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1
Peters, R. E., M. Heikenwälder e A. Knuth. "Expansion of umbilical cord blood mesenchymal stem cells". Journal of Clinical Oncology 27, n. 15_suppl (20 maggio 2009): 7103. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.7103.
Abstract (sommario):
7103 Background: Umbilical cord blood (UCB) is known to harbor 2 major types of stem cells, the hematopoietic stem cells (HSC) & the non-hematopoietic or mesenchymal stem cells (MSC). Under appropriate conditions, MSCs can give rise to cells of bone, fat, hepatic lineages, etc. Based on this potential, MSC hold promise for clinical applications in regenerative medicine. Methods: Stroma-free liquid culture: UCB cryopreserved mononuclear cells (MNC) were cultured in the presence of early growth factors: Flt-3 & SCF (25ng/ml), MGDF (10ng/ml) & human serum (10%). MNC derived adherent MSC were passaged at day 14 during HSC expansion & after enriching in MesenCult medium. Results: We developed a technology to generate & expand HSC & stromal/ MSC from all UCB units (5/5) at the same time using one culture system (stroma-free liquid culture). Following repeated passages, MSC count increased 357- 600-folds & CFU-Fibroblasts colonies (CFU-F) increased too (61–513 & 648–697) after 10 and 20 passages respectively. We used the CFU-F assay to demonstrate MSC activity in stromal cell formation in vitro. Phenotypically, MSC were negative for hematopoietic antigens (CD45, CD34 & CD14) & MHC class-II but >95% + for CD73, CD105, CD29, CD44 & MHC class I. To demonstrate MSC differentiation capacity in vitro, cells were incubated in various induction media to differentiate into adipocytes (fat)), osteoblasts (bone) and hepatocytes (liver) at passage 5. Following induction, positive staining with oil red O for cells of adipocyte and with alkaline phosphatase for cells of osteoblsts lineages was observed. The identity of hepatocytes was verified by the characteristic hexagonal hepatocytic shape as well as albumin, cytokeratin (CK) 18 and CK14 expression, as assessed by flow cytometry. Our data were corroborated by RT-PCR analysis. Conclusions: MSC described herein exhibit in vitro properties of multipotent stem cells. The established, stroma-free culture system facilitates expansion of MSC from all tested UCB units. Our data underline that it will be possible in the future to substitutes properly differentiated hepatocytes which might lead to efficient applications in patients suffering from various end stage liver disease. No significant financial relationships to disclose.
2
Prewitz, Marina, Friedrich Philipp Seib, Martin Bornhaeuser e Carsten Werner. "Engineering Biomimetic Culture Systems: Impact On Human Bone Marrow-Derived Stem Cells." Blood 114, n. 22 (20 novembre 2009): 3628. http://dx.doi.org/10.1182/blood.v114.22.3628.3628.
Abstract (sommario):
Abstract Abstract 3628 Poster Board III-564 The bone marrow (BM) harbours haematopoietic stem/progenitor cells (HSCs) in anatomically distinct sites (niches) where HSCs are subjected to regulatory cues such as cytokines, cell-cell contacts and extra-cellular matrix (ECM) all of which control stem cell fate. In particular mesenchymal stromal cells (MSCs) are an integral part of the bone marrow and are known to be key regulators of the HSC niche. We have previously shown that bio-artificial scaffolds can have a significant impact on the in vitro behaviour of MSCs. Here, we are therefore focussing on the role of (native) ECM within the MSC-HSC microenvironment by building on our previous findings and published data (Seib et al.,Tissue Eng Part A., 2009 in press). Thus the aim of the current study is (a) to identify niche-specific ECM components and (b) the use of such ECMs for in vitro culture of BM-derived stem cells. To mimic the natural ECM composition of the BM, different ECM types were generated from BM-derived cells using (a) Dexter cultures, (b) standard MSC cultures, (c) MSCs subjected to osteogenic differentiation. After 10 days of culture those MSC-derived ECMs were decellularised using 0.5% Triton-X and 20mM NH4OH leaving only the ECM behind (verified by scanning electron microscopy). Those ECMs were used as a substrate for a second culture of MSCs, which were analysed for their proliferation and differentiation potential. Cell-free ECM from standard MSC cultures improved MSC proliferation compared to cells grown on regular tissue culture plastic (TCP) over the period of 8 days. Most notably, all cell-free ECM preparations lead to a significant difference in the cytoskeletal arrangement of MSCs during the first 2 days of culture compared to TCP controls. Cultivation of MSCs on native ECM provided a guiding structure for those cells to grow into, and helped to maintain an elongated cell shape compared to substantial cell spreading on TCP (roundness 0.2 versus 0.5 and cell area of 2.2 versus 8.2mm2, respectively, p<0.001, n=60. A factor of 1 was set to equate to a perfect circle). Next, we investigate if native ECM could either directly improve HSC cultures or maximise MSC feeder characteristics. For the latter set of studies MSCs were initially cultured for 7 days on cell-free ECM (from standard MSC cultures) and subsequently co-cultured with human peripheral blood CD34+ HSCs in serum free medium supplemented with cytokines (Tpo, Flt3, and SCF at 10ng/ml). Following a 14 day culture period up to 3.5-fold more CD34+ cells were present in ECM co-cultures compared to TCP co-cultures that was accompanied with an overall expansion of CD45+ cells of 109-fold versus 35-fold, respectively. Our data suggest that ECM preparations derived from MSCs might be useful to accomplish better expansion of HSCs under defined culture conditions. In addition, this system permits the identification of bimolecular key components that can be utilized in the future design of simple and robust carrier systems for improved HSC maintenance in vitro. Figure HSC-MSC co-culture on preformed ECM substrates. (A) MSC-derived ECM (from standard MSC culture) following cell lysis (complete absence of cells). (B) Growth of a new set of MSCs on ECM substrates as shown in (A). (C) HSC-MSC co-culture on ECM substrates. Scale bars at 2μm. Arrow heads point out ECM structures. Figure HSC-MSC co-culture on preformed ECM substrates. (A) MSC-derived ECM (from standard MSC culture) following cell lysis (complete absence of cells). (B) Growth of a new set of MSCs on ECM substrates as shown in (A). (C) HSC-MSC co-culture on ECM substrates. Scale bars at 2μm. Arrow heads point out ECM structures. Disclosures: No relevant conflicts of interest to declare.
3
Robinson, Simon N., Paul J. Simmons, Nathalie Brouard, Shannon Kidd, Hong Yang, William K. Decker, Dongxia Xing et al. "Efficacy of ‘Off-the-Shelf’, Commercially-Available, Third-Party Mesenchymal Stem Cells (MSC) in Ex Vivo Cord Blood (CB) Co-Culture Expansion." Blood 110, n. 11 (16 novembre 2007): 4106. http://dx.doi.org/10.1182/blood.v110.11.4106.4106.
Abstract (sommario):
Abstract INTRODUCTION: Our previous studies have shown that clinically-relevant levels of hematopoietic stem and progenitor cell (HSPC) expansion are possible by ex vivo co-culture of cord blood (CB) mononuclear cells (MNC) with third-party bone marrow (BM)-derived mesenchymal stem cells (MSC) and growth factors.1 A recently activated M. D. Anderson protocol requires that BM from a haplo-identical family member be used for the de novo generation of sufficient MSC for subsequent co-culture, a process requiring ∼3 weeks. Time constraints, uncertainties associated with the identification of a suitable BM donor and potential variation in MSC performance make logistical execution of this strategy difficult. We therefore investigated the potential efficacy of ‘off-the-shelf’ commercially-available sources of MSC. Since MSC do not express HLA-II (DR) they are non-immunogenic, suggesting that this might be a valuable alternative strategy. We compared ex vivo CB HSPC expansion obtained following CB MNC co-culture with 2 commercially-available research-grade MSC isolated by density separation and plastic adherence (MSC#1, Cambrex, Walkersville, MD and MSC#2, Allcells, Emeryville, CA). A third MSC, isolated by Stro-12 selection (MSC#3, supplied by PJS) was also evaluated. METHODS: Two MDACC frozen CB units (CB#1&2) were thawed, washed and co-cultured with adherent monolayers from each MSC. Total nucleated cell (TNC) and HSPC (CD34+ cells and colony-forming units, CFU) numbers were measured at input (Day 0) and output (Day 14). RESULTS: TNC and HSPC numbers revealed that the 2 commercially-available research-grade MSC (MSC#1&2) supported ex vivo CB HSPC expansion. MSC TNC CB34+ CFU n/a - not available CB#1 #1 x 6 x23 n/a #2 x 3 x 8 x15 #3 x 6 x16 x23 CB#2 #1 x 7 x20 x31 #2 x 5 x10 x20 #3 x10 x16 x34 1 Robinson et al. x13 x14 x25 MSC#2 performed less well than MSC#1 for both CB units suggesting that variation may exist between individual MSC. These data suggest that the screening of clinical-grade MSC that perform optimally during ex vivo expansion co-culture might be warranted to best utilize this ‘off-the-shelf’ strategy. Data were similar to previous reports where TNC, CD34+ and CFU numbers were shown to increase approximately 13, 14 and 25-fold, respectively.1 Data were also similar for MSC#3, suggesting that the method used to isolate MSC does not appear to be an important variable for effective CB MNC/MSC co-culture. CONCLUSION: Although research-grade MSC were compared from different commercial sources, these data suggest that, in principle, commercially-available clinical-grade MSC might prove a valuable ‘off-the-shelf’ option, potentially reducing the time to therapy and addressing concerns associated with identifying a BM donor and variation in MSC performance. Future studies will evaluate FDA-compliant MSC that could be used clinically.
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Bucher, Christian, Amiq Gazdhar, Lorin M. Benneker, Thomas Geiser e Benjamin Gantenbein-Ritter. "Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System". Stem Cells International 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/326828.
Abstract (sommario):
Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved byin vitroelectroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.
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Tormin, Ariane, Jan Claas Brune, Stuart Walsh, Johan Richter, Xiaolong Fan e Stefan Scheding. "Human Primary Mensenchymal Stromal Progenitor Cells Are Highly Enriched in Both, the CD271+/CD146+ and CD271+/CD146− Bone Marrow Population with the Latter Acquiring CD146 Expression upon Culture in-Vitro". Blood 112, n. 11 (16 novembre 2008): 2422. http://dx.doi.org/10.1182/blood.v112.11.2422.2422.
Abstract (sommario):
Abstract Culture-derived mesenchymal stromal cells (MSC), which are attractive candidates for clinical cell therapy approaches, arise from primary MSC progenitor/stem cells in the bone marrow. Recently, several groups have reported markers (CD271, CD146, GD2, SSEA4, etc.) that allowed for an enrichment of CFU-F, i.e. primary MSC progenitors. However, the exact phenotype of the bona fide mesenchymal stem/progenitor cells has not yet not been sufficiently defined. We therefore aimed to investigate primary MSC in bone marrow subpopulations defined by the expression of CD271 and CD146, as both markers have been reported to contain all assayable CFU-F and stromal stem cells, respectively (Quirici et al., Exp. Hematol. 2002; Sacchetti et al, Cell, 2007). Utilizing multi-color flow cytometry, unfractionated human bone marrow mononuclear cells (BM-MNC) were found to contain 0.05 ± 0.05% CD271+/CD146+ cells, whereas 0.82 ± 0.43% and 0.71 ± 0.23% were single-positive for CD271 and CD146, respectively. CD271/CD146 subpopulations were FACS sorted from lineage-depleted BM-MNC (RosetteSep) and assayed for CFU-F content (n=3). CFU-F could not be detected in the CD271−/CD146− fraction. In contrast, CFU-F initiating cells were highly enriched in the CD271+/CD146+/CD45−/low fraction (1.1 ± 0.2 CFU-F per 10 plated cells), which corresponds to a ca. 400-fold enrichment compared to the entire lineage-depleted fraction (2.7 ± 3.4 CFU-F per 1 × 104 plated cells). Of note, CFU-F could also be assayed at high frequency from CD271+/CD146− cells (20.4 ± 22.6 CFU-F per 1 × 104 plated cells). Generally, CFU-F were not found in the CD271+/CD146+/CD45+ and the CD271−/CD146+ fractions, and were also not detectable within the whole CD271+/CD45+ population of unfractionated BM-MNC (n=4), which, however, gave rise to erythropoietic colonies. The two CFU-F enriched populations, i.e. CD271+/CD146+/CD45−/low and CD271+/CD146− cells, were then cultured under standard MSC growth conditions. MSC derived from both populations exhibited a typical MSC surface marker profile (CD105+, CD90+, CD73+, HLA-class I+, CD45−, CD34−, CD19−, CD14−, HLA-DR−) and typical MSC differentiation (adipocytes, osteoblasts, chondrocytes). Interestingly, MSC generated from CD271+/CD146− cells became positive for CD146 in culture and stable CD146 expression over time was observed for MSC from both populations (up to the 5th passage, average 82 ± 11%). In contrast, over the same culture period CD271 expression decreased with passage number and an average of only 10 ± 4% of the cultured cells remained positive for CD271. To further characterize the CFU-F enriched subpopulations, single cells from CD271+/CD146+/CD45−/low and CD271+/CD146−/CD45−/low cells were sorted into fibronectin-coated 96-well plates to investigate colony growth and differentiation potential. CFU-F frequencies in this assay were 4 per 96 seeded cells for both populations and all but one of the CD271+/CD146+/CD45−/low clones could be further expanded in culture. Subpopulation-derived clones were capable of typical MSC differentiation and MSC derived from CD271+/CD146−/CD45−/low clones–similar to the bulk cultures–became CD146 positive (89 ± 12%) after 2 passages, whereas here CD271 expression was not lost. Taken together, CD271+/CD146+/CD45−/low and CD271+/CD146−/CD45−/low bone marrow cells are highly enriched for primary MSC progenitor cells. The difference in CD146 expression, which disappears in culture, might relate different localizations of the primary cells in the marrow but might possibly also reflect functional differences, e.g. in stemness. Accordingly, experiments addressing in-situ location, in vivo differentiation potential, gene expression and surface-marker expression profiling of primary MSC are currently under way.
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Giallongo, Cesarina, Daniele Tibullo, Nunziatina Laura Parrinello, Piera La Cava, Giuseppina Camiolo, Nunzia Caporarello, Carmelina Daniela Anfuso et al. "Mesenchymal Stem Cells (MSC) from Patients with Multiple Myeloma Promote Myeloid Cells to Become Granulocytic-Myeloid-Derived Suppressor Cells (G-MDSC) with Immunosuppressive, Bone Resorption and Pro-Angiogenic Activity". Blood 128, n. 22 (2 dicembre 2016): 4458. http://dx.doi.org/10.1182/blood.v128.22.4458.4458.
Abstract (sommario):
Abstract Purpose A well-recognized feature of MM is the intimate relationship between plasma cells and bone marrow microenvironment, which is mainly composed of MSC, endothelial cells, immune cells and extracellular matrix. G-MDSC accumulate in the tumor microenvironment during tumor development promoting tumor growth and immunosuppression. Aim Analyzing MSC from MGUS, Smoldering myeloma (SMM) and MM patients in promoting G-MDSC generation. Methods Human peripheral blood mononucleated cells (PBMC) isolated from healthy subjects (HS) were cultured alone and with HS- (n=10), MGUS- (n=10), SMM- (n=4) or MM-MSC (n=14)(1:100). After 6 days, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype(CD11b+CD33+CD14-HLADR-) was confirmed by cytofluorimetric analysis. Results Only G-MDSC educated by SMM- and MM-MSC co-cultures (MSCed-G-MDSC) were able to suppress T cell proliferation when cultured with normal lymphocytes (p<0.001) compared to G-MDSC control (isolated from PBMC cultured in medium alone). SMM- and MM-MSCed-G-MDSC significantly up-regulated Arg1, NOS2, TNFα and CEBPA, a transcription factor promoting suppressive phenotype. Since also the angiogenic factor BV8 was significantly up-regulated, we next investigated the pro-angiogenic effect in vitro co-culturing MSCed-G-MDSC with Human Brain Microvascular Endothelial Cells (HBMEC) (1:2). After 5 h, we observed that MM-MSCed-G-MDSC were able to increase both tube length and number of branch points compared to G-MDSC control (p<0.05). Moreover, MM-MSCed-G-MDSC were able to digest bone matrix in vitro (p<0.01). Adding Bortezomib (5 nM), Lenalidomide (10 μM) or Pomalidomide (1 nM) during co-culture with MM-MSC, isolated G-MDSC showed a significant reduction of pro-angiogenic and bone resorption activity (p<0.05) but did not lose immunosuppressive ability. Conclusion MSC play a key role promoting tumor microenvironment transformation in SMM and MM patients.Indeed, only SMM- and MM-MSC and not MGUS-MSC are able to activate myeloid cells in G-MDSC with immunosuppressive, pro-angiogenic, and bone resorptionactivity. Disclosures No relevant conflicts of interest to declare.
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Maijenburg, Marijke W., Marion Kleijer, Kim Vermeul, Erik P. J. Mul, Floris P. J. van Alphen, C. Ellen van der Schoot e Carlijn Voermans. "Primary Bone Marrow-Derived MSC Subsets Have Distinct Wnt-Signatures Compared to Conventionally Cultured MSC and Differ in Their Capacity to Support Hematopoiesis in Vitro",. Blood 118, n. 21 (18 novembre 2011): 3414. http://dx.doi.org/10.1182/blood.v118.21.3414.3414.
