Flow cytometry and compensation of highly autofluorescent cells: the example of mesenchymal stem cells

Omentum mesenchymal stem cells (OMSCs) could be induced to differentiate into cell varieties under certain conditions. We studied differentiation of OMSCs induced by using placenta extract in NMRI mice. Mesenchymal stem cells (MSCs) were isolated from omentum and cultured with mice placenta extract. MSCs, were assessed after three passages by flow cytometry for CD90, CD44, CD73, CD105, CD34 markers and were recognized their ability to differentiate into bone and fat cell lines. Placenta extract dose was determined with IC50 test then OMSCs were cultured in DMEM and 20% placenta extract.The cell cycle was checked. OMSCs were assayed on 21 days after culture and differentiated cells were determined by flow cytometry and again processed for flow cytometry. CD90, CD44, CD73, CD105 markers were not expressed, only CD34 was their marker. OMSCs were morphologically observed. Differentiated cells are similar to the endothelial cells. Therefore, to identify differentiated cells, CD31 and FLK1 expression were measured. This was confirmed by its expression. G1 phase of the cell cycle shows that OMSCs compared to the control group, were in the differentiation phase. The reason for the differentiation of MSCs into endothelial cells was the sign of presence of VEGF factor in the medium too high value of as a VEGF secreting source.

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Effect of Silicon, Titanium, and Zirconium Ion Implantation on NiTi Biocompatibility

Advances in Materials Science and Engineering ◽

10.1155/2012/706094 ◽

2012 ◽

Vol 2012 ◽

pp. 1-16 ◽

Cited By ~ 19

Author(s):

L. L. Meisner ◽

A. I. Lotkov ◽

V. A. Matveeva ◽

L. V. Artemieva ◽

S. N. Meisner ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Ion Implantation ◽

Cell Counting ◽

High Dose ◽

Surface Layers ◽

Test Flow ◽

Ion Implanted

The objective of the work was to study the effect of high-dose ion implantation (HDII) of NiTi surface layers with Si Ti, or Zr, on the NiTi biocompatibility. The biocompatibility was judged from the intensity and peculiarities of proliferation of mesenchymal stem cells (MSCs) on the NiTi specimen surfaces treated by special mechanical, electrochemical, and HDII methods and differing in chemical composition, morphology, and roughness. It is shown that the ion-implanted NiTi specimens are nontoxic to rat MSCs. When cultivated with the test materials or on their surfaces, the MSCs retain the viability, adhesion, morphology, and capability for proliferationin vitro, as evidenced by cell counting in a Goryaev chamber, MTT test, flow cytometry, and light and fluorescence microscopy. The unimplanted NiTi specimens fail to stimulate MSC proliferation, and this allows the assumption of bioinertness of their surface layers. Conversely, the ion-implanted NiTi specimens reveal properties favorable for MSC proliferation on their surface.

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Isolation and Characterization of Human Synovial Fluid-Derived Mesenchymal Stromal Cells from Popliteal Cyst

Stem Cells International ◽

10.1155/2020/7416493 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15

Author(s):

Fang Li ◽

Jianglin Chen ◽

Mengjia Gong ◽

Yang Bi ◽

Chengchen Hu ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Synovial Fluid ◽

