfully purified MSCs, we learned that the coexistence of macrophages significantly reduced SOX9 and PDGFα (CD140a) expression by MSCs, regardless of whether they were derived from bone or bone marrow. This suggested that the presence of macrophages limits MSCs stem and progenitor activity. We also evaluated the functional aspects of these MSCs using our coculture system of MSCs and BM derived LSK (Lineage-/Sca-1+/c-Kit+) without any support of growth factors mainly to elucidate the hematopoietic cell support capacity of these MSCs. Interestingly, purified MSCs significantly enhanced reconstitution capacity of LSK cells in comparison to macrophage contaminated MSCs, especially for BM-MSCs. Macrophages also disrupted the reconstitution of stem/progenitor cells with BAC-MSC coculture even though mature blood cell reconstitution did not seem to be affected. Collectively, our novel ex vivo culture of primary MSCs can be useful to eliminate the artifacts of macrophage contributions to MSCs, which was observed in the conventional ex vivo cultures where high percentages of macrophage can persistently exist with MSCs possibly from CD45 negative macrophage progenitors. Moreover, the presence of macrophages and tissue source significantly alters the cell characteristics and fate of cultured MSCs, which could provide further understanding for MSC-macrophage crosstalk. In summary, our study highlights the importance of assessing the purity of primary MSC culture systems and heterogeneity based on tissue source, and they enable the isolation of highly purified MSCs to further study their role and macrophage crosstalk at homeostasis of BMME and in disease.

Effects of platelet-rich plasma on mesenchymal stem cells isolated from rat uterus.

BackgroundPlatelet-rich plasma (PRP), which represents a valuable source of growth factors, is increasingly being applied in regenerative medicine. Recent findings suggest the feasibility of using PRP in the treatment of infertility secondary to refractory thin endometrium. Mesenchymal stem/stromal cells (MSCs) of the endometrium are an essential cellular component responsible for extracellular matrix remodeling, angiogenesis, cell-to-cell communication, and postmenstrual tissue repair. Using a rat model, we examine the effects of autologous PRP on MSCs isolated from the uterus and compare them with the effects of autologous ordinary plasma (OP) and complete growth medium.MethodsMSCs were isolated from uterine tissues via enzymatic disaggregation. Flow cytometry immunophenotyping of the primary cell cultures was complemented by immunocytochemistry for Ki-67 and vimentin. The ability of MSCs to differentiate in osteo-, chondro-, and adipogenic directions was assessed using differentiation-inducing media. The levels of autophagy and apoptosis markers, as well as the levels of matrix metalloproteinase 9 (MMP9) and estrogen receptor α, were assessed by western blotting.ResultsAfter 24 h incubation, the proliferation index of the PRP-treated MSC cultures was significantly higher than that of the MSC cultures treated with complete growth medium. PRP treatment elevated production of LC3B protein, an autophagy marker, while OP treatment upregulated the expression of stress-induced protein p53 and extracellular enzyme MMP9. The results indicate practical relevance and validity for PRP use in the treatment of infertility.

Influences of Xeno-Free Media on Mesenchymal Stem Cell Expansion for Clinical Application.

Mesenchymal stem/stromal cells (MSCs) are multipotent somatic stem/progenitor cells that can be isolated from various tissues and have attracted increasing attention from the scientific community. This is due to MSCs showing great potential for incurable disease treatment, and most applications of MSCs involve tissue degeneration and treatment of immune- and inflammation-mediated diseases. Conventional MSC cultures contain fetal bovine serum (FBS), which is a common supplement for cell development but is also a risk factor for exposure to animal-derived pathogens. To avoid the risks resulting from the xenogeneic origin and animal-derived pathogens of FBS, xeno-free media have been developed and commercialized to satisfy MSC expansion demands for human clinical applications. This review summarized and provided an overview of xeno-free media that are currently used for MSC expansion. Additionally, we discussed the influences of different xeno-free media on MSC biology with particular regard to cell morphology, surface marker expression, proliferation, differentiation and immunomodulation. The xeno-free media can be serum-free and xeno-free media or media supplemented with some human-originating substances, such as human serum, human platelet lysates, human umbilical cord serum/plasma, or human plasma-derived supplements for cell culture medium. These media have capacity to maintain a spindle-shaped morphology, the expression of typical surface markers, and the capacity of multipotent differentiation and immunomodulation of MSCs. Xeno-free media showed potential for safe use for human clinical treatment. However, the influences of these xeno-free media on MSCs are various and any xeno-free medium should be examined prior to being used for MSC cultures.