Abstract (sommario):
Abstract Abstract 3414 Mesenchymal stromal cells (MSC) are of promising therapeutic use to suppress immunogenic responses following transplantation, and to support expansion of hematopoietic stem- and progenitors cells (HSPC) from small transplants derived for instance from cord blood. Culture-expanded MSC produce a wide variety and quantity of Wnt-proteins and the crucial role of Wnt-signaling in the hematopoietic stem cell niche is well established. However, studies addressing Wnt-signaling in MSC have (i) only focused on culture-expanded MSC and (ii) did not discriminate between phenotypically distinct subpopulations which are present in bulk cultures of expanded MSC. Recently we identified three new subpopulations of MSC in human bone marrow (BM) based on expression of CD271 and CD146: CD271brightCD146−, CD271brightCD146+, CD271−CD146+. These fractions co-express the “classical” MSC markers CD90 and CD105 and lack expression of CD45 and CD34 (Maijenburg et al, Blood 2010, 116, 2590). We and others demonstrated that the adult BM-derived CD271brightCD146− and CD271brightCD146+ cells contain all colony forming units-fibroblasts (Maijenburg et al, Blood 2010, 116, 2590; Tormin et al, Blood 2010, 116, 2594). To investigate how these primary subsets functionally compare to conventional, culture-expanded MSC, we investigated their Wnt-signature and hematopoietic support capacity. To this end, we sorted CD271brightCD146− and CD271brightCD146+ cells from human adult BM (n=3) and compared their Wnt-signatures obtained by Wnt-PCR array to the profiles from cultured MSC from the same donors. Fifteen genes were consistently differentially expressed in the two sorted uncultured subsets compared to their conventionally cultured counterparts. Expression of CCND1, WISP1 and WNT5B was strongly increased, and WNT5A was only detected in the conventionally cultured MSC. In contrast, WNT3A was exclusively expressed by sorted primary CD271brightCD146− and CD271brightCD146+ cells, that also expressed higher levels of JUN, LEF1 and WIF1. The differences in Wnt (target)-gene expression between CD271brightCD146− and CD271brightCD146+ cells were more subtle. The Wnt-receptors LRP6 and FZD7 were significantly higher expressed in CD271brightCD146+ cells, and a trend towards increased expression in the same subset was observed for CTNNB1, WNT11 and MYC. When the sorted subsets were cultured for 14 days (one passage), the differences in Wnt-related gene expression between the subsets was lost and the expanded sorted cells acquired an almost similar Wnt-signature as the MSC cultured from BM mononuclear cells from the same donors. The cultured subsets lost the expression of Wnt3a and gained the expression of Wnt5a, similar to the unsorted MSC cultured from the same donors in parallel. Despite the loss of a distinct Wnt-signature, co-culture experiments combining the sorted MSC subsets with human HSPC revealed that CD271brightCD146+ cells have a significantly increased capacity to support HSPC in short-term co-cultures (2 weeks) compared to CD271brightCD146− cells (p<0.021, n=3), which was analyzed in hematopoietic colony assays following co-culture. In contrast, a trend towards better long-term hematopoietic support (co-culture for 6 weeks) was observed on CD271brightCD146− cells. In conclusion, we demonstrate for the first time that primary sorted uncultured MSC subsets have a distinct Wnt-signature compared to cultured unsorted MSC and display differences in hematopoietic support. As it was recently shown that CD271brightCD146− and CD271brightCD146+ MSC localize to separate niches in vivo (Tormin et al, Blood 2011), our data indicate that the two MSC subsets are not necessarily distinct cell types and that the different Wnt-signature may be a reflection of these distinct microenvironments. Cell culturing for only one passage dramatically changed the Wnt-signature of the sorted MSC subsets, indicating that Wnt-signaling in in vitro expanded MSC does not resemble the Wnt-signature in their tissue resident counterparts in vivo. Disclosures: No relevant conflicts of interest to declare.
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Burk, Janina, Anna Sassmann, Cornelia Kasper, Ariane Nimptsch e Susanna Schubert. "Extracellular Matrix Synthesis and Remodeling by Mesenchymal Stromal Cells Is Context-Sensitive". International Journal of Molecular Sciences 23, n. 3 (3 febbraio 2022): 1758. http://dx.doi.org/10.3390/ijms23031758.
Abstract (sommario):
Matrix remodeling could be an important mode of action of multipotent mesenchymal stromal cells (MSC) in extracellular matrix (ECM) disease, but knowledge is limited in this respect. As MSC are well-known to adapt their behavior to their environment, we aimed to investigate if their mode of action would change in response to healthy versus pathologically altered ECM. Human MSC-derived ECM was produced under different culture conditions, including standard culture, culture on Matrigel-coated dishes, and stimulation with the pro-fibrotic transforming growth factor-β1 (TGFβ1). The MSC-ECM was decellularized, characterized by histochemistry, and used as MSC culture substrate reflecting different ECM conditions. MSC were cultured on the different ECM substrates or in control conditions for 2 days. Culture on ECM increased the presence of surface molecules with ECM receptor function in the MSC, demonstrating an interaction between MSC and ECM. In MSC cultured on Matrigel-ECM and TGFβ1-ECM, which displayed a fibrosis-like morphology, gene expression of collagens and decorin, as well as total matrix metalloproteinase (MMP) activity in the supernatant were decreased as compared with control conditions. These results demonstrated that MSC adapt to their ECM environment, which may include pathological adaptations that could compromise therapeutic efficacy.
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Koh, Benson, Nadiah Sulaiman, Mh Busra Fauzi, Jia Xian Law, Min Hwei Ng, Too Lih Yuan, Abdul Ghani Nur Azurah, Mohd Heikal Mohd Yunus, Ruszymah Bt Hj Idrus e Muhammad Dain Yazid. "A Three-Dimensional Xeno-Free Culture Condition for Wharton’s Jelly-Mesenchymal Stem Cells: The Pros and Cons". International Journal of Molecular Sciences 24, n. 4 (13 febbraio 2023): 3745. http://dx.doi.org/10.3390/ijms24043745.
Abstract (sommario):
Xeno-free three-dimensional cultures are gaining attention for mesenchymal stem cell (MSCs) expansion in clinical applications. We investigated the potential of xeno-free serum alternatives, human serum and human platelet lysate, to replace the current conventional use of foetal bovine serum for subsequent MSCs microcarrier cultures. In this study, Wharton’s Jelly MSCs were cultured in nine different media combinations to identify the best xeno-free culture media for MSCs culture. Cell proliferation and viability were identified, and the cultured MSCs were characterised in accordance with the minimal criteria for defining multipotent mesenchymal stromal cells by the International Society for Cellular Therapy (ISCT). The selected culture media was then used in the microcarrier culture of MSCs to determine the potential of a three-dimensional culture system in the expansion of MSCs for future clinical applications, and to identify the immunomodulatory potential of cultured MSCs. Low Glucose DMEM (LG) + Human Platelet (HPL) lysate media appeared to be good candidates for replacing conventional MSCs culture media in our monolayer culture system. MSCs cultured in LG-HPL achieved high cell yield, with characteristics that remained as described by ISCT, although the overall mitochondrial activity of the cells was lower than the control and the subsequent effects remained unknown. MSC microcarrier culture, on the other hand, showed comparable cell characteristics with monolayer culture, yet had stagnated cell proliferation, which is potentially due to the inactivation of FAK. Nonetheless, both the MSCs monolayer culture and the microcarrier culture showed high suppressive activity on TNF-α, and only the MSC microcarrier culture has a better suppression of IL-1 secretion. In conclusion, LG-HPL was identified as a good xeno-free media for WJMSCs culture, and although further mechanistic research is needed, the results show that the xeno-free three-dimensional culture maintained MSC characteristics and improved immunomodulatory activities, suggesting the potential of translating the monolayer culture into this culture system in MSC expansion for future clinical application.
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Kaminska, Agnieszka, Aleksandra Wedzinska, Marta Kot e Anna Sarnowska. "Effect of Long-Term 3D Spheroid Culture on WJ-MSC". Cells 10, n. 4 (24 marzo 2021): 719. http://dx.doi.org/10.3390/cells10040719.
Abstract (sommario):
The aim of our work was to develop a protocol enabling a derivation of mesenchymal stem/stromal cell (MSC) subpopulation with increased expression of pluripotent and neural genes. For this purpose we used a 3D spheroid culture system optimal for neural stem cells propagation. Although 2D culture conditions are typical and characteristic for MSC, under special treatment these cells can be cultured for a short time in 3D conditions. We examined the effects of prolonged 3D spheroid culture on MSC in hope to select cells with primitive features. Wharton Jelly derived MSC (WJ-MSC) were cultured in 3D neurosphere induction medium for about 20 days in vitro. Then, cells were transported to 2D conditions and confront to the initial population and population constantly cultured in 2D. 3D spheroids culture of WJ-MSC resulted in increased senescence, decreased stemness and proliferation. However long-termed 3D spheroid culture allowed for selection of cells exhibiting increased expression of early neural and SSEA4 markers what might indicate the survival of cell subpopulation with unique features.
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Jia, Yongsheng, Cuicui Zhang, Xiangqian Zheng e Ming Gao. "Co-cultivation of progenitor cells enhanced osteogenic gene expression and angiogenesis potential in vitro". Journal of International Medical Research 49, n. 4 (aprile 2021): 030006052110040. http://dx.doi.org/10.1177/03000605211004024.
Abstract (sommario):
Objectives The efficiencies of osteogenesis and angiogenesis present challenges that need to be overcome before bone tissue engineering can be widely applied to clinical uses. We aimed to optimize an in vitro culture system to enhance osteogenesis and angiogenesis. We investigated if hematopoietic stem cells (HSCs) promoted osteogenesis in vitro when co-cultured with mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Methods MSC/HSC, MSC/EPC/HSC, and MSC/EPC co-cultures were incubated for 21 days. Alkaline phosphatase (ALP) activity and calcium content were analyzed to assess mineralization. Expression levels of genes encoding osteogenesis-related proteins (ALP ( ALPL), collagen type IA ( COL1A1), osteocalcin ( BGLAP), and osteopontin ( OSTP)) were also evaluated by measuring mRNA levels at day 28. Angiogenesis was evaluated by tube-formation assay. Results COL1A1, OSTP, ALPL, and BGLAP genes were upregulated in MSC/HSC and MSC/EPC/HSC co-cultures compared with the MSC/EPC group. Upregulation was strongest in the MSC/EPC/HSC co-cultures. There were no significant changes in ALP levels and calcium content, but ALP activity was slightly higher and calcium content was relatively lower in the MSC/EPC and MSC/EPC/HSC groups. Conclusions Co-culture of MSCs with HSCs or EPCs/HSCs upregulated the expression of osteogenesis-related genes but did not affect the efficiency of osteogenesis.
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Hagen, Alina, Heidrun Holland, Vivian-Pascal Brandt, Carla U. Doll, Thomas C. Häußler, Michaela Melzer, Julia Moellerberndt, Hendrik Lehmann e Janina Burk. "Platelet Lysate for Mesenchymal Stromal Cell Culture in the Canine and Equine Species: Analogous but Not the Same". Animals 12, n. 2 (13 gennaio 2022): 189. http://dx.doi.org/10.3390/ani12020189.
Abstract (sommario):
Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-β1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications.
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Saccardi, Riccardo, Serena Urbani, Benedetta Mazzanti, Simone Dal Pozzo, Susanna Benvenuti, Alessandro Peri, Salvatore Frontera, Maurizio Genuardi e Alberto Bosi. "Human Bone Marrow MSC Transformation in Different Culture Conditions." Blood 108, n. 11 (16 novembre 2006): 4253. http://dx.doi.org/10.1182/blood.v108.11.4253.4253.
Abstract (sommario):
Abstract Human mesenchymal stem cells (MSC) have been isolated from different sources, expanded and characterized extensively. Their plasticity and immunomodulatory properties made these cells extremely promising in the fields of immunotherapy and regenerative medicine. Both safety and modality of preparation in a clinical setting are still be defined. We generated human MSC from normal donors bone marrow cells following 2 different isolation methods and culture conditions: white blood cells (buffy-coat) were plated in IMDM with gentamycin, 10% FBS and 2% Ultroser® G. Half of the complete medium was changed after one week and then the whole medium was added every 3–4 days. When approximately 80% of the flask surface was covered, the adherent cells were trypsinized and re-suspended in complete medium. mononucleated cells (MNC) purification by Ficoll density gradient separation and cultivation in DMEM-Low Glucose supplemented with 10% FBS and 1% antibiotic-antimycotic solution. Complete medium was changed after 3 days. When the cultures were near confluence, the cells were detached and replated. In both protocols, the isolated MSC (P0) were characterized by the number of Colony Forming Units-Fibroblasts (CFU-F), osteogenic and adipogenic differentiation, and immunophenotype based on the following monoclonal antibodies: CD34, CD45, CD14 (in order to quantify hematopoietic and monocytic contamination); CD29, CD44, CD166, CD90, CD73, HLA-DP DQ DR, HLA-ABC and CD105. We observed a spontaneous transformation of MSC in 6/29 cases (20%), irrespective of the isolation and expansion protocols and the cells showed a different morphologic, immunophenotypic, proliferative and cytogenetic pattern. In particular, cells assumed uniform cuboidal and round morphology and lost their spindle shaped aspect. MSC transformation usually occurred after the 4th passage in culture; only once it occurred after only one passage in culture (P1). Flow cytometric analysis showed a complete down regulation of MSC surface markers such as CD73, CD105, CD166, CD90, CD44, CD29, HLA ABC. The typical biparametric FSC/SSC distribution of RS cells and mMSC was lost as well. Transformed MSC showed very low expression of KDR, Ulex and vWF. Differentiation capability of the transformed cells was also lost and only small areas stained positive for classic differentiation tests (Oil Red O for adipogenic and Alizarin S for osteogenic differentiation). Cytogenetic analysis showed aneuploidy and a wide pattern of chromosomal aberrations. These cells also showed a high level of telomerase activity when compared with non-transformed MSC. Finally, their proliferation rate was greatly increased. CONCLUSIONS: we observed spontaneous transformation of MSC generated under various conditions. We speculate that this phenomenon might be related to the high proliferation rate in culture of cells still retaining a rather undifferentiated pattern. Further investigations about the transformation modality and its biological pathways are needed. The use of these cells in a clinical setting should be adequately evaluated for safety reasons.
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Stern-Straeter, Jens, Juritz Stephanie, Gregor Bran, Frank Riedel, Haneen Sadick, Karl Hörmann e Ulrich R. Goessler. "Skeletal Muscle Regeneration: MSC versus Satellite Cells". Otolaryngology–Head and Neck Surgery 139, n. 2_suppl (agosto 2008): P86. http://dx.doi.org/10.1016/j.otohns.2008.05.484.
Abstract (sommario):
Problem Differentiating stem cells into the myogenic linage in order to create functional muscle tissue is a challenging endeavour. In this work, adipose-derived mesenchymal stem cells (MSC) and satellite cells derived from muscle biopsies were compared regarding proliferation and myogenic differentiation potential under standardized cell culture conditions. This data was obtained in order to discover the most promising type of stem cell for regeneration of muscle tissue and to determine the optimal culture conditions for later clinical use. Methods Human MSC were isolated from adipose tissue, and primary human skeletal myoblasts were extracted from muscle biopsies by enzymatic digestion. Proliferation was analysed using the AlamarBlue® assay. Gene expression of marker genes – such as Myogenin, Myo D, Myf 5 and MHC – were analysed by RT-PCR. Immunostainings against desmin and sarcomeric-actin were performed as differentiation markers. Results MSC cell cultures showed a greater proliferation rate compared with satellite cell cultures. In both stem cell cultures, myogenic differentiation/heritage could be verified by immunostainings against the muscle-specific marker desmin. Gene expression and protein analysis revealed a more stable differentiation of human satellite cell cultures. Conclusion Characterization of both human MSC cultures and satellite cell cultures – and thereby an understanding of myogenesis – might lead to their clinical usage in skeletal muscle tissue engineering. The results in this study appear to indicate that human satellite cell cultures have a more stable differentiation under in vitro conditions and that they might offer a greater potential for skeletal muscle tissue engineering purposes. Significance Our study contributes to the understanding of myogenic differentiation of MSC and satellite cells and helps to improve culture systems for later clinical utilization.
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Cequier, Alina, Antonio Romero, Francisco J. Vázquez, Arantza Vitoria, Elvira Bernad, Sara Fuente, Pilar Zaragoza, Clementina Rodellar e Laura Barrachina. "Equine Mesenchymal Stem Cells Influence the Proliferative Response of Lymphocytes: Effect of Inflammation, Differentiation and MHC-Compatibility". Animals 12, n. 8 (11 aprile 2022): 984. http://dx.doi.org/10.3390/ani12080984.
Abstract (sommario):
Immunomodulation and immunogenicity are pivotal aspects for the therapeutic use of mesenchymal stem cells (MSCs). Since the horse is highly valuable as both a patient and translational model, further knowledge on equine MSC immune properties is required. This study analysed how inflammation, chondrogenic differentiation and compatibility for the major histocompatibility complex (MHC) influence the MSC immunomodulatory–immunogenicity balance. Equine MSCs in basal conditions, pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were co-cultured with either autologous or allogeneic MHC-matched/mismatched lymphocytes in immune-suppressive assays (immunomodulation) and in modified one-way mixed leukocyte reactions (immunogenicity). After co-culture, frequency and proliferation of T cell subsets and B cells were assessed by flow cytometry and interferon-ɣ (IFNɣ) secretion by ELISA. MSC-primed showed higher regulatory potential by decreasing proliferation of cytotoxic and helper T cells and B cells. However, MHC-mismatched MSC-primed can also activate lymphocytes (proliferative response and IFNɣ secretion), likely due to increased MHC-expression. MSC-chondro maintained their regulatory ability and did not increase their immunogenicity, but showed less capacity than MSC-primed to induce regulatory T cells and further stimulated B cells. Subsequent in vivo studies are needed to elucidate the complex interactions between MSCs and the recipient immune system, which is critical to develop safe and effective therapies.