Stromal Cells ◽

Pediatric Patients ◽

Cyst Fluid ◽

Popliteal Cyst ◽

Cell Marker ◽

Related Proteins

Mesenchymal stem cells (MSCs) are multipotent progenitor cells in adult tissues. The aim of this study is to isolate and identify synovial fluid-derived mesenchymal stromal cells (SF-MSCs) from the popliteal cyst fluid of pediatric patients. SF-MSCs were collected from the popliteal cyst fluid of pediatric patients during cystectomy surgery. After cyst fluid extraction and adherent culturing, in vitro morphology, growth curve, and cell cycle were observed. The expression of stem cell surface markers was analyzed by flow cytometry, and expression of cell marker protein was detected by immunofluorescence. SF-MSCs were cultured in osteogenic, adipogenic, and chondrogenic differentiation medium. The differentiation potential of SF-MSCs was analyzed by alkaline phosphatase (Alizarin Red), Oil Red O, and Alcian blue. Antibody detection of human angiogenesis-related proteins was performed compared with bone marrow mesenchymal stem cells (BM-MSCs). The results show that SF-MSCs from the popliteal cyst fluid of pediatric patients showed a shuttle appearance and logarithmic growth. Flow cytometry analysis revealed that SF-MSCs were negative for hematopoietic lineage markers (CD34, CD45) and positive for MSC markers (CD44, CD73, CD90, and CD105). Interstitial cell marker (vimentin) and myofibroblast-like cell marker alpha-smooth muscle actin (α-SMA) were positive. These cells could differentiate into osteogenic, adipogenic, and chondrogenic lineages, respectively. Several types of human angiogenesis-related proteins were detected in the cell secretory fluid. These results show that we successfully obtained SF-MSCs from the popliteal cyst fluid of pediatric patients, which have the potential to be a valuable source of MSCs.

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Human Umbilical Cord Mesenchymal Stem Cells Inhibit the Function of Allogeneic Activated Vγ9Vδ2 T Lymphocytes In Vitro

BioMed Research International ◽

10.1155/2015/317801 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 4

Author(s):

Xiaohuan Liu ◽

Ting Feng ◽

Tianxiang Gong ◽

Chongyang Shen ◽

Tingting Zhu ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

T Cells ◽

Mesenchymal Stem Cells ◽

T Cell ◽

Umbilical Cord ◽

Interleukin 2 ◽

Cell Contact ◽

Human Umbilical Cord ◽

Fas L

Background. Human umbilical cord mesenchymal stem cells (UC-MSCs) can regulate the function of immune cells. However, whether and how UC-MSCs can modulate the function of Vγ9Vδ2 T cells has not been fully understood. Methods. The PBMCs or Vγ9Vδ2 T cells were activated and expanded with pamidronate (PAM) and interleukin-2 (IL-2) with or without the presence UC-MSCs. The effects of UC-MSCs on the proliferation, cytokine expression, and cytotoxicity of Vγ9Vδ2 T cells were determined by flow cytometry. The effects of UC-MSCs on Fas-L, TRAIL-expressing Vγ9Vδ2 T cells, and Vγ9Vδ2 T cell apoptosis were determined by flow cytometry. Results. UC-MSCs inhibited Vγ9Vδ2 T cell proliferation in a dose-dependent but cell-contact independent manner. Coculture with UC-MSCs reduced the frequency of IFNγ+ but increased granzyme B+ Vγ9Vδ2 T cells. UC-MSCs inhibited the cytotoxicity of Vγ9Vδ2 T cells against influenza virus H1N1 infected A549 cells and also reduced the frequency of Fas-L+, TRAIL+ Vγ9Vδ2 T cells but failed to modulate the apoptosis of Vγ9Vδ2 T cells. Conclusions. These results indicated that UC-MSCs efficiently suppressed the proliferation and cytotoxicity of Vγ9Vδ2 T cells and modulated their cytokine production. Fas-L and TRAIL were involved in the regulation. Cell contact and apoptosis of Vγ9Vδ2 T cells were not necessary for the inhibition.

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135 Strategies for transfection of bovine mesenchymal stem cells with pBC1-anti-CD3 vector

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab135 ◽

2020 ◽

Vol 32 (2) ◽

pp. 194

Author(s):