Bone marrow-derived mesenchymal stem/stromal cells reverse the sensorial diabetic neuropathy via modulation of spinal neuroinflammatory cascades

BackgroundDiabetic neuropathy (DN) is a frequent and debilitating manifestation of diabetes mellitus, to which there are no effective therapeutic approaches. Mesenchymal stem/stromal cells (MSC) have a great potential for the treatment of this syndrome, possibly through regenerative actions on peripheral nerves. Here, we evaluated the therapeutic effects of MSC on spinal neuroinflammation, as well as on ultrastructural aspects of the peripheral nerve in DN-associated sensorial dysfunction.MethodsC57Bl/6 mice were treated with bone marrow-derived MSC (1 × 106), conditioned medium from MSC cultures (CM-MSC) or vehicle by endovenous route following the onset of streptozotocin (STZ)-induced diabetes. Paw mechanical and thermal nociceptive thresholds were evaluated by using von Frey filaments and Hargreaves test, respectively. Morphological and morphometric analysis of the sciatic nerve was performed by light microscopy and transmission electron microscopy. Mediators and markers of neuroinflammation in the spinal cord were measured by radioimmunoassay, real-time PCR, and immunofluorescence analyses.ResultsDiabetic mice presented behavioral signs of sensory neuropathy, mechanical allodynia, and heat hypoalgesia, which were completely reversed by a single administration of MSC or CM-MSC. The ultrastructural analysis of the sciatic nerve showed that diabetic mice exhibited morphological and morphometric alterations, considered hallmarks of DN, such as degenerative changes in axons and myelin sheath, and reduced area and density of unmyelinated fibers. In MSC-treated mice, these structural alterations were markedly less commonly observed and/or less pronounced. Moreover, MSC transplantation inhibited multiple parameters of spinal neuroinflammation found in diabetic mice, causing the reduction of activated astrocytes and microglia, oxidative stress signals, galectin-3, IL-1β, and TNF-α production. Conversely, MSC increased the levels of anti-inflammatory cytokines, IL-10, and TGF-β.ConclusionsThe present study described the modulatory effects of MSC on spinal cord neuroinflammation in diabetic mice, suggesting new mechanisms by which MSC can improve DN.

Exposing mesenchymal stem cells to chondroitin sulphated proteoglycans reduces their angiogenic and neuro-adhesive paracrine activity

The multifactorial complexity of spinal cord injuries includes the formation of a glial scar, of which chondroitin sulphated proteoglycans (CSPG) are an integral component. Previous studies have shown CSPG to have inhibitory effects on endothelial and neuronal cell growth, highlighting the difficulty of spinal cord regeneration. Mesenchymal stem/stromal cells (MSC) are widely used as a cell therapy, and there is mounting evidence for their angiogenic and neurotrophic paracrine properties. However, in vivo studies have observed poor engraftment and survival of MSC when injected into SCI. Currently, it is not known whether increasing CSPG concentrations seen after SCI may affect MSC; therefore we have investigated the effects of CSPG exposure to MSC in vitro. CSPG-mediated inhibition of MSC adhesion was observed when MSC were cultured on substrates of increasing CSPG concentration, however MSC viability was not affected even up to five days of culture. Culture conditioned medium harvested from these cultures (primed MSC CM) was used as both culture substrata and soluble medium for EA.hy926 endothelial cells and SH-SY5Y neuronal cells. MSC CM was angiogenic, promoting endothelial cell adhesion, proliferation and tubule formation. However, exposing MSC to CSPG reduced the effects of CSPG-primed MSC CM on endothelial cell adhesion and proliferation, but did not reduce MSC-induced endothelial tubule formation. Primed MSC CM also promoted neuronal cell adhesion, which was reduced following exposure to CSPG. There were no marked differences in neurite outgrowth in MSC CM from CSPG primed MSC cultures versus control conditions, although non-primed MSC CM from the same donors was found to significantly enhance neurite outgrowth. Taken together, these studies demonstrate that MSC are resilient to CSPG exposure, but that there is a marked effect of CSPG on their paracrine regenerative activity. The findings increase our understanding of how the wound microenvironment after SCI can mitigate the beneficial effects of MSC transplantation.

Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.

While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25–400 MSCs and 10 umbilical cord blood (CB)-derived CD34+ progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34+ cell expansion outcomes. By contrast, a substantial increase in CD34+CD38− cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34+CD38− cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34+CD38− cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34+ cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.

Avoidance of Maternal Cell Contamination and Overgrowth in Isolating Fetal Chorionic Villi Mesenchymal Stem Cells from Human Term Placenta.

Human placenta is rich in mesenchymal stem/stromal cells (MSC), with their origin widely presumed fetal. Cultured placental MSCs are confounded by a high frequency of maternal cell contamination. Our recent systematic review concluded that only a small minority of placental MSC publications report fetal/maternal origin, and failed to discern a specific methodology for isolation of fetal MSC from term villi. We determined isolation conditions to yield fetal and separately maternal MSC during ex vivo expansion from human term placenta. MSCs were isolated via a range of methods in combination; selection from various chorionic regions, different commercial media, mononuclear cell digest and/or explant culture. Fetal and maternal cell identities were quantitated in gender‐discordant pregnancies by XY chromosome fluorescence in situ hybridization. We first demonstrated reproducible maternal cell contamination in MSC cultures from all chorionic anatomical locations tested. Cultures in standard media rapidly became composed entirely of maternal cells despite isolation from fetal villi. To isolate pure fetal cells, we validated a novel isolation procedure comprising focal dissection from the cotyledonary core, collagenase/dispase digestion and explant culture in endothelial growth media that selected, and provided a proliferative environment, for fetal MSC. Comparison of MSC populations within the same placenta confirmed fetal to be smaller, more osteogenic and proliferative than maternal MSC. We conclude that in standard media, fetal chorionic villi‐derived MSC (CV‐MSC) do not grow readily, whereas maternal MSC proliferate to result in maternal overgrowth during culture. Instead, fetal CV‐MSCs require isolation under specific conditions, which has implications for clinical trials using placental MSC. Stem Cells Translational Medicine 2017;6:1070–1084

Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates.

Three-dimensional (3-D) aggregate culturing is useful for investigating the functional properties of mesenchymal stem/stromal cells (MSCs). For 3-D MSC analysis, however, pre-expansion of MSCs with two-dimensional (2-D) monolayer culturing must first be performed, which might abolish their endogenous properties. To avoid the need for 2-D expansion, we used prospectively isolated mouse bone marrow (BM)-MSCs and examined the differences in the biological properties of 2-D and 3-D MSC cultures. The BM-MSCs self-assembled into aggregates on nanoculture plates (NCP) that have nanoimprinted patterns with a low-cellular binding texture. The 3-D MSCs proliferated at the same rate as 2-D-cultured cells by only diffusion culture and secreted higher levels of pro-angiogenic factors such as vascular endothelial growth factor and hepatocyte growth factor (HGF). Conditioned medium from 3-D MSC cultures promoted more capillary formation than that of 2-D MSCs in an in vitro tube formation assay. Matrigel-implanted 3-D MSC aggregates tended to induce angiogenesis in host mice. The 3-D culturing on NCP induced alpha-fetoprotein (AFP) expression in MSCs without the application of AFP- or endodermal-inducible factors, possibly via an HGF-autocrine mechanism, and maintained their differentiation ability for adipocytes, osteocytes, and chondrocytes. Prospectively isolated mouse BM-MSCs expressed low/negative stemness-related genes including Oct3/4, Nanog, and Sox2, which were not enhanced by NCP-based 3-D culturing, suggesting that some of these cells differentiate into meso-endodermal layer cells. Culturing of prospectively isolated MSCs on NCP in 3-D allows the analysis of the biological properties of more closely endogenous BM-MSCs and might contribute to tissue engineering and repair.