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Kim, G. A., H. J. Oh, J. Kim, T. H. Lee, J. H. Lee, S. H. Oh e B. C. Lee. "293 EFFECT OF MEDIUM TYPE FOR CULTURE OF ADIPOSE-DERIVED MESENCHYMAL STEM CELLS ON PRE-IMPLANTATION DEVELOPMENT OF CLONED EMBRYOS". Reproduction, Fertility and Development 25, n. 1 (2013): 294. http://dx.doi.org/10.1071/rdv25n1ab293.
Abstract (sommario):
Mesenchymal stem cells (MSC) have been known as useful donor cells for somatic cell nuclear transfer (SCNT). It has been suggested that the culture condition of donor cells causes different results on preimplantation development of SCNT embryos. In this study, we investigated the patterns of gene expression of adipose-derived mesenchymal stem cells (ad-MSC) in different culture media (DMEM and RKME), and examined the effect of ad-MSC, with the gene expression changed, used as donor cells on the preimplantation development of cloned embryos. Canine ad-MSC were isolated from fat tissue of 3-year-old female beagle and were cultured in DMEM supplemented with 10% fetal bovine serum (MSC-DMEM) and RKME (MSC-MSC) provided from RNL Bio Corp. (Seoul, Korea). Total RNA was extracted from ad-MSC cultured in each culture medium. After synthesising cDNA of each sample, quantitative RT-PCR was done according to the Takara Bio Inc. guidelines and using the 7300 Real Time PCR Cycler System (Applied Biosystems, Carlsbad, CA, USA). The level of all tested gene transcription was normalized to β-actin expression levels. The relative quantification of gene expression was analysed by the 2–ΔΔCt method. The data from all experiments were analysed by Student’s t-test using a statistical analysis GraphPad Prism 4.02 (GraphPad Software Inc., San Diego, CA, USA). Significance was determined at P < 0.05. The stemness, the reprogramming-related gene expression level of donor cells of MSC-DMEM and MSC-MSC were compared. In order to confirm the effect of MSC cultured in 2 different culture media on somatic cell nuclear transfer, we performed interspecies somatic cell nuclear transfer (iSCNT). The enucleated bovine oocytes were injected, respectively, with donor cells of MSC-DMEM and MSC-MSC, and were fused by electrofusion. The iSCNT embryos were cultured in modified SOF at 38.5°C for 7 days in an atmosphere of 5% CO2 and 5% O2, and the developmental ability of iSCNT embryos was observed under the microscope. The MSC-MSC contained a significantly higher amount of Sox2, Nanog, Oct4, Stella, HDAC1, DNMT1, and MeCP2 than the MSC-DMEM, whereas the amount of Rex1 was not different in either MSC-MSC or MSC-DMEM. In the development ability of iSCNT embryos, MSC-DMEM embryos resulted in a 16-cell embryo formation rate that was higher than that of MSC-MSC embryos (9.09 and 5.30%, respectively; P < 0.05). However, the blastocyst formation rate was not different between MSC-DMEM embryos and MSC-MSC embryos (4.5 and 3.2%, respectively; P > 0.05). These results demonstrate that the gene expression of ad-MSC can be modified, by culture media, into a state where reprogramming is easily done. Even so, ad-MSC with gene expression changed by culture medium did not influence the developmental ability of blastocysts. In conclusion, the alteration of gene-related stemness and reprogramming in canine ad-MSC would not be able to effectively control reprogramming in SCNT. This study was supported by RDA (#PJ0089752012), RNL Bio (#550-20120006), IPET (#311062-04-1-SB010), Research Institute for Veterinary Science, and Nestlé Purina Korea.
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Ludwig, Annette, Rainer Saffrich, Volker Eckstein, Angela Lenze, Anke Diehlmann, Anthony D. Ho e Patrick Wuchter. "Plerixafor Abrogates the Supportive Function of MSC for Self-Renewal of Human Hematopoietic Stem Cells",. Blood 118, n. 21 (18 novembre 2011): 3408. http://dx.doi.org/10.1182/blood.v118.21.3408.3408.
Abstract (sommario):
Abstract Abstract 3408 The CXCR4-SDF1α axis plays an important role in maintaining the stemness of human hematopoietic stem cells (HSC). In the present study we established a surrogate model for the bone marrow niche by culturing HSC on a feeder-layer of human mesenchymal stromal cells (MSC) and investigated the proliferation and differentiation behaviour upon perturbation by Plerixafor. HSC (CD34+ cells) were isolated from umbilical cord blood by fluorescent activated cell sorting (FACS). MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were stained with carboxyfluorescein succinimidyl ester (CFSE) and cultured on MSC feeder-layer for 6 days. For evaluating the influence of the culture medium on MSC cultures, three different media conditions (M1-M3) were used. Medium 1 (M1) contained 2% fetal calf serum (FCS), medium 2 (M2) contained 10% FCS and medium 3 (M3) contained GMP-grade human platelet lysate (hPL). Proliferation of HSC was calculated by analyzing the distribution of the CFSE dye (measured at day 1 and day 6). Plerixafor was added in concentrations of 0.1, 1.0 and 10.0 μM. On day 6, HSCs were harvested and analyzed by flow cytometry for CD34, CD38 and CXCR4 expressions in relationship to the cell division rate. When co-cultured with MSC, the division kinetics of HSC was increased, while the proportion of CD34+ cells remained significantly higher compared to HSC without MSC. Accordingly, more CD38− HSC were found after 6 days upon co-culture with MSC. All three MSC preparations supported self-renewing proliferation of HSC, whereas MSC M1 induced the strongest effect. This underlines that co-culture with MSC has a significant supportive function for hematopoiesis. The additional exposure to Plerixafor in the co-culture system partially reversed this effect in a dose-dependent manner: division rate of HSC and the proportion of CD34+ and CD38− cells were reduced with higher concentrations of Plerixafor. The reduction of self-renewing proliferation by Plerixafor was not observed in controls consisting of HSC without MSC. Plerixafor also rendered the CXCR4 receptors undetectable on the surface of CD34+ cells for up to 6 days, most probably due to a persisting blockade of the antibody-binding site. Human HSC co-cultured with MSC showed an increased cell division rate and produced a higher proportion of CD34+/CD38− cells. Different MSC culture media were systematically analysed in this setting and subtle differences in the supportive function could be observed. The addition of Plerixafor neutralized the effects of MSC, leading to an earlier loss of “stemness” and to lineage-commitment of HSC, thus providing evidence for the role of the CXCR4/SDF1α axis in terms of supportive function of MSC for self-renewal of HSC. Disclosures: Ho: Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Ito, Sawa, A. John Barrett, Andre Larochelle, Nancy F. Hensel, Keyvan Keyvanfar e J. Joseph Melenhorst. "Long Term Maintenance of Myeloid Leukemia Stem Cell-Like Populations Cultured with Mesenchymal Stromal Cells (MSC)". Blood 120, n. 21 (16 novembre 2012): 3546. http://dx.doi.org/10.1182/blood.v120.21.3546.3546.
Abstract (sommario):
Abstract Abstract 3546 Because MSC support the growth and the differentiation of normal hematopoietic stem cells we hypothesized that MSC might also support leukemia cells, in particular leukemia stem cells (LSC) in vitro. We cultured blast cells from patients with acute myelogenous leukemia (AML) in liquid medium to study persistence of stem-cell-like and differentiated leukemia cell populations by flow cytometry, with and without MSC and additional growth factors. Cryopresrerved peripheral blood mononuclear cells (PBMC) were obtained from 6 AML patients (mean Age 47, range 23–74). Leukemia blasts were isolated by sorting live (propidium iodide (PI)-negative) CD34+ lineage (CD2+, CD3+, CD14+ and CD19+) -negative cells using a FACS ARIA II cell sorter (BD). Sorted blasts (2.5 ×105 cells) were co-cultured with an equal number of irradiated MSC derived from healthy donor bone marrow in RPMI medium supplemented with 10% human serum, with or without a human cytokine (CYTO) mixture (50 ng/ml interleukin 3, 150 ng/ml stem cell factor, and 150ng/ml Flt-3 ligand). MSC were replenished every two weeks. The phenotype of cultured cells was analyzed weekly using fluorescently-conjugated monoclonal antibodies against CD34, CD38, and CD45, plus the lineage panel and a dead cell exclusion dye Cell cycle analysis with Hoeschst 33342 and Pyronin Y was performed on cells co-stained with CD34, CD45 and PI. Primary leukemia samples were phenotypically heterogeneous with respect to proportions of cells (co-)staining for CD34 and CD38 as previously reported: three samples showed CD34+CD38- predominance (LSC-like leukemia), and three were CD34+CD38+ (common myeloid progenitor (CMP)-like leukemia). LSC-like leukemia maintained viable CD34+CD38- cells for at least 6 weeks when co-cultured with MSC alone, in contrast to cultures with cytokines or medium only which showed rapid decline in the LSC populations and no prolonged maintenance of viable cells (p=0.0005) (Figure, left panel). CMP-like leukemia maintained their CD34+CD38+ phenotype when co-cultured with MSC alone but persistence of this subset was not significantly different from the other culture conditions (p=0.5) and no culture remained viable after 4 weeks (Figure, right panel). Cell cycle analysis showed that co-culture with MSC maintained CD34+ blasts in G0 significantly more than other culture conditions (P<0.0001). We conclude that MSC support the maintenance of a leukemia stem cell phenotype in a long- term (6 week) in vitro culture system. The differential capacity of MSC to support LSC- like and CMP- like leukemia may be associated with the different frequency of leukemia initiating cells within each leukemic blast population. NSG mice xenotranplant model experiments are ongoing to confirm this hypothesis. Co-culture of LSC with MSC represents a simple approach to maintain LSC in vitro and could be utilized to screen the drug targeting LSCs. Further study of the effect of MSC on LSC would elucidate a potential mechanism whereby the marrow microenvironment serves as a reservoir of persisting leukemia after remission induction chemotherapy. Disclosures: No relevant conflicts of interest to declare.
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Chitteti, Brahmananda Reddy, Bradley Poteat, Sonia Rodriguez Rodriquez, Nadia Carlesso, Melissa A. Kacena e Edward F. Srour. "Interactions of the Cellular Components of the Hematopoietic Niche". Blood 112, n. 11 (16 novembre 2008): 3563. http://dx.doi.org/10.1182/blood.v112.11.3563.3563.
Abstract (sommario):
Abstract Hematopoietic Stem Cell (HSC) self-renewal and multilineage differentiation potential is governed by multiple intrinsic and extrinsic parameters. Collectively, these parameters dictate the fate of HSC and underscore the heterogeneity observed within phenotypically defined groups of stem cells. While cell cycle status and the genetic profile of HSCs are critical intrinsic modulators of cell fate, interactions with cytokines, growth factors, and cellular elements of the hematopoietic niche (HN) are key extrinsic regulators of stem cell function. We examined the impact of cellular elements of the HN on stem cell fate and maintenance by analyzing the combined effect of calvaria-derived osteoblasts (OB) and mesenchymal stromal cells (MSC) on cultured murine HSC. Murine bone marrow-derived KSL cells were co-cultured with OB alone, MSC alone, or with mixtures of OB and MSC at different ratios for one week. Cultures were supplemented with SCF, Fl-3, Tpo, IL-3, IL-6, IGF1 & OPN. OB alone, maintained the functional properties of cultured HSCs significantly better than MSC thus corroborating the importance of OB in the overall competence of the HN. On day 7, the fold-increase in the number of LSK cells was 1473 ± 291 in OB cultures, 561 ± 159 in MSC cultures, and 603 ± 263 in OB+MSC cultures (n= 4 for all 3 groups). During the same 7 day-period, the number of CFU in progeny cells expanded 74 ± 15 fold in OB cultures, 23 ± 2 fold in MSC cultures, and 27 ± 15 in OB+MSC cultures (n=3 for all groups). The substantial increase in KSL progeny in OB cultures on day 7 was accompanied by a high percentage of cells in active phases of cell cycle (% G0/G1 = 72.5 ± 7.0, n=3) compared to their counterparts in MSC or OB+MSC cultures. In addition, co-culture of KSL cells with OB resulted in an unexpected higher maintenance of the Sca-1+Lin- phenotype (26.5% ± 2.8%) relative to MSC cultures (4.6% ± 1.0%) and OB+MSC cultures (11.7% ± 1.8%; n=3 for all). Only some of these results were reproduced when KSL cells were cultured in OB-conditioned medium suggesting that cell-to-cell contact may be essential for the observed activities. To assess the in vivo potential of LSK cells maintained in these cultures, the 10-day expansion equivalent of 1,000 LSK cells were competitively transplanted in lethally irradiated congenic mice and chimerism was monitored for the next 4 months. At 1 and 2 months post-transplantation, the level of chimerism sustained by LSK cells maintained in OB cultures for 10 days surpassed or was slightly lower than that observed with freshly isolated LSK cells (72.7% vs 59.7% and 57.4% vs 74.7%, respectively) suggesting that OB culture conditions effectively expanded short-term repopulating cells. At 4 months post-transplantation, mice receiving freshly isolated LSK cells were 83.6% ± 1.8% chimeric compared to 53.7% ± 16.1% for mice transplanted with cells from OB cultures and 31.9% ± 21.4% for mice receiving cells from OB+MSC cultures. Overall, these data suggest that OB-LSK interactions promote the maintenance of both short-term and long-term repopulating cells while MSC suppress the OB-mediated activity. To investigate the mechanism of OB-mediated maintenance of stem cell phenotype and function, we examined Notch signaling using Real-Time Q-PCR on cells maintained in culture for 7 days. Relative to the expression in KSL cells, expression of Notch 2 was elevated in OB cultures and suppressed over 2-fold in cultures of MSC and OB+MSC. Similarly, the expression of Jagged 1 and 2, Delta 1 and 4, Hes 1 and 5, Deltex, and SKP2 was increased in OB cultures and suppressed in MSC and OB+MSC cultures. Collectively, these data illustrate that cell-to-cell contact between OB and KSL cells promotes the in vitro maintenance of long-term and short-term repopulating cells and suggest that this stem cell function-promoting activity is induced in part by the upregulation of Notch-mediated signaling between HSCs and osteoblasts. The suppressive effect imparted by MSC on stem cell maintenance compared to cultures of OB alone suggest that these two cellular elements of the HN have opposite effects on the fate and function of stem cells.
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Rühle, Alexander, Marie Lies, Maren Strack, Ramon Lopez Perez, Birgit Bieber, Andreas R. Thomsen, Peter Bronsert et al. "Human Mesenchymal Stromal Cells Do Not Cause Radioprotection of Head-and-Neck Squamous Cell Carcinoma". International Journal of Molecular Sciences 23, n. 14 (12 luglio 2022): 7689. http://dx.doi.org/10.3390/ijms23147689.
Abstract (sommario):
Radiotherapy of head-and-neck squamous cell carcinoma (HNSCC) can cause considerable normal tissue injuries, and mesenchymal stromal cells (MSCs) have been shown to aid regeneration of irradiation-damaged normal tissues. However, utilization of MSC-based treatments for HNSCC patients undergoing radiotherapy is hampered by concerns regarding potential radioprotective effects. We therefore investigated the influence of MSCs on the radiosensitivity of HNSCCs. Several human papillomavirus (HPV)-negative and HPV-positive HNSCCs were co-cultured with human bone marrow-derived MSCs using two-dimensional and three-dimensional assays. Clonogenic survival, proliferation, and viability of HNSCCs after radiotherapy were assessed depending on MSC co-culture. Flow cytometry analyses were conducted to examine the influence of MSCs on irradiation-induced cell cycle distribution and apoptosis induction in HNSCCs. Immunofluorescence stainings of γH2AX were conducted to determine the levels of residual irradiation-induced DNA double-strand breaks. Levels of connective tissue growth factor (CTGF), a multifunctional pro-tumorigenic cytokine, were analyzed using enzyme-linked immunosorbent assays. Neither direct MSC co-culture nor MSC-conditioned medium exerted radioprotective effects on HNSCCs as determined by clonogenic survival, proliferation, and viability assays. Consistently, three-dimensional microwell arrays revealed no radioprotective effects of MSCs. Irradiation resulted in a G2/M arrest of HNSCCs at 96 h independently of MSC co-culture. HNSCCs’ apoptosis rates were increased by irradiation irrespective of MSCs. Numbers of residual γH2AX foci after irradiation with 2 or 8 Gy were comparable between mono- and co-cultures. MSC mono-cultures and HNSCC-MSC co-cultures exhibited comparable CTGF levels. We did not detect radioprotective effects of human MSCs on HNSCCs. Our results suggest that the usage of MSC-based therapies for radiotherapy-related toxicities in HNSCC patients may be safe in the context of absent radioprotection.
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Zhang, Xiaohong, Masako Hirai, Susana Cantero, Rodica Ciubotariu, Allen Hirsh, Hitoshi Satoh, Izuru Yokomi et al. "Reevaluation of Factors Leading to the Successful Isolation of Mesenchymal Stem Cells From Human Umbilical Cord Blood and Their Differentiation to Mesenchymal Lineages." Blood 114, n. 22 (20 novembre 2009): 3232. http://dx.doi.org/10.1182/blood.v114.22.3232.3232.