F. B. Duarte ◽

S. N. Báo ◽

M. Brígido ◽

J. M. Araújo ◽

E. d. O. Melo ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Calf Serum ◽

Genetic Material ◽

Cell Receptor ◽

Wharton’S Jelly ◽

Control Group ◽

Wharton's Jelly ◽

Transgenic Cells

Cells from different origins behave differently regarding the incorporation of exogenous genetic material and the formation of transgenic cells. In this context, the objective of this study was to verify the potential of transfection of bovine mesenchymal stem cells from Wharton's jelly and adipose tissue, comparing two transfection protocols, using Lipofectamine LTX and Plus or Xfect reagents, with the integration of humanized anti-CD3. Skin fibroblasts were used as a control group. Humanized anti-CD3 is a monoclonal antibody that interacts with the CD3 molecule of the T-cell receptor, leading to the suppression of T-cells. This antibody is considered an option in the treatment of human autoimmune diseases and against the rejection of transplanted organs. Humanized anti-CD3 was used in this work for the production of bovine transgenic cells that, in the future, will be used in the development of bioreactor animals. In all steps of this study, cell types were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics, in an incubator at 39°C with 5% CO2 in air with saturated humidity. All cells were plated at 5×105 into 24-well culture dishes and co-transfected with vector pBC1-anti-CD3-IRES-FEO and pEF-NEO-GFP using Lipofectamine LTX with reagent Plus or Xfect. Forty-eight hours after transfection, neomycin was added in each treatment and cells were cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton's jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than in fibroblasts, for both the Xfect reagent (20.057±1.620.7 and 10.601±702.86, respectively, P<0.05) and for LTX (19.590±113.84 and 10.518±442.65 respectively, P<0.05). These results, associated with the evaluation of epifluorescence, demonstrated that adipocytes presented a better response to transfection than did other cells, independent of the kit used. Performing PCR on co-transfected adipocytes and fibroblasts demonstrated the presence of anti-CD3, making this approach feasible in future experiments. Southern blotting analysis is being performed to confirm DNA integration. Financial support was provided by Fundação de Amparo à Pesquisa do Distrito Federal (FAPDF); Embrapa MP1.

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Mesenchymal Stem Cells Immunosuppressed IL-22 in Patients with Immune Thrombocytopenia via Soluble Cellular Factors

Journal of Immunology Research ◽

10.1155/2015/316351 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Mei Wu ◽

Hongfeng Ge ◽

Shue Li ◽

Hailiang Chu ◽

Shili Yang ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Immune Thrombocytopenia ◽

Therapeutic Strategy ◽

Alizarin Red ◽

Cellular Factors ◽

Oil Red O ◽

Proliferation Suppression

Mesenchymal stem cells are immunoregulation cells. IL-22 plays an important role in the pathogenesis of immune thrombocytopenia. However, the effects of mesenchymal stem cells on IL-22 production in patients with immune thrombocytopenia remain unclear. Flow cytometry analyzed immunophenotypes of mesenchymal stem cells; differentiation of mesenchymal stem cells was observed by oil red O and Alizarin red S staining; cell proliferation suppression was measured with MTS; IL-22 levels of cell-free supernatants were determined by ELISA. Mesenchymal stem cells inhibited the proliferation of activated CD4+T cells; moreover, mesenchymal stem cells immunosuppressed IL-22 by soluble cellular factors but not PGE2. These results suggest that mesenchymal stem cells may be a therapeutic strategy for patients with immune thrombocytopenia.

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Human Bone Marrow Mesenchymal Stem Cells Enhance Extramedullary Engraftment and Proliferation of Human AML Blasts in NOD-SCID Mice.

Blood ◽

10.1182/blood.v106.11.2312.2312 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2312-2312

Author(s):

Dean A. Lee ◽

William C. Choi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Peripheral Blood ◽