Mesenchymal Stem/Stromal Cells in Liver Fibrosis: Recent Findings, Old/New Caveats and Future Perspectives.

Mesenchymal stem/stromal cells (MSCs) are progenitors which share plastic-adherence capacity and cell surface markers but have different properties according to their cell and tissue sources and to culture conditions applied. Many recent publications suggest that MSCs can differentiate into hepatic-like cells, which can be a consequence of either a positive selection of rare in vivo pluripotent cells or of the original plasticity of some cells contributing to MSC cultures. A possible role of MSCs in hereditary transmission of obesity and/or diabetes as well as properties of MSCs regarding immunomodulation, cell fusion and exosome release capacities are discussed according to recent literature. Limitations in methods used to track MSCs in vivo especially in the context of liver cirrhosis are addressed as well as strategies explored to enhance their migratory, survival and proliferation properties, which are known to be relevant for their future clinical use. Current knowledge regarding mechanisms involved in liver cirrhosis amelioration mediated by naïve and genetically modified MSCs as well as the effects of applying preconditioning and combined strategies to improve their therapeutic effects are evaluated. Finally, first reports of GMP guidelines and biosafety issues in MSCs applications are discussed.

High incidence of contaminating maternal cell overgrowth in human placental mesenchymal stem/stromal cell cultures: a systematic review.

Placenta is a readily accessible translationally advantageous source of mesenchymal stem/stromal cells (MSCs) currently used in cryobanking and clinical trials. MSCs cultured from human chorion have been widely assumed to be fetal in origin, despite evidence that placental MSCs may be contaminated with maternal cells, resulting in entirely maternally derived MSC cultures. To document the frequency and determinants of maternal cell contamination in chorionic MSCs, we undertook a PRISMA-compliant systematic review of publications in the PubMed, Medline, and Embase databases (January 2000 to July 2013) on placental and/or chorionic MSCs from uncomplicated pregnancies. Of 147 studies, only 26 (18%) investigated fetal and/or maternal cell origin. After excluding studies that did not satisfy minimal MSC criteria, 7 of 15 informative studies documented MSC cultures as entirely fetal, a further 7 studies reported cultured human chorionic MSC populations to be either maternal (n=6) or mixed (n=1), whereas 1 study separately cultured pure fetal and pure maternal MSC from the same placenta. Maternal cell contamination was associated with term and chorionic membrane samples and greater passage number but was still present in 30% of studies of chorionic villous MSCs. Although most studies assume fetal origin for MSCs sourced from chorion, this systematic review documents a high incidence of maternal-origin MSC populations in placental MSC cultures. Given that fetal MSCs have more primitive properties than adult MSCs, our findings have implications for clinical trials in which knowledge of donor and tissue source is pivotal. We recommend sensitive methods to quantitate the source and purity of placental MSCs.