Abstract (sommario):
Abstract Abstract 3232 Poster Board III-169 Introduction Cord blood derived mesenchymal stem cells (CB-MSC) have been identified as an alternative cell source to bone marrow derived mesenchymal stem cells (BM-MSC) and adipose tissue derived mesenchymal stem cells (AT-MSC) for use in regenerative medicine. However, the low frequency of these cells in cord blood (CB) has led to conflicting reports of its efficacy and this, in turn, has been the main reason limiting their clinical use thus far. We searched for critical factors determining successful isolation of CB-MSC from more than 300 units of CB donated to two public CB banks using a range of different collection methods for CB. We applied several processing and culture methods to identify an optimal method for isolating CB-MSC. Proliferative, in vitro differentiation ability and immunosuppressive ability of CB-MSC were compared with BM- and AT-MSC. CB-MSC cultured with scaffolds were transplanted to nude mice. Additionally, chromosomal stability of CB-MSC after long-term culture was analyzed. Materials and Methods CB was collected after obtaining informed consent at two collection facilities: either while the placenta was in utero, or after the delivery of the placenta (ex utero). The mononuclear cells (MNC) were isolated by Ficoll-Paque (FP) density gradient centrifugation or other methods and subjected to a colony forming unit-fibroblast (CFU-F) assay. Their ability to differentiate into osteoblasts, chondorocytes, and adipocytes was tested in vitro and in vivo. Specific genes for differentiation to the mesoderm lineage were identified by RT-PCR. Immunosuppression by CB-MSC was tested by addition of cells to phytohemagglutinin (PHA) activated human T cells and to mixed lymphocyte reactions. Karyotypes of expanded CB-MSC were analyzed. Osteogenesis and chondrogenesis of CB-MSC in vivo were examined by transplantation of CB-MSC with scaffolds (β-TCP block, collagen sponge) subcutaneously to nude mice. Results CB-MSCs capable of proliferating were isolated from 121 units of 307 units of CB (63.1 ± 20.7 ml w/o anticoagulant). Two critical factors contributing to the success rate of isolating CB-MSC were: interval between collection of CB and processing of cells, and CB volume. When the interval was less than 2 hours there was a marked increase in success, S, according to the equation S=0.55*t-0.4316, (R2>0.99, n=81). There was also a more modest increase in S from increasing volume: S=0.0034*V (ml) + 0.2244, (R2>0.85, n=249). When both volume was higher than 90 ml and time was shorter than 5 hours, the success rate increased to 84.6%. The mean number of clonies from the units was calculated to be 1.59 ± 1.48 CFU /108 MNC (n=40) and 2.7 ± 2.3 CFU/CB unit. Variation in isolation and culture methods of did not improve the success rate. Most CB-MSC isolated grew rapidly and proliferated at more than 40 PDL (>15 passages), whereas BM-MSC and AT-MSC stopped proliferating at about 10 PDL. The CB-MSC showed higher differentiation ability to chondrocytes more than BM-MSC and AT-MSC. In vivo osteogenesis and chondrogenesis were observed when CB-MSC cultured with scaffolds were transplanted subcutaneously to nude mice. CB-MSC suppressed proliferation of lymphocytes stimilated allogeneically (mixed lymphocyte reaction) and by PHA as the dose of cells increased similar to finding with BM-MSC and AT-MSC. Gene expression related to the differentiation to the mesenchymal lineage indicated that CB-MSC can differentiate towards osteoblasts and chondrocytes. CB-MSC derived cell lines maintained normal karyotypes when the cells were cultured up to 40 PDL. Conclusions Among several factors possibly responsible for success in isolating CB-MSC, time between delivery and processing was decisive and volume was also critical. Even though the frequency of CB-MSC was much lower initially than BM-MSC, the high proliferation rate of these cells should allow expansion to cell numbers adequate for clinical use. High proliferation rate combined with high differentiation capability and the karyotype stability after long culture, indicate that CB-MSC should be a potential practical source of MSC for regenerative medicine. Disclosures No relevant conflicts of interest to declare.
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Segunda, Moisés N., Carlos Díaz, Cristian G. Torres, Víctor H. Parraguez, Mónica De los Reyes e Oscar A. Peralta. "Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC)". Animals 13, n. 2 (16 gennaio 2023): 318. http://dx.doi.org/10.3390/ani13020318.
Abstract (sommario):
Although spermatogonial stem cells (SSC) constitute primary candidates for in vitro germ cell (GC) derivation, they are scarce and difficult to maintain in an undifferentiated state. Alternatively, mesenchymal stem cells (MSC) are also candidates for GC derivation due to their simplicity for culture and multipotential for transdifferentiation. The aim of the present study was to compare the GC differentiation potentials of bull peripheral blood-derived MSC (PB-MSC) and SSC using an in vitro 3D co-culture system with Sertoli cells (SC). Samples of PB-MSC or SSC co-cultures with SC were collected on days 0, 7, 14 and 21 and analyzed for pluripotency, GC and mesenchymal marker expression. Co-culture of PB-MSC+SC resulted in down-regulation of NANOG and up-regulation of OCT4 at day 7. In comparison, co-culture of SSC+SC resulted in consistent expression of NANOG, OCT4 and SOX2 at day 14. During co-culture, SSC+SC increased the expression of DAZL, PIWIL2, FRAGILIS and STELLA and activated the expression of STRA8, whereas co-culture of PB-MSC+SC only increased the expression of DAZL and PIWIL2. Thus, co-culture of bull PB-MSC+SC and SSC+SC in 3D SACS results in differential expression of pluripotency and GC markers, where bull SSC display a more robust GC differentiation profile compared to PB-MSC.
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Kotobuki, Noriko, Koji Ioku, Daisuke Kawagoe, Daishiro Nomura, Hirotaka Fujimori, Seishi Goto e Hajime Ohgushi. "In Vitro Osteogenic Activity of Rat Mesenchymal Cells Cultured on Transparent β-Tricalcium Phosphate Ceramics". Key Engineering Materials 284-286 (aprile 2005): 663–66. http://dx.doi.org/10.4028/www.scientific.net/kem.284-286.663.
Abstract (sommario):
We have cultured mesenchymal cells (MSC) on various types of ceramic disks and used these tissue-engineered ceramics for hard tissue regeneration. In this approach, observation of cultured cell morphology is important even if culture substrata are calcium phosphate ceramics, which usually show bioactive nature. However, due to the opaque nature of the ceramics, cells observation is very difficult. Here, we demonstrate light microscopic observation of rat MSC cultured on transparent β-tricalcium phosphate ceramics (β-TCP). The culture was performed in osteogenic medium. Thus, the cell differentiated into bone-forming osteoblasts, which fabricated a mineralized matrix on the ceramic disks. Microscopic observation revealed that the cascade of osteogenic differentiation after attachment/proliferation of MSC on the ceramic disks was similar to that on a culture grade polystyrene dish. These results confirmed the excellent property of β-TCP for MSC culture leading to hard tissue regeneration.
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Ding, Wei, Grzegorz S. Nowakowski, Jennifer L. Abrahamzon, Linda E. Wellik, Asish K. Ghosh, Charla R. Secreto, Kay L. Medina, Tait D. Shanafelt, Clive S. Zent e Neil E. Kay. "Crosstalk between Chronic Lymphocytic Leukemia (CLL) B-Cells and Marrow Stromal Cells: Implication for CLL B-Cell Activation and Survival." Blood 110, n. 11 (16 novembre 2007): 337. http://dx.doi.org/10.1182/blood.v110.11.337.337.
Abstract (sommario):
Abstract It is believed that malignant cells “condition” the microenvironment to facilitate tumor cell survival. We hypothesized that crosstalk between CLL B-cells and marrow stromal cells impacts both cell types bi-directionally and ultimately contributes to leukemic cell apoptotic resistance. To test this hypotheses, bone marrow stromal cells from core bone biopsies from CLL patients were isolated and cultured using methods we have previously described (Leuk Res 2007 31(7):899). Subsequently, we determined the impact of co-culture on CLL B-cell features including apoptosis and CD38 expression. In addition, we evaluated the release of angiogenic cytokines on co-culture and signal events in the stromal cells. Immunophenotyping demonstrated that cultured bone biopsy derived stromal cells were CD73+, CD105+, CD146+, CD14−, CD45−, CD34−, HLA-DR-, suggesting they were mesenchymal stem cells (MSC). Co-culture of these MSC with CLL B-cells protected CLL B-cells from both spontaneous apoptosis (SA) and drug-induced (fludarabine and chlorambucil) apoptosis (DA). For SA, the mean survival of CLL B-cells with or without co-culture of MSC for 5 days were 56.9 ± 10.0 and 7.7 ±3.7 (p<0.05), respectively. When CLL B cells were treated with fludarabine or chlorambucil, the fraction of CLL cells tightly adherent to MSC (TA-CLL) showed higher survival than a less adherent but viable fraction of CLL B-cells. The mean survival of TA-CLL cells treated with 10 μM of fludarabine for 48 hours in the presence of MSC were 67.5 ± 3.6 vs 29.8 ± 11.1 without MSC (P<0.05), respectively. When CLL cells with evidence for CD38 expression were co-cultured with MSC, both the percentage of CD38 positive cells and level of expression of CD38 per cell were up-regulated (mean fold change: CD38 percentage, 2.7, p<0.05; CD38 MFI, 1.9, p<0.05) after 2 weeks. In contrast, the CD38 percentage and expression were not changed in cells with minimal CD38 expression when these CLL B-cells were co-cultured with MSC. In addition, co-culture of MSC with CLL cells induced rapid ERK and AKT phosphorylation (within 30 min) in the MSC on immunoblot analysis. When CLL B cells and MSCs were cultured in transwells, the activation of ERK and AKT in MSC occurred at similar levels, indicating that activation of MSC was mediated by soluble factors. In addition, co-culture led to increased secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as well as a decrease of thrombospondin-1 (TSP-1) in the culture medium. These findings confirm that co-culture of CLL B-cells and MSC culminates in “angiogenic switch.” Taken together, these results strongly suggest interactions between MSC and CLL B cells are a bi-directional process. In leukemic cells, the interaction not only protects against spontaneous and drug induced apoptosis but also leads to an increase in CD38 expression consistent with an activated status. In MSC, the interaction leads to activation of ERK and AKT. Co-culture also facilitates angiogenic switching. These results underscore the dynamic and complex nature of the interactions between bone marrow stromal cells and CLL B-cells. Further studies are needed to dissect how crosstalk between CLL B-cells and MSC relates to disease progression, and determines whether these interactions can be targeted with therapeutic intent.
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Ichii, Michiko, Kenji Oritani, Takafumi Yokota, Isao Takahashi, Takahiro Shirogane, Norimitsu Saitoh, Rie Tanigawa, Satomi Kasai e Yuzuru Kanakura. "Establishment of Stroma-Free Cultures for Human B Lymphopoiesis: Roles of High Cell Density Condition and Mesenchymal Stem Cell-Secreted Factors." Blood 110, n. 11 (16 novembre 2007): 1420. http://dx.doi.org/10.1182/blood.v110.11.1420.1420.
Abstract (sommario):
Abstract Background: Due to lacking of appropriate culture systems, it has been difficult to analyze mechanisms of human B lymphocytes. However, we recently found that human mesenchymal stem cells (MSC) have high ability to support human B lymphopoiesis. Although many investigators have believed that stromal layers are essential for supporting the differentiation of human B lymphocyte progenitors, this hypothesis is not a case. By manipulating our co-culture system, we have successfully established stroma-free suspension cultures, which produce CD10+ B cells from human umbilical cord blood (CB) CD34+ cells. Methods: Our stroma-free culture of CB CD34+ cells was performed in QBSF® medium with 10% FCS in the presence of 10 ng/ml stem cell factor (SCF) and 5 ng/ml Flt3-ligand (FL) with or without 5 ng/ml IL-7. Results: Even when co-cultures were separated from MSC with membrane filters, they could produce CD10+ cells. Moreover, addition of MSC supernatant to the cultures permitted CD34+ cells to emerge CD10+ cells in the absence of MSC. Therefore, the cell-cell contact between MSC and B lymphocyte progenitors was not essential. For stroma-free human B cell cultures with MSC supernatant, QBSF® was the most suitable and serum components were essential while depending on their lot. Although addition of thymic stromal lymphopoietin or IL-7 increased the production of CD10+ cells, neutralizing antibodies for them showed no effect. Addition of Hemokinin-1 antagonist diminished, albeit to a limited degree, the production of CD10+ cells. Addition of neutralizing antibody for CXCR4 had no effect. Therefore, MSC supernatant contains some supportive factors except for them. When cultures without MSC or their supernatant stared at 1x104 cells/ml, they could successfully produce CD10+ cells. The density of the cultured cells was critical. The production of CD10+ cells was not detected when CD34+ cells were seeded at low density (1x103/ml). Moreover, when the cultured cells were diluted and adjusted at 1x104/ml weekly, the emergence of CD10+ cells was not observed while the production of CD33+ myeloid cells was enhanced. Therefore, surrounding hematopoietic cells seemed to be required to support human B lymphopoiesis. Our suspension cultures of 1x104 CD34+ cells in the presence of SCF, FL, and IL-7 without any stromal materials generated approximately 0.5–1x106 CD10+ cells at 4 week. CD33+ cells were first expanded within 2 weeks, and then CD10+ cells appeared. When the cultured cells were transplanted into NOD/SCID/γcnull mice, they reconstituted both myeloid and B lymphoid lineages. Therefore, some cultured cells maintain stem cell character. Conclusions: We have established stroma-free suspension cultures, which effectively produce CD10+ B cells from CB CD34+ cells. High cell density condition can in part substitute for stromal layers in supporting human B lymphopoiesis although the addition of MSC supernatant enhances the production of CD10+ cells. Our suspension culture does not use any stromal cells, which produce many positive or negative regulators for human B lymphopoiesis. This simplicity proposes that this culture system is useful in a variety of fields such as the screening direct effects of drugs influencing on human B lymphocyte development and the evaluation of progenitors in patients with B-cell malignancies as well as the cloning of human B lymphocyte-supportive molecules.
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Brandhorst, Heide, Daniel Brandhorst, Anju Abraham, Samuel Acreman, Simen W. Schive, Hanne Scholz e Paul R. V. Johnson. "Proteomic Profiling Reveals the Ambivalent Character of the Mesenchymal Stem Cell Secretome: Assessing the Effect of Preconditioned Media on Isolated Human Islets". Cell Transplantation 29 (1 gennaio 2020): 096368972095233. http://dx.doi.org/10.1177/0963689720952332.
Abstract (sommario):
Previous studies in rodents have indicated that function and survival of transplanted islets can be substantially improved by mesenchymal stem cells (MSC). The few human islet studies to date have confirmed these findings but have not determined whether physical contact between MSC and islets is required or whether the benefit to islets results from MSC-secreted proteins. This study aimed to investigate the protective capacity of MSC-preconditioned media for human islets. MSC were cultured for 2 or 5 days in normoxia or hypoxia before harvesting the cell-depleted media for human islet culture in normoxia or hypoxia for 6–8 or 3–4 days, respectively. To characterize MSC-preconditioned media, proteomic secretome profiling was performed to identify angiogenesis- and inflammation-related proteins. A protective effect of MSC-preconditioned media on survival and in vitro function of hypoxic human islets was observed irrespective of the atmosphere used for MSC preconditioning. Islet morphology changed markedly when media from hypoxic MSC were used for culture. However, PDX-1 and insulin gene expression did not confirm a change in the genetic phenotype of these islets. Proteomic profiling of preconditioned media revealed the heterogenicity of the secretome comprising angiogenic and antiapoptotic as well as angiostatic or proinflammatory mediators released at an identical pattern regardless whether MSC had been cultured in normoxic or hypoxic atmosphere. These findings do not allow a clear discrimination between normoxia and hypoxia as stimulus for protective MSC capabilities but indicate an ambivalent character of the MSC angiogenesis- and inflammation-related secretome. Nevertheless, culture of human islets in acellular MSC-preconditioned media resulted in improved morphological and functional islet integrity suggesting a disbalance in favor of protective factors. Further approaches should aim to eliminate potentially detrimental factors to enable the production of advanced clinical grade islet culture media with higher protective qualities.
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Jarocha, Danuta, Ewa Lesko, Mariusz Z. Ratajczak e Marcin Majka. "Comparison of Different Strategies of MSC Isolation Revels Advantage To Expand MSC Directly from Purified CD105+ and CD271+ Cells." Blood 108, n. 11 (16 novembre 2006): 2566. http://dx.doi.org/10.1182/blood.v108.11.2566.2566.