Human Mesenchymal Stem Cells ◽

Tumor Burden ◽

Scid Mice ◽

Mouse Bone Marrow ◽

Mouse Tumor

Abstract BACKGROUND: Inoculation of human normal or leukemic myeloblasts into sublethaly irradiated NOD/SCID mice often results in persistent low-level engraftment (< 5%), but significant proliferation (≥ 5-fold expansion) rarely occurs. Most malignant samples that engraft and proliferate are of FAB M4 subtype and exhibit rapid extramedullary growth at the site of injection without significant marrow or spleen involvement. We hypothesized that low engraftment and proliferation of less mature FAB subtypes results from an increased requirement of these cells for a marrow environment of cytokine and contact-dependent growth and survival factors not adequately provided across species by the mouse bone marrow stroma. Here we show that the subcutaneous injection of minimally-differentiated human mesenchymal stem cells (MSC) in a Matrigel matrix creates an artificial human marrow environment resulting in improved survival and proliferation of human myeloblasts. METHODS: Human leukemic myeloblasts were obtained from the marrow or peripheral blood of 14 newly diagnosed pediatric patients under an IRB-approved collection and banking protocol. MSC were obtained from sterile filters following processing of human marrow from healthy donors or from the NIH-funded MSC bank at Tulane University. 6-to-12 week old NOD-SCID mice were injected IV with 5x106 AML blasts via the retro-orbital sinus (N=38), subcutaneously in 0.5mL Matrigel (N=18), or subcutaneously with 5x105 MSC in 0.5mL of Matrigel (N=14). Mice were euthanized when evidence of tumor burden was present. Peripheral blood, bone marrow, spleen, and subcutaneous nodules were obtained for flow immunophenotyping, FISH, and histopathology. Percent engraftment was determined by flow cytometry for human CD33-APC and mouse H2Kd-PE. RESULTS: Median time from injection to necropsy was 12.5 weeks. 18% died of spontaneous murine thymomas. No animals died of progressive human AML if myeloblasts were injected IV or subcutaneously with Matrigel, and all had < 5% involvement of bone marrow, spleen, and blood. Six animals injected with AML and MSC (43%) developed visible tumors at a median of 8.5 weeks. These tumors were easily reduced to single cell suspensions of > 98% CD33+ by flow cytometry, with mean estimated recovery of 1.3x108 human myeloblasts per mouse tumor (mean 36-fold expansion, range 4 to 52-fold). For cases in which the AML and MSC were derived from subjects of disparate gender, the origin of the cells (leukemic donor vs. MSC donor) was validated by FISH for human X/Y chromosomes. Histopathology of the resulting mass revealed the central development of a stromal chondroid matrix similar to trabecular bone. Marrow, spleen, and blood for all these animals contained < 5% human myeloblasts. CONCLUSIONS: Here we describe an effective method for expanding immature human leukemic myeloblasts in the NOD/SCID mouse. These findings suggest that less mature myeloblasts require human MSC for survival and proliferation and appear to lack significant homing to or expansion in mouse marrow even in the presence of a significant ectopic tumor burden. This is a useful technique for expanding human AML cells for research, may be a model for more broad-based patient-oriented testing of chemotherapeutic and biologic therapies for AML, and represents a novel animal model for studying the stromal interactions and growth requirements of malignant and non-malignant myeloid precursors.

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Research of HIF-1 Mediated SDF-1/CXCR4 Axis on the Directional Migration and Neural Differentiation of Mesenchymal Stem Cells

Blood ◽

10.1182/blood.v116.21.4699.4699 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4699-4699

Author(s):

Qin Yu ◽

Jie Lin ◽

Lizhen Liu ◽

Peipei Li ◽

XiaoBo Xuan ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Western Blotting ◽