Detection of spontaneous tumorigenic transformation during culture expansion of human mesenchymal stromal cells

Human mesenchymal stem/stromal cells (MSCs) have been explored in a number of clinical trials as a possible method of treating various diseases. However, the effect of long-term cell expansion invitro on physiological function and genetic stability is still poorly understood. In this study, MSC cultures derived from bone marrow and liver were evaluated for the presence of aberrant cells following long-term expansion. In 46 independent cultures, four batches of transformed MSCs (TMCs) were found, which were all beyond the culture period of five weeks. These aberrant cells were first identified based on the appearance of abnormal cytology and the acquirement of growth advantage. Despite common MSC markers being diminished or absent, TMCs remain highly susceptible to lysis by allogenic natural killer (NK) cells. When transplanted into immunodeficient mice, TMCs formed sarcoma-like tumors, whereas parental MSCs did not form tumors in mice. Using a combination of high-resolution genome-wide DNA array and short-tandem repeat profiling, we confirmed the origin of TMCs and excluded the possibility of human cell line contamination. Additional genomic duplication and deletions were observed in TMCs, which may be associated with the transformation event. Using gene and microRNA expression arrays, a number of genes were identified that were differentially expressed between TMCs and their normal parental counterparts, which may potentially serve as biomarkers to screen cultures for evidence of early transformation events. In conclusion, the spontaneous transformation of MSCs resulting in tumorigenesis is rare and occurs after relatively long-term (beyond five weeks) culture. However, as an added safety measure, cultures of MSCs can potentially be screened based on a novel gene expression signature.

Cellular Kinetics of Perivascular MSC Precursors

Mesenchymal stem/stromal cells (MSCs) and MSC-like multipotent stem/progenitor cells have been widely investigated for regenerative medicine and deemed promising in clinical applications. In order to further improve MSC-based stem cell therapeutics, it is important to understand the cellular kinetics and functional roles of MSCs in the dynamic regenerative processes. However, due to the heterogeneous nature of typical MSC cultures, their native identity and anatomical localization in the body have remained unclear, making it difficult to decipher the existence of distinct cell subsets within the MSC entity. Recent studies have shown that several blood-vessel-derived precursor cell populations, purified by flow cytometry from multiple human organs, give rise to bona fide MSCs, suggesting that the vasculature serves as a systemic reservoir of MSC-like stem/progenitor cells. Using individually purified MSC-like precursor cell subsets, we and other researchers have been able to investigate the differential phenotypes and regenerative capacities of these contributing cellular constituents in the MSC pool. In this review, we will discuss the identification and characterization of perivascular MSC precursors, including pericytes and adventitial cells, and focus on their cellular kinetics: cell adhesion, migration, engraftment, homing, and intercellular cross-talk during tissue repair and regeneration.

Study of the Quantitative, Functional, Cytogenetic and Immunoregulatory Properties of Bone Marrow Mesenchymal Stem Cells in Patients with B-Cell Chronic Lymphocytic Leukemia