Abstract (sommario):
Abstract The potential for multilineage differentiation together with the ability to expand in cultures are the reasons why Mesenchymal Stem Cells (MSC) are considered to be the population of stem cells for potential treatment for variety of disorders (e. g. Osteogenesis Imperfecta, Myocardium Infarction, GvHD). MSC are isolated from the bone marrow mononuclear cells (MNC) based on their adhesive properties. There have been few attempts to isolate MSC directly based on the expression of selected surface antigens, but these isolation strategies were not compared with “the gold standard” procedure which is still selection by plastic adherence. Nevertheless, it is obvious that a presence of different populations of cells “contaminating” MSC in adherent cell cultures (e.g., endothelial cells, macrophages, dendritic cells) may affect expansion of MSC. In this study we proposed new isolation strategies of bone marrow MSC based on RosetteSep Isolation Kit (Stem Cells Technologies Inc., Vancouver, Canada) and immunomagnetic isolation of CD105+ or CD271+ cell populations (Miltenyi Biotec, Germany). Four fractions of bone marrow mononuclear cells i) non-purified MNC, ii) MNC enriched in MSC by RosetteSep Isolation Kit, iii) sorted CD105+ and iv) sorted CD271+ cells were cultured for three passages. Subsequently, we evaluated i) number of CFU-F colonies, ii) expression of selected surface antigens (CD105, CD166, CD44, CD73, CD45, CD34), iii) in vitro osteogenic differentiation of expanded cells and iv) changes in the expression of genes related to osteogenesis (RQ-PCR). We found that the mean number of CFU-F colonies counted on the 9th day of culture was 26 (range 14,5–41,4), 49 (range 21,2–97,1), 105 (range 36,5–221) and 148 (range 55,3–211) per 107 MNC for non-purified MNC, MNC enriched in MSC by RosetteSep Isolation Kit, purified CD105+cells and purified CD271+cells, respectively. After 3rd passage the phenotype of cells was similar as we observed a comparable percentage of cells positive (over 90%) for CD105, CD166, CD44, CD73 and negative (below 5%) for CD45, CD34 surface antigens in all fractions. The RQ - PCR analysis of mRNA level of osteogenic (osteocalcin, PTHR, α1collagen), adipogenic (lipoprotein lipase, leptin, PPARγ2) and chondrogenic (aggrecan1) genes in all four populations revealed that MSC isolated by means of expression of 105 and CD271 antigens had higher level of mRNA for all assessed genes except for lipoprotein lipase and α1collagen prior to differentiation. After 30 days of osteogenic differentiation RQ - PCR analysis was repeated and compared with that before differentiation. We noticed an increased level of mRNA for osteocalcin and PTHR (markers of osteogenic differentiation) in all four populations, with the highest expression in MSC derived from non-purified MNC. However, this fraction had also the highest mRNA level of PPARγ2, lipoprotein lipase, and aggrecan genes (adipogenic and chondrogenic lineage respectively). Since the highest number of CFU-F was derived from purified CD105+ and CD271+ cells as well as these two populations seem to be the most homogenous based on RQ-PCR data, these cell fractions should be employed to expand most efficiently MSC for potential therapeutic purposes. Our data suggest that, non-MSC cells present in MNC and RosetteSep cultures may negatively affect both the expansion efficiency and differentiation along desired MSC lineage.
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Reese, Jane S., Luis A. Solchaga, Karen T. Lingas, Hillard M. Lazarus e Stanton L. Gerson. "Improved Culture Expansion of Human Mesenchymal Stem Cells (MSCs) Using Fibroblastic Growth Factor-2 for the Treatment of Graft Versus Host Disease (GVHD)." Blood 110, n. 11 (16 novembre 2007): 1207. http://dx.doi.org/10.1182/blood.v110.11.1207.1207.
Abstract (sommario):
Abstract Allogeneic hematopoietic stem cell (HSC) transplantation is an effective therapy for a number of diseases. However, graft-versus-host disease (GVHD) remains a significant obstacle to the successful outcome of this procedure. We have demonstrated in Phase I clinical trials that co-transplantation of culture expanded human MSCs during allogeneic HSC transplantation can facilitate engraftment without increasing the risk of GVHD. MSC have been shown to have immunomodulatory activity, and decrease T-cell interferon (IFN)-γ production. More recently, clinical studies have suggested that MSC infusion can also reduce the severity of GVHD. Based on these data, we have initiated a Phase I clinical trial (CWRU 3Y03) using allogeneic same-sibling donor MSC infusion of 1–6 × 106 culture expanded MSCs per kg for the treatment of acute or chronic GVHD of clinical grade II – IV after sibling donor HSC transplant. One of the main hurdles to overcome in MSC infusion protocols is the expansion of single donor MSCs to achieve the prescribed cell dose in a specified time frame. Here we report that the addition of recombinant human FGF-2 to the culture medium expedites and enhances MSC expansion capacity in normal adult donors cultured under clinically relevant conditions. In pre-clinical studies, MSCs from 13 normal donors (median age 28.5; range 21 – 40) were cultured in standard growth medium with or without FGF-2. The cells were expanded for up to 8 passages. MSCs expanded in the presence of FGF-2 exhibited shorter population doubling times and achieved a greater number of population doublings (31 ± 3) than those expanded in standard conditions (23 ± 2). These FGF-stimulated MSCs exhibited the same phenotype and immunomodulatory potential as MSCs grown in conventional medium. For each patient enrolled on CWRU 3Y03, donor MSCs were harvested, culture expanded and cryopreserved until indicated. Twenty-three culture expansions were attempted from 21 different donors (18 without FGF; 3 with FGF; median donor age 52, range 38 – 67). From the cultures grown in the absence of FGF, 11 donors were expanded to an infusion dose of 0.5 – 2.4 × 106 cells/kg (based on patient weight) with a mean of 161 ± 54 × 106 MSCs at harvest and a median cell expansion time of 41 days (range 23 – 66). Nine cultures failed to reach a minimum cell dose of 0.5 × 106 cells/kg during an 8-week culture period. The three MSC cultures grown in the presence of FGF -2 (R&D systems) were successful and reached infusion doses of 1.65 – 2.4 × 106 cells/kg. In these 3 cultures, the mean number of MSCs was 135 × 106 and the median day to harvest was 28 (range 27 – 41). The immunomodulatory potential of these three MSC preparations was tested in vitro in an IFN-γ EliSpot assay in which they inhibited IFN-γ production by 87.6 ± 5.4%. Cells from one donor that failed to expand in media without FGF-2 reached 171 × 106 (2.8 × 106/kg) MSCs in 27 days when MSCs from a second marrow harvest from the same donor were cultured in medium containing FGF-2, suggesting that FGF-2 supplementation may rescue a culture that may be otherwise unable to expand. Thus, FGF-2 can facilitate MSC culture expansion for clinical use while retaining their immunomodulatory function.
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Peters, Rowayda Elias, Melissa Mock, Peter Meier, Christoph Renner, Alexander Knuth e Bruno Stieger. "Differentiation of Non-Adherent Hematopoietic Stem Cells from Umbilical Cord Blood Cells into Adherent Hepatocytic Lineage." Blood 108, n. 11 (16 novembre 2006): 2578. http://dx.doi.org/10.1182/blood.v108.11.2578.2578.
Abstract (sommario):
Abstract The liver consists of a variety of different cell types, the vast majority of which are hepatocytes. It has been shown that adult, as well as umbilical cord blood (UCB) mesenchymal stem cells (MSC) are capable of differentiation into mesenchymal and non-mesenchymal tissues including hepatocytes. Only a limited number of UCB units (20%) such as frozen, unfractionated and concentrate leukocytes induced by hydroxyethyl starch volume reduced UCB units can generate MSC. In light of this, we have developed the technology to generate potent MSC from all frozen UCB samples tested (3/3). In the present study, the incubation of thawed out UCB mononuclear cells (MNC) and non-selected CD34+ cells in the presence of SCF (25ng/ml), FLt-3 (25ng/ml), MGDF (10ng/ml) & IL-6 (20ng/ml) and 10% human serum in stroma-free liquid culture not only generated long term expansion of transplantable UCB hematopoietic stem cells (non-adherent) (Peters et al., 2004), but also long-term expansion of MSC (adherent cells). Adherent cells were enriched by medium changes followed by trypsinization and subsequent culture and passing on fibronectin in the presence of 10% human serum and 30ng/ml bFGF. The stroma-free liquid cultures were tested for the production of hematopoietic stem cells (HSC), as well as MSC over a period of 15 weeks; MSC increased throughout expansion of HSC. We used the CFU-Fibroblast assay to demonstrate MSC activity in stromal cell formation in vitro. Expanded MSC between passages 3–5 were plated using MesenCult™ (Stem Cell Technologies). After 14 days incubation at 37oC, 5% CO2, very large and small CFU-fibroblast colonies generated from 105 MNC/ml (initiated on Day 0 in stroma-free liquid culture) were continuously detected throughout expansion. In order to test the capacity of MSC to differentiate into hepatocytes, cells were cultured in DMEM supplemented with dexamethasone and 25ng/ml HGF, the latter of which was replaced with 50ng/ml oncostatin M after incubation day 14. The cells were able to expand to cell lines and took on the common hexagonal shape characteristic of hepatocytes; this parameter was used as a primary screening criterion. In addition, the hepatocyte-like cells were further assessed by reverse transcription-polymerase chain reaction and found to express mRNA for albumin, as well as two hepatocyte-specific bile salt transport proteins, basolateral bile transport system NTCP and the canalicular bile salt export pump BSEP. These data demonstrate the presence of pluripotent stem cells in UCB with the capacity to differentiate into a hepatocytic lineage.The research in this area has great potential, both with respect to future application in patients with end-stage liver disease, as well as for learning more about the role of MSC in new clinical cellular therapies.
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Thilakavathy, Karuppiah. "ID3013 Comparative characterization of murine Bone marrow mesenchymal stem cells cultured using two different supplements". Biomedical Research and Therapy 4, S (5 settembre 2017): 25. http://dx.doi.org/10.15419/bmrat.v4is.233.
Abstract (sommario):
Preclinical studies on mesenchymal stem cells (MSC) have allowed the cells to be considered as a promising candidate for cellular therapy. The mouse is the most widely used species for studying the characteristics of MSC. In recent years, conflicting data were reported regarding growth kinetics, surface marker profile, differentiation capacity, genetic instability or malignant transformation and so forth, that may be a result of a range of factors. One of the factors probably is the culture medium formulation. Here we have made a comparative characterization of bone marrow-derived mesenchymal stem cells (mBM-MSC), under the same experimental conditions, cultured using two common supplements, fetal bovine serum (FBS) and MesenCultTM Stimulatory Supplement (MSS). mBM-MSC isolated from the tibias of C57BL/6 mice were cultured and expanded in Dulbecco’s Modified Eagle’s Medium supplemented with either 15% FBS or 15% MSS. Clonogenic potential, population doubling time, immunophenotyping, differentiation immunosuppression potentials and chromosome analysis of early and late passage of mBM-MSC were assessed.
The findings showed that the immunophenotype and differentiation potential of mBM-MSC were similar when cultured using these supplements irrespective of passages. Variations were seen in clonogenic, growth, proliferation rate and immunosuppression potential of the mBM-MSC. This study also revealed that prolonged culture will disrupt their genetic stability regardless of the supplements used. The genetically mutated mBM-MSC were also found to maintain their stemness characteristics and immunosuppression potential.
In conclusion, culture medium formulation causes variations in the cultured MSC and may influence downstream investigation findings.
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Melzer, Catharina, Juliane von der Ohe, Hannah Otterbein, Hendrik Ungefroren e Ralf Hass. "Changes in uPA, PAI-1, and TGF-β Production during Breast Cancer Cell Interaction with Human Mesenchymal Stroma/Stem-Like Cells (MSC)". International Journal of Molecular Sciences 20, n. 11 (28 maggio 2019): 2630. http://dx.doi.org/10.3390/ijms20112630.
Abstract (sommario):
The interactions of cancer cells with neighboring non-malignant cells in the microenvironment play an important role for progressive neoplastic development and metastasis. Long-term direct co-culture of human MDA-MB-231cherry breast cancer cells with benign human mesenchymal stroma/stem-like cells (MSC) MSC544GFP stably expressing mCherry and eGFP fluorescence proteins, respectively, was associated with the formation of three-dimensional (3D) tumor spheroids in vitro. The quantification of the breast tumor marker urokinase plasminogen activator (uPA) in mono-cultured MDA-MB-231 cells revealed an approximately 14-fold enhanced expression when compared to five different normal human MSC mono-cultures. Moreover, uPA levels in 3D tumor spheroids remained elevated 9.4-fold above the average of five different human MSC cultures. In contrast, the expression of the corresponding plasminogen activator inhibitor type-1 (PAI-1) declined by 2.6-fold in the breast cancer cells and was even further reduced by 3.2-fold in the MDA-MB-231cherry/MSC544GFP 3D co-culture spheroids when compared to the various MSC populations. The supportive data were obtained for the production of TGF-β1, which is an important growth factor in the regulation of tumor growth and metastasis formation. Whereas, TGF-β1 release in MDA-MB-231cherry/MSC544GFP co-cultures was elevated by 1.56-fold as compared to MSC544 mono-cultures after 24 h; this ratio further increased to 2.19-fold after 72 h. Quantitative PCR analyses in MSC544 and MDA-MB-231 cells revealed that MSC, rather than the breast cancer cells, are responsible for TGF-β1 synthesis and that TGF-β1 contributes to its own synthesis in these cells. These findings suggested potential synergistic effects in the expression/secretion of uPA, PAI-1, and TGF-β during the co-culture of breast cancer cells with MSC.
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Ramos, Teresa L., Luis Ignacio Sánchez-Abarca, Rosón Beatriz, Concepción Rodríguez Serrano, Alba Redondo, Rebeca Ortega, Ángel Hernández-Hernández et al. "HDAC8 Overexpression in Mesenchymal Stromal Cells from JAK2+ myeloproliferative Neoplasms: A New Therapeutic Target?" Blood 126, n. 23 (3 dicembre 2015): 2831. http://dx.doi.org/10.1182/blood.v126.23.2831.2831.
Abstract (sommario):
Abstract JAK2-V617F mutation in hematopoietic stem cells (HSC) is a common finding in myeloproliferative neoplasms (MPNs). Although alterations in the hematopoietic microenvironment have been described in these entities, information on the functional and genetic characteristics of bone marrow (BM) derived mesenchymal stromal cells (BM-MSC) from JAK2+ MPNs patients is scarce. The aim of the current study was to characterize and compare BM-MSC from 24 MPNs patients with JAK2V617F mutation (14 BM-MSC from essential thrombocythemia-ET and 10 BM-MSC from polycythemia vera-PV) with those from 14 healthy donors-HD. For this purpose BM-MSC expansion, multilineage differentiation, apoptosis, inmunophenotyping, gene expression profiling, RT-PCR and Western Blot analysis were performed. Compared with HD, BM-MSC from MPNs patients showed similar morphology and differentiation capacity, but an increased proliferation rate with less apoptosis cells. BM-MSC from MPNs expressed comparable levels of CD73, CD44, CD90 and CD166, whereas they were negative for hematopoietic markers. The median expression of CD105 was lower in BM-MSC from MPNs patients (p <.05) when compared with BM-MSC from HD. Gene expression profile of BM-MSCs from 8 JAK2V617F (4 PV/4 TE) patients, and from 10 HD showed a total of 169 genes that were differentially expressed in BM-MSC from MPNs patients compared to HD. RT-PCR was performed in two genes to confirm these results, demonstrating that HDAC8 and CXCL12 genes were up-regulated. To analyze whether these changes in MPNs-MSC conferred an alteration in their functional capacity, co-cultures with CD34+ cells from MPNs and BM-MSC were performed. A significant increase in the CFU-GM clonogenic supporting capacity of MPNs-MSC when compared with HD-MSC was observed. To evaluate whether a Histone deacetylase (HDAC) inhibitor could modify the behavior of MPNs-MSC an HDAC8 specific inhibitor, PCI-34051 was used. A decrease in HDAC8 gene (RT-PCR) and protein (WB analysis) expression was observed in BM-MSC from MPNs treated with PCI-34051 at a concentration of 25µM for 48 hours. HDAC8-selective inhibition also induced a cell cycle arrest in the MPNs BM-MSC with an increase of the proportion of apoptotic cells. To assess the impact of this inhibition on the capacity of MPNs-MSC to support hematopoiesis, BM mononuclear cells (BM-MNC) were co-cultured in transwell for 48 hours with PCI-34051-treated and non-treated BM-MSC. After co-culture, cell viability, clonogenic (CFU-GM) assays and TP53 expression were analyzed. A decrease in cell viability (p=0.028) and CFU-GM (p=0.018) was demonstrated when BM-MNC from MPNs had been in culture with MPNs BM-MSC treated with the HDAC8 inhibitor, as well as an increase in TP53 expression. These results suggest that MPNs-MSC display different proliferative rate, MSC markers, gene expression profile and HDAC8 overexpression compared to HD-MSC. The inhibition of HDAC8 expression by its specific inhibitor decreases the capacity of the stroma to support hematopoietic cells from MPNs patients, suggesting that HDAC8 may be a potential therapeutic target in this setting. Disclosures Sánchez-Guijo: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau.
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Lane, Thomas A., Davina Garls, Ellen Mackintosh, Sanjivan Kohli e Steven C. Cramer. "Feasibility of Rapidly Generating Sufficient Autologous Human Marrow Stromal Cells for Cellular Therapy." Blood 110, n. 11 (16 novembre 2007): 1218. http://dx.doi.org/10.1182/blood.v110.11.1218.1218.
Abstract (sommario):
Abstract Marrow stromal cells (MSC) are increasingly employed for human cellular therapy protocols. In some cases, it may be advantageous to use autologous MSC, but the feasibility of quickly generating sufficient MSC for autologous therapy has not been established. We investigated the feasibility of rapidly generating large numbers of autologous, early passage, MSC as a prerequisite for the clinical investigation of MSC in human ischemic stroke. Bone marrow (25–35 mL) was collected from 8 healthy volunteers under an IRB-approved protocol; a mononuclear cell (mnc) fraction prepared by ficol density gradient, a portion of the mnc fraction was suspended in low glucose DMEM with 20% pre-screened FBS and plated in T175 culture flasks at 0.2–0.4 × 106 mnc/cm2; maintained at 37 C in a 5% CO2 incubator; non-adherent cells were removed at 48 h and a portion of the cells were passaged when 80% confluent into additional flasks (12–14 days) at 5700 cells/cm2 and MSC were harvested with trypsin at 21 days. The MSC were washed x3 and resuspended in sterile saline for enumeration and suitability testing. MSC were identified according to ISCT criteria (Dominici M, Cytotherapy2006;8(4):315), including plastic adherence, morphology, growth characteristics, and phenotype by flow cytometry (CD73, CD90, CD105 positive; CD14, CD34, CD45, HLA-DR negative) and differentiation into adipocytes. The expected total cell yield was calculated based upon actual cell yields minus the volume of cells required for testing based upon a 70 kg patient. Results: In 8 experiments using different donors, a median of 40 (range 20–225) × 106 mnc were available for plating from the original marrow. The median mnc recovery after ficol density gradient was 37% (range 23–130), but the median CFU-F recovery after ficol from the original marrow was 96% (94–96). The calculated MSC yield after primary culture was a median of 25 (10–53) × 106 cells. The calculated yield of MSC after 1st passage at 21 days was a median of 2.1 (0.9–4.4) × 106 MSC/kg (70 kg patient weight), and the median cell viability was 98.5% (92–97.9). At the end of 1st passage all samples yielded negative gram stains and cultures for mycoplasma, bacteria, fungal growth and the median endotoxin level was 0.027 EU/mL (0.02 – 0.10). The viability of the harvested MSC was well maintained in saline at 1 × 106 MSC/mL for 24 h at 4 C (85% +/− 9%) but not after 48 h (37% +/− 9%) or if stored at room temperature. The effects of supplementation with 5% human serum albumin on MSC storage for 24 h at 4 C were variable, ranging from 27% to 95% viability, depending on the albumin manufacturer (3 mfrs; n=4 each). Conclusion: These studies suggest that sufficient MSC that are suitable for autologous human cellular therapy can be rapidly and reliably generated under GTP conditions, that the resultant MSC may be preserved for up to 24 h if stored at 4 C in either saline or pre-screened human serum albumin.