Protein Level ◽

Neural Differentiation ◽

Directional Migration ◽

Protein Levels ◽

Cxcr4 Mrna ◽

Cxcr4 Axis

Abstract Abstract 4699 Introduction: Mesenchymal stem cells (MSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. At present, the mechanisms of targeted therapy of MSCs are known to relate with the hypoxia-inducible factor-1 (HIF-1) and its regulated biological axis stromal cell-derived factor-1/chemokine receptor 4 (SDF-1/CXCR4). SDF-1 and CXCR4 have a wide distribution in various cells and tissues, which plays an important role in the development of immune system, circulatory system and central nervous system. We thus inferred that SDF-1/CXCR4 may participate in the differentiation as well as the migration of stem cells. Our research aims to explore the effect of HIF-1α and its mediated SDF-1/CXCR4 axis on the directional migration and neural differentiation of MSCs, which may lead to a break in the efficiency and target distribution of MSCs therapy. Method: (1) Influence of hypoxia, CXCR4 antagonist (AMD3100) and SDF-1α on the proliferation of rat Mesenchymal Stem Cells (rMSCs): rMSCs were isolated from bone marrow of rats, and expanded in vitro. The growth feature of rMSCs exposed to hypoxia (PO2=1%) or normoxia was identified by growth curve, while the effect of AMD3100 (5ug/ml) and SDF-1α (10ng/ml and 100ng/ml) on the proliferation ability of rMSCs was detected by cell counting kit-8. (2) Effect of hypoxia on the expression of HIF-1α, CXCR4 and SDF-1α: Firstly, we used RT-PCR, western blotting and flow cytometry to detect the expression of HIF-1α and CXCR4 mRNA and protein levels in rMSCs which treated with hypoxia for 0h, 6h, 12h, 24h, 48h and 72h. Secondly, HIF-1α, SDF-1α mRNA and protein level in the hippocampus of rats which suffered hypoxia- ischemia for 1d, 3d, 5d, 7d, 14d and 21d were also detected by the same assays. (3) Research of HIF-1α and SDF-1/CXCR4 axis on the migration of rMSCs: We first detected the change of CXCR4 mRNA and protein levels in rMSCs treated with AMD3100 (5ug/ml) and SDF-1α (10ng/ml) by RT-PCR, western blotting and flow cytometry, and then studied SDF-1/CXCR4 axis on the migration of rMSCs using Transwell assay. (4) Effect of HIF-1α and SDF-1/CXCR4 axis on the differentiation of rMSCs: protein level of NSE and GFAP as well as positive rate of neural-induced rMSCs which have been pretreated with AMD3100 (5ug/ml) were detected by western blotting and immunocytochemistry. Results: Persistent hypoxia promoted the proliferation of rMSCs, while AMD3100 and SDF-1α at the concentration mentioned above had no effect. Compared to normal control, the protein expression of HIF-1α in rMSCs increased in hypoxic condition while the mRNA of HIF-1α did not change. Furthermore, the mRNA and protein level of CXCR4 both increased in rMSCs exposed to hypoxia for 6h and 12h, and the results confirmed by flow cytometry. We found HIF-1α mRNA was stably expressed in hippocampus, and increased significantly in hypoxia-ischemia brain damaged (HIBD) rats in a time dependent manner, which reached the peak on 7d. As expected, SDF-1α mRNA in hippocampus of HIBD rats was higher than that of normal control group, which reached the peak on 7d (P<0.01) and stably expressed till 21d, while the protein level is mainly in concordance. Moreover, CXCR4 mRNA was extremely up-regulated in rMSCs treated with SDF-1α (10ng/ml), however, in 5 ug/ml AMD3100 treated rMSCs, which decreased markedly (P<0.01), and the results were confirmed by western blotting and flow cytometry assays (P<0.05). Transwell assay manifested that SDF-1α had obvious chemotaxis to rMSCs. Protein level and positive cell number of NSE and GFAP were extremely down-regulated in rMSCs which pretreated with 5ug/ml AMD3100. Conclusion: Increased expression of HIF-1α led to the up-regulation of SDF-1/CXCR4 axis, and rMSCs displayed chemotaxis migration ascribed to the receptor-ligand interactions of SDF-1α and CXCR4, suggesting that HIF-1 and its mediated SDF-1/CXCR4 axis are of great significant on the directional migration of rMSCs. We also showed that CXCR4 antagonisation reduced the neural differentiation capabilities of rMSCs, thus suggested that SDF-1/CXCR4 axis may deeply involve in the neural differentiation of rMSCs. Disclosures: No relevant conflicts of interest to declare.

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Identification of mesenchymal stem cell differentiation state using dual-micropore microfluidic impedance flow cytometry

Analytical Methods ◽

10.1039/c6ay01377e ◽

2016 ◽

Vol 8 (41) ◽

pp. 7437-7444 ◽

Cited By ~ 13

Author(s):

Hongjun Song ◽

Jenna M. Rosano ◽

Yi Wang ◽

Charles J. Garson ◽

Balabhaskar Prabhakarpandian ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Cell Differentiation ◽

Mesenchymal Stem Cell ◽

Electrical Impedance ◽

Stem Cell Differentiation ◽

Non Invasive ◽

Mesenchymal Stem Cell Differentiation

A dual-micropore-based microfluidic electrical impedance flow cytometer for non-invasive identification of the differentiation state of mesenchymal stem cells.

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