Abstract 3861The microenvironment in both the bone marrow (BM) and lymph nodes has clearly been implicated in the biology of B-chronic lymphocytic leukemia (B-CLL). The non-hematopoietic components of the BM microenvironment originate from a rare population of multipotent progenitor cells, currently referred to as mesenchymal stem/stromal cells (MSCs). The latter have been shown to support hematopoiesis and to affect B cell proliferation and differentiation, thereby setting the stage for studying MSCs' putative role in CLL pathogenesis. However, this particular field of research has not been extensively investigated and the question as to whether patient MSCs differ from their normal counterparts has not been properly answered. The aim of the present study is to explore whether BM-derived MSCs from CLL patients harbor intrinsic abnormalities, which in turn might contribute to the pathophysiology of the disease.BM MSCs were thus isolated from 11 patients with B-CLL (Rai stage 0-III) and 16 age- and sex-matched healthy individuals and their quantitative, functional and cytogenetic characteristics were comparatively assessed. BM MSCs were expanded and re-seeded for a total of 6 passages.Adherent cells from both study groups displayed the same spindle-shape morphology and showed a similar expression of CD29, CD44, CD73, CD90 and CD105 while being negative for CD14, CD34 and CD45. Even though MSC cultures could be established and serially replated from all CLL patients, their growth rate over passages was significantly reduced compared to cultures generated from normal individuals (P < 0.0001). These findings were further substantiated by the MTT assay according to which the number of live cells, at a representative passage (P2), remained significantly lower in CLL patients compared to controls (P <0.0001). The survival characteristics of CLL-derived MSCs at P2 were further evaluated using flow cytometry and 7AAD staining. A statistically significant increase in the proportion of both early and late apoptotic cells was thus documented in B-CLL-derived MSCs as compared to their normal counterparts (P=0.0007 and P=0.0045, respectively). These data suggest that the impaired proliferative potential of CLL-derived MSCs can be attributed, at least in part, to increased cell apoptosis. The frequency of MSCs within the BM mononuclear cell (BMMC) fraction, estimated via the standard CFU-F assay, was significantly reduced in patients, as compared to normal controls (P=0.0039) and this might also be explained by the increased apoptotic rate of CLL-derived MSCs, as well as by BM infiltration by the malignant cells. Regarding MSC differentiation capacity, patient MSCs exhibited normal osteogenic and adipogenic potential as evidenced by cytochemical staining (Von Kossa and Oil Red, respectively) and also by the quantification of osteogenesis (RUNX2, ALP)- and adipogenesis (PPARG, aP2)-related specific gene expression. Furthermore, CLL-derived MSCs did not differ from their normal counterparts in the ability to suppress mitogen-induced T-cell proliferative responses. Since B-CLL cells undergo spontaneous apoptosis in vitro, unless cultured on direct contact with marrow stroma, we evaluated whether ex vivo expanded patient MSCs confer a survival advantage to the malignant clone. Yet, MSCs from both study groups exerted a similar anti-apoptotic effect on CLL cells. CLL-derived MSCs exhibited aberrant production of cytokines that are crucial for B-cell differentiation, survival and apoptosis. More specifically, they secreted significantly lower levels of SDF-1 (P=0.002) and TGF-β1 (P<0.001), as compared to their normal counterparts. Furthermore, CLL-derived MSC supernatants were shown, for the first time, to contain significantly higher levels of APRIL (P=0.0002) and lower levels of BAFF (P=0.0036), as compared to normal MSCs. Finally FISH analysis demonstrated that CLL-derived MSCs did not share any of the cytogenetic abnormalities with CLL cells, thereby suggesting that they are not part of the leukemic clone.In conclusion, ex-vivo expanded B-CLL-derived MSCs harbor intrinsic qualitative and quantitative abnormalities that may be implicated in disease development and/or progression. We anticipate that our observations will contribute to delineating B-CLL biology and will hopefully provide important clues for the design of appropriate microenvironment-targeted therapies. Disclosures:No relevant conflicts of interest to declare.

3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Mesenchymal stem/stromal cells (MSC) are rapidly becoming a leading candidate for use in tissue regeneration, with first generation of therapies being approved for use in orthopaedic repair applications. Capturing the full potential of MSC will likely require the development of novel in vitro culture techniques and devices. Herein we describe the development of a straightforward surface modification of an existing commercial product to enable the efficient study of three dimensional (3D) human bone marrow-derived MSC osteogenic differentiation. Hundreds of 3D microaggregates, of either 42 or 168 cells each, were cultured in osteogenic induction medium and their differentiation was compared with that occurring in traditional two dimensional (2D) monolayer cultures. Osteogenic gene expression and matrix composition was significantly enhanced in the 3D microaggregate cultures. Additionally, BMP-2 gene expression was significantly up-regulated in 3D cultures at day 3 and 7 by approximately 25- and 30-fold, respectively. The difference in BMP-2 gene expression between 2D and 3D cultures was negligible in the more mature day 14 osteogenic cultures. These data support the notion that BMP-2 autocrine signalling is up-regulated in 3D MSC cultures, enhancing osteogenic differentiation. This study provides both mechanistic insight into MSC differentiation, as well as a platform for the efficient generation of microtissue units for further investigation or use in tissue engineering applications.