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Kollar, K., E. Seifried e R. Henschler. "Therapeutic potential of intravenously administered human mesenchymal stromal cells". Hämostaseologie 31, n. 04 (2011): 269–74. http://dx.doi.org/10.5482/ha-1158.
Abstract (sommario):
SummaryMesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in preclinical and clinical studies. The study of MSC migration following systemic infusion of exogenous MSC is difficult. The challenges facing these efforts are due to a number of factors, including defining culture conditions for MSC, the phenotype of cultured MSC, the differences observed between cultured MSC and freshly isolated MSC. However, even if, MSC populations consist of a mixture of stem and more committed multipotent progenitors, it remains probable that these cell populations are still useful in the clinic as discussed in this review.
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Bassi, Giulio, Luciano Pacelli, Cedric Mènard, Francesco Bifari, Luc Sensebé, Frederic Deschaseaux, Fabien Guilloton et al. "Microporous Biphasic Calcium Phosphate Granules (MBCP®) Retain Immunological Properties of Bone Marrow-Derived Mesenchymal Stromal Cells and Promote Osteoblastic Differentiation". Blood 118, n. 21 (18 novembre 2011): 1924. http://dx.doi.org/10.1182/blood.v118.21.1924.1924.
Abstract (sommario):
Abstract Abstract 1924 Bone is among the most frequently transplanted tissue with about 1 million procedures annually in Europe. Allografts and autografts account for more than 80% of total graft volume, despite their considerable disadvantages, including the risk of disease transfer and immunologic rejection, limited supply of bone, costs and complications. Significant growth opportunities exist for synthetic bone grafts in association with mesenchymal stromal cells (MSC) from autologous or allogeneic sources as alternatives to biological bone grafts in orthopaedic and maxillofacial surgery. The objective of REBORNE is to perform clinical trials using advanced biomaterials and cells triggering bone healing in patients. To reach this goal, five phase I clinical studies with 20 patients have been planned in 12 clinical Centers spread in 8 European countries. Aim of the Immunological Unit of Reborne is to assess the MSC immunomodulatory properties in presence of the biomaterial used as scaffold for MSC delivery. All the functional experiments were performed in parallel, by comparing the effects of standard culture conditions and three-dimensional culture setting using MBCP (Biomatlante). Material and methods: Bone marrow MSC were provided from REBORNE Consortium Centers. To perform proliferation assays, different immune effector cells (T, B and NK cells) were stained with CFSE according to manufacturer's protocol. Active caspase-3 cell staining was used for survival quantification of immune effector cells after co-culture experiments. Differentiation potential was evaluated by culturing MSC with two different media containing either bone morphogenetic protein 4 (BMP4) or dexamethasone. After three weeks, osteogenic differentiation was quantified by qRT-PCR, alkaline phosphatase activity and alizarin red staining. Results: We found that primed MSC, pre-treated with the inflammatory cytokines IFNg and TNFa, displayed upregulation of HLA-ABC, CD54, CD106 and de novo expression of HLA-DR, both in standard culture conditions and in association with MBCP. Immune effector cells could be cultured and collected even in presence of MBCP and no significant differences were found between standard- (MSC + effector cells) and 3D-coculture conditions (MSC + MBCP + effector cells), in terms of immune effector cell proliferation. In both experimental conditions MSC suppressed T and NK cell proliferation (% suppression: MSC + T = 68.4; MSC + MBCP + T = 62.4; MSC + NK = 17.5; MSC + MBCP + NK = 20.2) and increased B cell proliferation (MSC + B = +13%; MSC + MBCP + B = +12.3%). In addition, immune effector cells viability was not affected by MBCP and MSC co-culture increased their survival even in presence of MBCP; in fact, in each culture condition the percentage of inhibition of T, B and NK cell apoptosis was higher than 20% in comparison to immune effector cells cultured without MSC. Dexamethasone and BMP4 were capable of inducing MSC differentiation into osteoblast-like cells, as confirmed by qRT-PCR analysis. We demonstrated that BMP4-based medium led to fully differentiated osteoblasts (Osterix+, RUNX2+, DLX5+ and alkaline-phosphatase+). Moreover, MBCP was more efficient in increasing osteoblastic differention as compared to standard culture conditions, as shown by the higher expression of Osteocalcin and Osterix. These data show that the association of MBCP and MSC does not affect MSC properties and suggest that it could be a treatment of choice of bone defects instead of allograft and autograft transplantation. Disclosures: No relevant conflicts of interest to declare.
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Auletta, Jeffery J., Basabi Maitra, Emese Szekely e Omer N. Koc. "Toll-Like Receptor Agonists Induce Secretion of Immunomodulatory Soluble Factors from Human Mesenchymal Stem Cells." Blood 106, n. 11 (16 novembre 2005): 1394. http://dx.doi.org/10.1182/blood.v106.11.1394.1394.
Abstract (sommario):
Abstract Human mesenchymal stem cells (MSCs) suppress T-cell activation and proliferation by inducible expression and secretion of soluble inhibitory factors. We have previously shown that MSCs require activation by antigen-presenting cells (APCs) to secrete these factors. Toll-like receptors (TLRs) and their associated agonists have critical roles in APC activation, maturation and function. Therefore, we investigated whether TLR agonists induce cytokine and chemokine production from MSCs and if such soluble factors mediate inhibition of T-cell alloreactivity. Human MSCs (CD45-CD105+CD90+CD80-CD73+HLA−I+) were expanded from normal volunteer bone marrow aspirate specimens. MSCs were characterized morphologically by light microscopy, phenotypically by flow cytometry and functionally by ex vivo cell culture stimulation and inhibition of T-cell IFN-γ production. Cytokine and chemokine induction of third and fourth-passage MSCs co-cultured in triplicate with established TLR agonists were measured and compared to soluble factor induction from human IL-1β stimulated MSCs. TLR agonists included formalin-fixed Staphylococcus aureus Cowan A strain (SAC, TLR-2), Pam3CysSerLys4 (Pam3Cys, TLR-2), polyinosine:polycytidylic acid (poly I:C, TLR-3), Salmonella enteriditis lipopolysaccharide (LPS, TLR-4), R848 (TLR7/8) and unmethylated CpG oligodeoxynucleotides 1826 and 2216 (TLR-9). 24h MSC-culture supernatants were assessed for levels of soluble factors using standard and multiplex ELISA and for inhibition of T-cell alloreactivity using an established mixed lymphocyte reaction (MLR) IFN-γ ELISPOT. MLR was also performed in the presence of TLR agonists alone and agonist-stimulated MSCs. In general, TLR-MSC supernatants contained 2 to 5-fold lower levels of inducible IL-6, IL-8, IL-10 and RANTES than IL-1β-MSC supernatants. LPS- and poly I:C-MSC supernatants had comparable levels of inducible factors as IL-1β-MSC supernatant. Neither stimulation with IL-1β nor TLR agonists induced phenotypic changes in MSCs, as measured by surface expression of MSC markers as well as activation markers (HLA-DR, CD40, CD40L, CD80, HLA-I and HLA-II). However, supernatant from TLR-MSC cultures (CpG 1826 and poly I:C) and from IL-1β-MSC cultures did inhibit T-cell IFN-γ production. For example, percent IFN-γ inhibition using supernatant from MSC-CpG 1826 culture versus supernatant from IL-1β-MSC culture was 46.5% ± 22.8 and 66.8% ± 13.5, respectively (mean ± SEM, n=3 separate experiments involving different donor MSCs). Likewise, TLR- and IL-1β-stimulated MSCs themselves inhibited T-cell IFN-γ. Together, these results demonstrate that ex vivo culture with TLR agonists activates human MSCs to inhibit ex vivo T-cell alloreactivity likely via inducible soluble factors. Optimization of cell culture conditions is needed to define the soluble factors mediating this inhibitory effect. Our results suggest the presence of a potentially novel immunomodulatory interface at which TLR agonists are uniquely positioned to influence immune effector cell and mesenchymal stem cell responses.
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Chu, Pat PY, Arun Prasath, Zhihong Li, Sudipto Bari, Justina ML Ang, Xiubo Fan, Chengcheng Zhang, Harvey F. Lodish e William YK Hwang. "Mesenchymal Stromal Cell Co-Culture Enhances Insulin-Like Growth Factor Binding Protein 2 - Augmented Ex Vivo Cultures of Human Cord Blood in a Viability Supporting, Contact-Dependent Process Involving the Rescue of Cells from Early Apoptosis". Blood 112, n. 11 (16 novembre 2008): 211. http://dx.doi.org/10.1182/blood.v112.11.211.211.
Abstract (sommario):
Abstract The use of cord blood (CB) for adult transplantation is limited by the number of hematopoietic stem (HSC) and progenitor cells (HPC) residing in the CB. Successful ex vivo expansion of HSC and HPC may overcome this limitation. An ex vivo expansion system (Zhang et. al., 2008, Blood) based on the additional of Angiopoietin-like 5 and/or Insulin-like Growth Factor Binding Protein 2 (IGFBP2) to a standard cytokine cocktail of stem cell factor, thrombopoietin and FLT3-ligand has recently been reported to expand CB HSC by ~20-fold in a SCID-repopulating cell (SRC) assay. In this study, we further investigated if co-culture of the CB cells with mesenchymal stromal cells (MSC) would enhance the ex vivo expansion of CB in IGFBP2-augmented cultures. Using thawed unselected CB cells, IGFBP2-augmented cultures were able to expand CD45(+)/CD34(+) cells 49-fold and retain in vitro colony-forming units granulocyte-macrophage (CFU-GM) colony size with a concomitant 15-fold increase in number. Under the same conditions, co-culture with MSC resulted in a further 2.9-fold enhancement of CD45(+)/CD34(+) cell expansion (144-fold expansion) and a 2.2-fold enhancement of CFU-GM expansion (33- fold expansion). The numbers reflect true expansion of cell numbers as no prior CD34/ CD133 selection was employed. We further studied the mechanism of MSC enhancement of HSC and HPC expansion. Rather than an increase in cellular proliferation rate, the MSC enhancement effect was associated with 2- to 5-fold increase in viability of a population of lymphocyte-like cells [FS(low) SS(low) CD45(+)] in CB, as determined by annexin-V/7AAD staining. Moreover, direct contact with MSC was necessary for optimal expansion and viability support as shown by use of conditioned media and transwell assays. Thawed mononucleated CB cells exhibited a high level of positivity for Annexin-V, which translated to subsequent 7AAD positivity during ex vivo expansion. MSC co-culture resulted in reversion of Annexin-V positivity within 3–6 hours, and reached a maximum on day 3 (80–90% viability with MSC versus 40–60% without, as defined by double negative staining for Annexin-V and 7AAD). Concomitantly, the loss of mitochondrial membrane potential as determined by JC-1 was also reduced by MSC co-culture. There was a reduction of Caspase 9 activity (12.7% with MSC versus 23.8% without) at day 3 and a reduction in Caspase 3 and 7 activity (5.5% with MSC versus 15.5% without) at day 7 with MSC co-culture. In further experiments, an increase of lysosomal activity in CB, as determined by acridine orange staining, was found to be reduced in MSC co-culture. The use of a non-viable MSC layer did not support CB viability and subsequent expansion. We hypothesized that cytosolic transfer of nutrient or other survival factors from MSC to CB could be a potential mechanism responsible for the rescue of thawed CB from loss of viability and subsequent enhancement in CB ex vivo expansion. Using genetically modified stromal cells expressing with the fluorescent protein containing mitochondria targeting sequence (DsRed-mito) and time-lapse (>10 hours) confocal imaging, we observed potential cytosolic transfer activities between the stromal cells. In conclusion, IGFBP2-augmented cultures result in successful CB expansion, which is further enhanced by MSC co-culture in a viability supporting, contact-dependent process that could involve reversal of cells from early apoptosis possibly mediated through cytosolic transfer of nutrient or cellular material. Further in vivo experimentation is in progress to verify the effect of our combined MSC-IGFBP2 culture system on HSC expansion.
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Arrivabene Neves, Camila, Lucilene dos Santos Silva, Camila Ernanda Sousa de Carvalho, Marina Silva Carvalho, José Lindenberg Rocha Sarmento, Tânia Vasconcelos Cavalcante, Mônica Arrivabene et al. "Culture of goat preantral follicles in situ associated with mesenchymal stem cell from bone marrow". Zygote 28, n. 1 (18 novembre 2019): 65–71. http://dx.doi.org/10.1017/s0967199419000686.
Abstract (sommario):
SummaryThis study aims to develop an in vitro co-culture system of in situ goat preantral follicles with bone marrow-derived mesenchymal stem cells (BM-MSC), evaluating the influence of these cells on follicular growth, rate of activation and morphologically normal follicles. Fragments of ovarian cortex were cultured for 1 or 7 days in the presence of BM-MSC (BM-MSC+) and absence of BM-MSC (BM-MSC−). Histological sections of the fragments were analysed and data were obtained regarding morphological classification, survival rate of morphologically normal follicles and rate of follicular activation. Culture medium on days 1 and 7 was also sampled for nitrite concentration and reduced glutathione activity. There was a reduction (P < 0.05) in the percentage of morphologically normal follicles in the BM-MSC+ compared with the fresh control only on the seventh day of culture. When comparing treatments, on the seventh day of culture, a higher rate of morphologically normal preantral follicles was observed in BM-MSC+ (P < 0.05). In both treatments, primordial and developing follicle rates were similar to the fresh control (P > 0.05). When comparing treatments with each other, as well as with the fresh control, no differences were observed in follicular diameter (P > 0.05) or nitrite concentration (P > 0.05). The concentration of reduced glutathione was lower on the seventh day of co-culture in both treatments (P < 0.05). In conclusion, co-culture had no influence on follicular or oocyte development. However, it was critical to maintain the survival of preantral follicles during 7 days of culture.
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Wobus, Manja, Jing Duohui, David M. Poitz, Katrin Müller, Rainer Ordemann, Gerhard Ehninger e Martin Bornhäuser. "Hypoxia Alters the Main Characteristics of the Hematopoietic Stem and Progenitor Cell Microenvironment in Vitro". Blood 118, n. 21 (18 novembre 2011): 4803. http://dx.doi.org/10.1182/blood.v118.21.4803.4803.
Abstract (sommario):
Abstract Abstract 4803 Background: Hematopoietic stem and progenitor cells (HSPC) are located in a specialized microenvironment, called the stem cell niche, where their stem cell phenotype and differentiation are tightly regulated via interactions with the supporting mesenchymal stromal cells (MSC). These niches have been shown to be localized in regions with a lower oxygen tension which may also impact on the functional properties of MSC. For a better understanding to what extent hypoxia contributes to the establishment of an undifferentiated niche microenvironment that prevents inopportune differentiation of HSPC, we investigated MSC/HSPC co-cultures as well as MSC single cultures under low oxygen conditions. Design and Methods: Distribution, functional and phenotypical characteristics of CD34+ HSPC in hypoxic co-cultures (0.5% O2) were analyzed by flow cytometry. The effect of co-culture medium on the HSPC migration potential was tested in a transwell assay. The secretion of vascular endothelial growth factor A (VEGF-A), stromal-derived factor 1 (SDF-1), IL-6 and IL-8 by MSC was determined using ELISA whereas the expression of cell surface molecules was detected by flow cytometry. Moreover, the MSC proliferation as well as adipogenic and osteogenic differentiation was compared between hypoxic and normoxic culture conditions. Results: In the hypoxic co-culture, the adhesion of HSPC to the MSC layer was inhibited, whereas HSPC transmigration beneath the MSC layer was favoured. Increased VEGF-A secretion by MSC under hypoxic conditions, which enhanced the permeability of the MSC monolayer, was responsible for this effect. Furthermore, VEGF expression in hypoxic MSC was induced via hypoxia-inducible factor (HIF) signalling. Whereas IL-6 and IL-8 secretion were increased, SDF-1 expression by MSC was down-regulated under hypoxic conditions in a HIF-independent manner. The MSC immunophenotype which is characterized by expression of CD73, CD90, CD105, and CD166 was not significantly changed by hypoxia. Interestingly, a significant decrease of CD146 mRNA and protein expression levels was observed. The MSC proliferation was not significantly affected by lower oxygen tension. Culture of MSC in adipogenic induction medium for 14 days under hypoxia resulted in a reduced appearance of adipocyte-like cells containing lipid droplets and almost 50 % lower mRNA levels of fatty acid binding protein 2. The ALP activity as readout for osteogenic differentiation was decreased between 10% and 60% in hypoxic MSC. Conclusions: Low oxygen tension reduces the in vitro differentiation capacity and alters the cytokine secretion profile of primary human MSC. These functional changes may favour the homing and maintenance of quiescent HSC simulating the physiologically hypoxic niche conditions in vitro. Disclosures: No relevant conflicts of interest to declare.
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Chen, Guanghua, Ting Yang, Man Qiao, Huiwen Liu e Wu Depei. "Comparative Study of the Biological Characteristics of Serum-Free and Fetal Bovine Serum-Contained Medium Cultured Umbilical Cord-Derived Mesenchymal Stem Cells". Blood 120, n. 21 (16 novembre 2012): 4737. http://dx.doi.org/10.1182/blood.v120.21.4737.4737.
Abstract (sommario):
Abstract Abstract 4737 Objective: To compare the difference of biological characteristics between human umbilical cord-derived mesenchymal stem cells (UC-MSC) cultured by serum free medium and fetal bovine serum-contained complete medium and to create a xenogeneic protein-free UC-MSC culture system. Methods: Healthy human umbilical cord segments were digested with collagenase. Umbilical cord-derived mesenchymal stem cells were cultured by serum free MesenCult-XF medium and FBS-based αMEM complete medium. We analysed the morphology, immunophenotype, expansion potential, trilineage differentiation potential, karyotype and immunosuppression of early passage of UC-MSC. Results: The average cell diameters of UC-MSC in suspension cultured by serum free medium and FBS-based medium are 26 (18–39) μm and 35 (20–61) μm, respectively. Cell expansion folds with serum free medium and FBS-based medium were (5.2±0.2) and (3.5±0.1) in the first five passage, respectively. The expansion potential of MSCs was significantly higher with serum free medium compared to FBS-based medium (P<0.05). A panel of markers as CD29, CD44, CD90, CD73, CD105 and HLA-ABC were expressed by human UC-MSC. Hematopoietic lineage markers CD34, CD45 and HLA-DR were not detectable on UC-MSC. The cpm were (4.57±0.14)×104, (2.04±0.16)×104 and(0.42±0.04)×104 when serum free medium cultured MSCs were added to the cultures at ratios MSCs/T cell of 1:100, 1:10 and 1:5. While the cpm were (4.57±0.14)×104, (2.04±0.16)×104 and(0.42±0.04)×104when serum free medium cultured UC-MSCs were added to the cultures. The immunosuppressive potential of serum free medium-cultured UC-MSC was higher than that of serum-contained medium cultured UC-MSC at three different ratios MSC/T cell (P<0.05). Conclusion Compare with serum-contained medium cultured early passage of UC-MSC, the cell diameter of serum free medium cultured MSCs was smaller and the expansion potential was higher. No xenogeneic proteins were presented in UC-MSC preparation when UC-MSC was cultured with serum free medium. Human UC-MSC suppresses T-cell proliferation in a dose-dependent manner. The immunosuppressive potential of UC-MSC was higher when cultured in serum free medium compared with FBS-based medium. Disclosures: No relevant conflicts of interest to declare.
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Jiang, Xuejie, Dan Xu, Fang Chen, Ling Jiang, Guopan Yu, Fanyi Meng, Xiaoli Liu e Qifa Liu. "BM-MSC Hhip Induced Microenvironment Protection from Chemotherapy in AML Via Ptch/Smo/Gli Pathwa". Blood 128, n. 22 (2 dicembre 2016): 5235. http://dx.doi.org/10.1182/blood.v128.22.5235.5235.
Abstract (sommario):
Abstract Introduction. The mechanism of drug resistance is complicated in acute myeloid leukemia (AML), bone microenvironment provides protection niche for leukemia stem cells (LSC), and considered as an internal environment for AML cells to escape from chemotherapeutics cytotoxicity, but mechanism of drug resistance induced by microenvironment mesenchymal stem cell (MSC) isn't completely clear. In prophase study, it was found that Ptch/Smo/Glis pathway was the key in the network of AML drug resistance. Smo inhibition improved the survival of AML-bearing mice. Low expression of HHIP was found in refractory AML MSC and bone morrow liquid, overexpression of Smo and Gli-1 was verified in AML cells, and negatively correlated with prognosis in AML patients. Chemotherapeutic drug sensitivity was decreased in AML cells after co-cultured with AML MSC in vitro. Methods. Bone morrow samples from AML patients and normal donors were collected to culture MSC cells. HL60/ADM and Kasumi-1 cells as well as HL60、KG-1cells were treated with daunomycin (DNR) or cytosine arabinoside (Ara-C) when cocultured with MSC. Cell cycle and apoptosis were determined by flow cytometry. The expression of HHIP was determined by confocal image and western blotting. Smo and Gli-1 activity was determined by western blotting. Result. We took advantage of AML cells cocultured with MSC to imitate leukemic microenvironment in vitro. Co-culture with AML BM-MSC decreased the sensitivity to DNR or Ara-C compared with normal BM-MSC in HL60/ADM or Kasumi-1 cells. Flow cytometry analysis showed that cells in S phase and percentage of apoptosis cells were increased after co-culture with AML MSC. Western blotting also determined that low expression of HHIP was detected in refractory AML BM-MSC, Smo and Gli-1 expression were increased in HL60/ADM or Kasumi-1 cells after co-cultured with MSC. Confocal analysis also confirmed that the combination of HHIP and Ptch was decreased in AML MSC co-culture, Smo/Gli-1 pathway was activated through decreased inhibition of Shh in refractory AML cells co-cultured with MSC. Samples from AML patients also demonstrated that HHIP expression in AML BM-MSC was lower than that in normal BM-MSC, especially in refractory AML samples. HHIP expression in BM-MSC was negatively related to Smo and Gli-1 activity. Clinic data also showed that AML patients with overexpression of HHIP had a worse prognosis. Conclusion. Our study demonstrated expression of BM-MSC HHIP was negatively related to activity of Ptch/Smo/Glis in AML. Low expression of BM-MSC HHIP resulted in activating Ptch/Smo/Gli pathway, and indicated drug resisitance and bad prognosis in AML. This project is connected with basic research and clinic, and provides support for diagnosis and targeted therapy in AML. Disclosures No relevant conflicts of interest to declare.
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Roderburg, Christoph, Anke Diehlmann, Frederik Wein, Anne Faber, Ulf Krause, Maria Frankhauser, Anja Seckinger, Anthony D. Ho e Wolfgang Wagner. "The Hematopoietic Supportive Potential of Human Mesenchymal Stromal Cells Is Associated with Expression of Cadherins." Blood 108, n. 11 (16 novembre 2006): 1402. http://dx.doi.org/10.1182/blood.v108.11.1402.1402.
Abstract (sommario):
Abstract Self renewal and differentiation of hematopoietic stem cells (HSC) are governed by interaction with the supportive microenvironment of the bone marrow. Secreted factors as well as specific cell adhesion proteins are involved in this interaction. As an in vitro model system, the hematopoietic microenvironment can be mimicked by supportive mesenchymal stromal cells (MSC). We have compared the supportive potential of human MSC from bone marrow (BM) isolated under two different culture conditions (BM-MSC-M1 and BM-MSC-M2), from adipose tissue (AT) and umbilical cord blood (CB) that were all cultivated as described before (Wagner et al. Exp Hematol.2005;11:1402–1416.). As controls we have used the human fibroblast cell line HS68 and the murine fetal liver cell line AFT024. CD34+ cells were isolated from human cord blood and cultured in direct contact with irradiated stromal cells. After four, seven and twelve days the immunophenotype of the hematopoietic cells was analyzed by flow cytometry. Many progenitor cells cultured on BM-MSC or AFT024 maintained a primitive phenotype of CD34+/CD38- cells whereas the proportion of these cells was reduced upon cultivation with CB-MSC and cells cultured on AT-MSC and HS68 displayed a significantly higher expression of CD38 and lower expression of CD34. Furthermore, long term culture initiating-cell (LTC-IC) assays were performed on the different feeder layer. LTC-IC frequency was significantly higher on BM-MSC that were isolated under the two different culture conditions (BM-MSC-M1 1,15 ±0.11%; BM-MSC-M2 1.14±0.08%) and on CB-MSC (1.10±0.13%) compared to AT-MSC (0.32±0.09%) and HS68 (0.67±0.12%). We have compared gene expression profiles of BM-MSC-M1, BM-MSC-M2, CB-MSC, AT-MSC and HS68 by cDNA microarray analysis (51,144 different cDNA clones of the RZPD3 Unigene Set). Differential expression of various genes correlated with the observed differences in supportive potential. Among these were adhesion proteins like N-cadherin, cadherin11, fibronectin1, various integrins (ITGA1, ITGA5 and ITGB1) and VCAM1 as well as secreted proteins including osteonectin, CTGF and SDF1. Westerblot analysis verified on protein level that cadherin11, N-cadherin, and ITGB1 were highly expressed on BM-MSC as compared to AT-MSC and HS68 fibroblasts. In conclusion MSC from human bone marrow or from umbilical cord blood support to a significantly higher degree the maintenance and proliferation of primitive hematopoietic progenitors than MSC derived from adipose tissue. This affinity correlated with up-regulation of cadherin11, N-cadherin and intergrin-beta1 on BM-MSC and CB-MSC.
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Renda, Maria Concetta, Emanuela Fecarotta, Giovanna Schillaci, Filippo Leto, Giuseppina Calvaruso, Giuseppa Maria Garofalo, Angela Piazza et al. "Mesenchymal Fetal Stem Cells (FMSC) from Amniotic Fluid (AF): Expansion and Phenotypic Characterization". Blood 126, n. 23 (3 dicembre 2015): 4758. http://dx.doi.org/10.1182/blood.v126.23.4758.4758.
Abstract (sommario):
Abstract Introduction. Mesenchymal stem cells (MSCs) are multipotent precursors able to differentiate into bone, cartilage and fat. In humans, the MCS were isolated from bone marrow, cord blood, peripheral blood, adipose tissue and the amniotic fluid (AF) in the second trimester of pregnancy (FA-MSC). The FA-MCS have immunosuppressive properties, extensive proliferative potential and self-renewal, and express the fetal stem transcription factor Oct4. The purpose of this work was to isolate and characterize cells isolated from human FA as alternative source of multipotent MSC for regenerative medicine and transplantation. Materials and Methods. Expansion. The FA cells (extracted from the first three milliliters of FA collected during diagnostic amniocentesis and can not be used for prenatal diagnosis), were cultured in serum free medium specific to the MSC (STEM CELL). Phenotypic analysis. On the FA-expanded MSC the presence of mesenchymal markers CD106, CD146 and CD105, and non-mesenchymal markers CD45 and CD34 have been evaluated by flow cytometry. The fetal origin of the FA-expanded MSC was verified by QF-PCR comparing FA-MSC DNA and maternal mononuclear DNA. Stemness analysis. The stemness, potential before and after expansion, was evaluated by expression analysis(RT-PCR) of Oct4 and Nanog gene. cDNA from adult peripheral blood and cDNA from chorionic villus at 10 weeks of gestation were used as control. The specific primers for the analysis of Oct4 and Nanog genes were designed in the laboratory on the basis of the gene maps (GENE ATLAS). Results and Conclusions. After 8 days of culture FA cells have produced CFU-F colonies. At 15 days of culture started the proliferation of fibroblasts, and at 28 days of culture the fibroblasts have reached 80% confluency. Phenotypic analysis. The flow cytometry assay allowed cultures immunophenotypic characterization. The flow cytometry analysis of the FA-MSC showed the presence of mesenchymal markers CD146, and CD105 and the absence of hematopoietic/endothelial markers CD34 and CD45. The QF-PCR confirmed the fetal origin of the expanded FA-MSC. Using medium serum-free adherence method, it has been developed a reproducible protocol for the isolation and expansion of AF-MSC. Phenotypic analysis results, confirmed the nature of the mesenchymal cells expanded AF. The presence of expression of Nanog (a homeobox playing a key role in embryonic development and in maintaining the pluripotency of stem cells), and Oct4 (a transcription factor involved in stem cells pluripotency and self-renewal maintenance) confirmed the stamness persistence of AF-MSCs after expansion. The expanded AF-MSCs could be used in different therapeutic fields: bone marrow transplantation in adults, transplant "in utero" and gene therapy for congenital blood diseases, treatment of chronic inflammatory bowel disease. The use of AF-MSCs also can open new perspectives for research in regenerative medicine because of their possible de-differentiation by "ex vivo" procedures. Disclosures No relevant conflicts of interest to declare.
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Wuchter, Patrick, Rainer Saffrich, Stefan Giselbrecht, Patrick Horn, Anthony D. Ho e Eric Gottwald. "Understanding The Marrow Niche: Advanced 3D Model System Allows Functional Analysis Of The Interaction With Human Hematopoietic Progenitor Cells". Blood 122, n. 21 (15 novembre 2013): 2462. http://dx.doi.org/10.1182/blood.v122.21.2462.2462.
Abstract (sommario):
Abstract We previously demonstrated that “stemness” of human hematopoietic progenitor cells (HPC) was maintained in a co-culture setting with a monolayer of human mesenchymal stromal cells (MSC). To simulate and monitor the marrow microenvironment of the HPC niche more precisely we have established a 3D co-culture system based on a proprietary KITChip. The KITChip was developed by the Karlsruhe Institute of Technology (KIT) and represents a unique microchip with defined microwell cavities for 3D cell cultures. Sample acquisition was approved by the local Ethics Committee and informed written consent was obtained from all subjects. MSC were derived from human bone marrow of healthy voluntary donors, and HPC were isolated from umbilical cord blood. Cells were mixed in suspension in a ratio of 3:2 (3x105 MSC and 2x105 HPC) and inoculated into the KITChip, which was subsequently mounted into a microbioreactor. This closed loop setup allowed precise control of medium flow and oxygen saturation. After 1 to 5 days of co-culture, the two cell populations were analyzed by immunostaining, RT2-PCR and colony formation assay. MSC form a complex 3D mesh in the microcavities of the KITChip and were maintained stable for up to 6 weeks. We have demonstrated that HPC were distributed three-dimensionally inside this MSC mesh and could be kept viable in this environment for at least 14 days. A defined proportion of CD34+ HPC adhered to the MSC in the microcavities and built up direct cellular connections to the surrounding MSC. By means of RT2-PCR, we could demonstrate that throughout the whole culture period of 14 days a subpopulation of CD34+/p21+/CXCR4+ cells was maintained in the 3D-environment more efficiently than compared to conventional co-culture with MSC monolayer. This was confirmed by Western blotting after the isolation of both cell populations from the chip. The colony formation assay revealed that the plasticity of the HPC cultivated in the 3D KITChip was nearly the same as that of freshly isolated HPC at day 0, whereas HPC co-cultured on MSC monolayer showed a significant decrease in stem cell plasticity. Further analysis under hypoxic conditions (5% O2) indicated that gene expressions of CD33, CD34, CD38 and CD44 were markedly reduced, while those of CD90, CD105, c-Kit, p21, SDF-1 and Angpt-1 remained stable compared to normoxic culture conditions. This novel model system allows analysis of the major determinants of the niche and the impact of a 3D microenvironment on vital stem cell functions. Early HPC were maintained more efficiently and showed a superior plasticity potential when cultured in the 3D KITChip as compared to conventional 2D co-culture systems. Current studies are in process to define the functional significance of the observed changes in gene expression pattern under hypoxic conditions, which resembles the physiologic milieu of the marrow. Disclosures: Wuchter: ETICHO: Consultancy, Honoraria; Sanofi: Honoraria for lectures Other. Ho:Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.
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Abdul-Aziz, Amina M., Manar S. Shafat, Matthew J. Lawes, Kristian M. Bowles e Stuart A. Rushworth. "Protein Kinase C-ß Dependent IL-8 Release Promotes Acute Myeloid Leukemia Blast Cell Survival in Co-Cultures with Bone Marrow Stromal Cells". Blood 126, n. 23 (3 dicembre 2015): 3064. http://dx.doi.org/10.1182/blood.v126.23.3064.3064.
Abstract (sommario):
Abstract Introduction: Acute myeloid leukemia (AML) cells exhibit a high level of spontaneous apoptosis when cultured in vitro but have a prolonged survival time in vivo indicating that the tissue microenvironment plays a critical role in promoting AML cell survival. Knowledge of the complexity of the bone marrow microenvironment is increasing especially with respect to the bone marrow mesenchymal stromal cells [BM-MSC] which are considered a major protective cell type. Other studies have demonstrated the ability of BM-MSC to protect leukemia cells from spontaneous and chemotherapy-induced apoptosis. Increasing evidence suggests the existence of crosstalk between leukemia cells and BM stromal cells to create a leukemia-promoting environment. Recently our group and others have shown that this crosstalk is achieved by a complex communication system that involves multiple bidirectional signals which enhance AML survival and proliferation. Here we report a novel interaction between AML blasts and BM-MSC which benefits AML proliferation and survival. Methods: To investigate the interaction between primary AML blasts and BM-MSC we isolated AML and BM-MSC from the same patient and used an autologous in vitro culture assay to analyze the cytokine profile. Conditioned medium was collected from cultures of primary human AML alone or cultured with autologous BM-MSC and analyzed using Proteome Profiler Human XL Cytokine Array and target specific ELISAs. Real-time PCR was also used to verify the array data. MIF-Receptor inhibitors (SB 225002- CXCR2, AMD3100 - CXCR4 and CD74 blocking antibody - CD74) and signaling kinase inhibitors (LY294002- PI3K/AKT, PD098059 - MAPK, Ro-31-8220 - PKC) were used for initial determination of MIF signaling pathways in BM-MSC. Specific PKC isoform inhibitors (Go6976-PKCα/ß and enzastaurin -PKCß) were then used to determine isoform specific activation. Western blot and siRNA were used to confirm the role of AML derived MIF in regulating downstream BM-MSC signaling pathways including MAPK, PI3K/AKT, and PKC. Results: We initially examined the cytokine profile in cultured human AML compared to AML cultured with autologous BM-MSC or BM-MSC alone and found that MIF was highly expressed by primary AML and that IL-8 was increased in AML/BM-MSC co-cultures. The observed changes in IL-8 were confirmed by ELISA assays. RT-PCR was used to measure MIF and IL-8 gene expression from RNA extracted from primary AML or BM-MSC cultured alone or in combination. Results confirmed that MIF is highly expressed at the RNA and protein level by AML blasts and IL-8 transcription and cytokine release was upregulated in BM-MSC in response to co-culture with AML. Next we found that recombinant MIF increases IL-8 mRNA and protein expression in BM-MSC. Moreover, the MIF inhibition by, ISO-1, inhibits AML induced IL-8 expression and secretion by BM-MSC. Next we sought to determine which kinase signaling cascade is activated by MIF. We used a panel of protein kinase inhibitors and found that the pan-PKC inhibitor Ro-31-8220 completely inhibits AML and MIF induced IL-8 mRNA at sub micromolar concentrations. To further identify the specific PKC isoform responsible for linking AML induced MIF to IL-8 we used PKC isoform specific inhibitors (Go6976 and enzastaurin) which significantly inhibited MIF induced IL-8 expression and protein in BM-MSC. The introduction of PKCß siRNA dramatically inhibited MIF induced IL-8 mRNA expression in BM-MSC confirming that PKCß regulates AML induced BM-MSC derived IL-8 expression. Finally, inhibition of AML/BM-MSC co-cultures with the PKCß inhibitor enzastaurin inhibits BM-MSC induced AML survival in vitro. Conclusions: These results reported here show a novel bidirectional survival mechanism between AML blasts and BM-MSC. Furthermore this work identifies the PKC-ß-IL8 pathway in the BM-MSC of patients with AML as a novel target for future treatment strategies. Disclosures No relevant conflicts of interest to declare.
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Podestà, Marina, Daniela Cilloni, Elena Ponomareva, Clara Cassanelli, Claudia Cossu, Federica Benvenuto, Paola Anserini et al. "Dissecting the Hematopoietic Niche to Improve Transplant: Hedgehog and Wnt Pathways Activation in Fetal Mesenchymal Cells (MSC) Are Associated with Better Support to Hematopoietic Progenitors and Higher Proliferative Capacity Compared to Adult MSC". Blood 118, n. 21 (18 novembre 2011): 4809. http://dx.doi.org/10.1182/blood.v118.21.4809.4809.
Abstract (sommario):
Abstract Abstract 4809 Background. Mesenchymal Stem Cells (MSC) are among major players of the hematopoietic niche. In addition, they have shown to be useful in clinical studies both as immune-modulators as well as in regenerative medicine. However, a major obstacle for proving their usefulness is to obtain a clinically effective cell dose, since often less than 10̂6 /kg of adult MSC have been used. Thus, their potential efficacy can be either disregarded or not formally proved. In addition, forcing in vitro the proliferative potential of adult BM derived MSC (BM-MSC) implies the risk of generating chromosomal abnormalities. The aim of the study is to investigate the potential use of fetal derived MSC (FT-MSC) which could provide means to overcome the above limits. In addition, we attempted to identify some molecular mechanisms regulating the proliferative potential of FT-MSC and their supportive capacity of hematopoietic progenitors. Methods: Specimens from 5 fetal samples were collected after mechanically induced abortion (median gestational age 11 weeks). FT-MSC were obtained from kidneys and femurs and adult MSC from normal BM. After 15 days of culture adherent cells were sub-cultured until 80% confluence. Immunophenotype and karyotype were evaluated. The telomere length was evaluated using the Telomere PNA system. FT-MSC were injected into NOD/SCID mice to rule out any tumorigenic potential. The immunosuppressive activity of FT-MSC and adult BM-MSC was evaluated by standard assays. To quantify the supportive capacity of FT-MSC we weekly measured the colony output using normal BM MNC layered over confluent FT-MSC compared to stromal cell line MS-5 and to BM-MSC. Protein expression of the key regulators of Hedgehog and Wnt pathways were investigated in FT-MSC and BM-MSC by immunofluorescence using monoclonal antibodies against Indian Hedghog (IHh), Sonic Hedgehog (SHh), Desert Hedgehog (DHh) and b-catenin. Fluorescent signal was detected by confocal microscope. Results. MSC successfully grew from 100% of fetal samples. FT-MSC maintained the typical MSC markers (CD73, CD105, CD44, CD106, CD166, HLA class I, negative for HLA class II) and possessed the same capacity to inhibit T cell proliferation as BM-MSC. FT-MSC compared to adult BM-MSC showed a higher proliferative potential: they reached the fifteenth generation and had a median cumulative population doubling of 26 (range 4–47) while BM-MSC grew until the sixth passage showing a median cumulative population doubling of 4 (range 2–5) (p<0.05 at the fourth, fifth and sixth passage). This might be ascribed to both Hedghog and Wnt pathways which were highly activated in FT-MSC compared to BM-MSC. The mean value of SHh protein expression was 11,5 RU (relative units) compared to 5,5 RU in BM-MSC and b-catenin was 17 RU in FT-MSC vs 3.7 RU in adult. Differently from BM-MSC, FT-MSC maintained a stable karyotype without any sign of tumorigenic potential in the mouse model and stable telomere length during culture. By contrast, 5 out of 32 adult MSC developed genetic abnormalities during in vitro expansion. Importantly, FT-MSC display a better support of hematopoiesis compared to BM-MSC or MS-5 cell line, in fact, during five weeks of culture normal bone marrow samples produced an overall colony number higher when FT-MSC were used as feeder layer compared to BM-MSC or MS-5 (p= 0.07 for both). IHh was significantly higher in FT-MSC compared to BM-MSC (121 RU vs 9 RU). This is in keeping with what already reported by Kobune et al. (Blood 2006) regarding the role of IHh in supporting hematopoiesis and provides the molecular basis for the high supportive capacity of FT-MSC. In conclusion, FT-MSC seem to be candidate for being used to support hematopoiesis in transplant settings. FT-MSC overcome the limitation imposed by adult MSC providing a remarkable yield in culture and they show a reassuring safety profile. FT-MSC show a peculiar profile of Hedgehog and Wnt activation possibly responsible for their biological properties described in this study. Disclosures: Saglio: Novartis Pharmaceutical: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau; Pfizer: Consultancy.
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Lima-Neto, J. F., G. H. M. Araujo, M. L. Alvarenga, C. F. Moya-Araujo, I. D. P. Blanco, T. S. Rascado, C. C. Macedo, T. I. Wodewotzky, M. A. Alvarenga e F. C. Landim-Alvarenga. "389 CULTURE OF EQUINE INNER CELL MASS USING EQUINE MESENCHYMAL CELLS AS A FEEDER LAYER". Reproduction, Fertility and Development 22, n. 1 (2010): 351. http://dx.doi.org/10.1071/rdv22n1ab389.
Abstract (sommario):
Stem cells can originate from embryo, fetus, and adults tissues. Adult stem cells are present in almost all organs and are responsible for tissue regeneration and repair. The mesenchymal stem cells (MSC) are multipotent cells present in the bone marrow. The plasticity of these cells allows them to be used in cell therapy because they have the potential to differentiate into several tissues with mesenchymal origin. On the other hand, embryonic stem cells (ESC) have the ability to differentiate into the 3 embryonic tissues (endoderm, mesoderm, and ectoderm) generating all kinds of tissue in the living organism. Embryonic stem cells can be maintained in their undifferentiated state when cultured in vitro in the presence of the leukemia inhibitory factor (LIF) and over a feeder layer composed normally by mouse fibroblasts. Because of the small number of articles studying equine ESC, the current experiment aimed to compare the utilization of equine fibroblasts and equine MSC as feeders for inner cell mass (ICM) culture. Eighteen blastocysts were collected on Day 7 after ovulation from 5 fertile mares that had been artificially inseminated. The embryos were placed in a petri dish with Dulbecco’s phosphate buffered saline + 10% FCS, and the ICM was mechanically removed using 2 insulin needles. The ICM was then transferred to one well of 24 well plates containing either an equine fibroblast monolayer (9 embryos) or a MSC monolayer (9 embryos). Both feeders layers were inactivated with 10 μg mL-1 mitomycine C, and the cell concentration was 140 000 cells/well. The culture media utilized was the DMEM/F12 with 20% FCS, 1% essential amino acids, 1% nonessential amino acids, 0.1 mM β-mercaptoetanol, 10 ng mL-1 LIF, penicillin, streptomycin, and amphotericin B. The ICM was cultured during 5 days for the adherence to the plate, and the media was change every 2 days. Data was analyzed by ANOVA. Results indicated no significant differences between the use of equine fibroblasts or MSC as feeder layer to support equine ICM in culture. The expansion of the ICM cells was observed after 10 to 12 days in culture in 44.4% (4/9) of ICM cultured in MSC monolayer and 55.5% (5/9) of the ICM cultured over a fibroblast layer. The formation of embryoid body-like structures were observed after the second passage (5-7 days in culture) in 50% (2/4) of embryos using MSC feeders and in 40% of embryos (2/5) for which fibroblasts were used. Cultures were then positively marked with the Oct 4 antibody for characterization of the undifferentiated lineage. The results of the current experiment showed that although further patronization of the culture system is still needed, both fibroblasts and MSC are suitable as feeders for the culture of equine ICM when aiming to establish ESC lineage in vitro. Financial support was provided by FAPESP and CNPq.
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Rojewski, Markus, Natalie Fekete, Daniel Fuerst, Philippe Bourin, Ramin Lotfi, Volker Mailaender, Gerlinde Schmidtke-Schrezenmeier, Luc Sensebé e Hubert Schrezenmeier. "GMP-Grade Large-Scale Expansion of Bone-Marrow- (BM) Derived Human Mesenchymal Stem/Stroma Cells (MSC): Comparison of Efficacy of Different Expansion Systems and Role of Cytokines/Chemokines". Blood 116, n. 21 (19 novembre 2010): 337. http://dx.doi.org/10.1182/blood.v116.21.337.337.
Abstract (sommario):
Abstract Abstract 337 Background: MSC cells can differentiate into different tissues and exhibit non-HLA-restricted immunosuppressive properties. They are promising candidates for cellular therapy. Therapeutic use requires large-scale GMP-grade expansion of MSC. Several protocols have been published. Here we systematically compare different expansion procedures with particular emphasis on role of cytokines/chemokines in the expansion medium. Materials and Methods: Bone marrow (BM) was obtained by aspiration from the iliac crest of healthy donors after informed consent and IRB approval. BM aspirate (anticoagulated with heparin (500 U/ml)) was incubated without manipulation in 5-chamber stacks (CellStacks; Corning) in a medium free of animal components (α-MEM (Lonza) with 10% human platelet lysate (hPL)). In the single-step protocol 1.2×104 MNC/ cm2 were seeded. Non-adherent cells were washed off after 72–96 hrs. Partial medium exchange (40%) was performed twice a week (wk). After 11 days MSC were harvested by incubation with recombinant trypsin (TrypZean, Lonza). In the two-step protocol 5×104 leukocytes/cm2 were seeded in 2-chamber stacks. Non-adherent cells were removed after 72–96 hours and complete medium exchange was performed twice/wk. Cells were harvested after 10 days and the harvest was seeded in a 2nd culture at a density of 0.4×104 MSC/cm2. This 2nd culture was harvested after 5 days. Cytokines/chemokines in hPL and in culture medium during the course of expansion was measured by Milliplex MAP Kit (Millipore Corp). Surface marker expression was measured on FACSAria and FACScan. Results: Higher number of MSC could be achieved in cultures with hPL compared to fetal calf serum. hPL was equally effective in supporting MSC proliferation if prepared from apheresis platelet concentrates (PC), buffy coat-derived pooled PC in plasma or pooled PC in additive solution. hPL contained large amounts of PDGF-AB/BB (790 ng/ml; mean of 3 batches of hPL from buffy coat-derived pooled PC), PDGF-AA (266 ng/ml), RANTES (2706 ng/ml), sCD40L (27 ng/ml), GRO (11 ng/ml), sVCAM (2511 ng/ml), sICAM (188 ng/ml). During culture, sCD40L declined rapidly to very low levels. Concentration of PDGF-AA, RANTES and sICAM remained almost stable. In contrast, PDGF-AB/BB declined to low levels (<0.007 ng/ml) in MSC expansion culture whereas concentration remained stable under the same conditions in the absence of MSC. Decline was associated with MSC numbers in the expansion. BM samples from healthy donors (n=4) were split in order to perform paired comparison of single-step vs. two-step expansion protocol. In the single-step protocol 16.3×103±5.8×103 MSC/μl BM seeded were harvested after 11±0 days. In the two-step protocol 12.0×103±4.4×103 MSC/μl BM were harvested after 10 days at the end of passage 0 and 104.0×103±60.4×103 after 5±1 days at the end of passage 1. The overall consumption of medium in the single-step protocol was substantially higher than in the two-step protocol. Phenotype of MSC from the two culture systems did not significantly differ regarding standard markers (positive for CD73, CD90, CD105, HLA-class I; neg. for CD45, CD3, CD34, HLA-DR). However, in passaging experiments we could demonstrate that proportion of MSC positive for CD49a, CD71, CCR4/CD194, CD349 and MSCA-1 decreased whereas proportion of cells positive for c-kit/CD117, CCR3/CD193, CXCR4 and CD200 increased. Conclusion: hPL-based system allows efficient expansion of MSC up to a total number >1×109 MSC from a 15 ml BM aspirate in 2–3 wks with only one passaging step. hPL is a rich source of cytokines, some of which (PDGF-AB/BB) seem to be consumed during expansion and arrive at very low concentrations at the end of the expansion culture. A two-step system provides higher number of MSC per BM cells seeded and requires less medium/culture vessels. Phenotype and differentiation capacity does not differ between single- or two-step culture. However, further passaging goes along with substantial changes of the phenotype. Previous conflicting results regarding chemokine expression of MSC might be due to differences in ex-vivo culture period. Given that chemokine receptor expression affects in-vivo behaviour of cells, MSC harvested after initial expansion (passage 0 or 1) substantially differ from older cells, emphasizing the need to highly standardize all parameters of expansion. (Supported by EU 7th Framework Programme, Projects CASCADE and REBORNE). Disclosures: Fekete: Institute of Clinical Transfusion Medicine and Immunogenetics: Employment. Fuerst:Institute of Clinical Transfusion Medicine and Immunogenetics: Employment. Schrezenmeier:Institute of Clinical Transfusion Medicine and Immunogenetics: Employment.
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Cesarz, Zoe, e Kenichi Tamama. "Spheroid Culture of Mesenchymal Stem Cells". Stem Cells International 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/9176357.
Abstract (sommario):
Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.
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Rink, E., H. French, E. Watson, C. Aurich e F. X. Donadeu. "224 CHARACTERIZATION OF EQUINE ENDOMETRIAL-DERIVED MESENCHYMAL STROMAL CELLS". Reproduction, Fertility and Development 28, n. 2 (2016): 243. http://dx.doi.org/10.1071/rdv28n2ab224.
Abstract (sommario):
Equine mesenchymal stromal cells (MSC) are mainly harvested from bone marrow and adipose tissue, requiring surgical procedures. Although human endometrium is known to harbor mesenchymal precursor cells, the presence of MSC in equine endometrium, a dynamic tissue, has not been investigated. This study reports for the first time the culture and characterisation of MSC from equine endometrium compared with equine bone marrow (BM)-derived MSC. Samples of equine endometrium (n = 6) and BM (n = 3) were collected postmortem. Endometrial tissue was digested using a dissociation medium containing collagenase I and DNase type I, and CD227 (mucin-1)-bound magnetic beads were utilised to separate epithelial (CD227+) from stromal (CD227–) cell fractions. Red blood cells from BM samples were excluded using a density gradient. All cell fractions were cultured in DMEM/F-12 containing 10% fetal bovine serum. After expansion, colony-forming unit (CFU) assay at passage 2, trilineage differentiation (adipogenic, chondrogenic, osteogenic), and flow cytometry analysis at passage 3/4 were performed for CD227– fractions and BM-MSC. Descriptive statistical analysis and 2-tailed t-test was performed with IBM SPSS Statistics 22 (SPSS Inc./IBM, Chicago, IL, USA). Both isolated cell fractions were plastic adherent and grew well under standard MSC culture conditions, although endometrial CD227– cells attached quicker to culture plasticware than did BM-MSC. The CFU assay at passage 2 showed no significant difference in cloning efficiency (CE) between BM-MSC (20.78 ± 2.86%) and CD227– (24.89 ± 3.04%) cell lines (P = 0.36). Flow cytometry showed the expression of MSC markers (CD29, CD44, CD90, CD105) and perivascular markers (CD146, NG2) but almost no expression of haematopoietic markers (CD34, CD45) in both cell lines (Table 1). No statistically relevant difference was seen except for the higher expression of NG2 in BM-MSC (P = 0.054). Trilineage differentiation was successfully induced in both cell lines. In conclusion, we showed the presence of putative MSC in equine endometrium. We successfully isolated and cultured these cells, which display comparable characteristics in MSC criteria as well-established BM-derived MSC. These endometrial-derived MSC may provide a convenient source for veterinary regenerative therapies in equine reproduction. Table 1.Flow cytometry marker expression (mean ± standard error) at passage 4