In Vivo Myeloperoxidase Imaging and Flow Cytometry Analysis of Intestinal Myeloid Cells

2016 ◽  
pp. 161-167 ◽  
Author(s):  
Jan Hülsdünker ◽  
Robert Zeiser
Keyword(s):  
Flow Cytometry ◽  
Myeloid Cells ◽  
Flow Cytometry Analysis

scholarly journals Cisplatin Increased the Expression of PD-1 and PD-L1 by YAP1 in Hepatocellular Carcinoma

2021 ◽  
Author(s):  
Liyuan Hao ◽  
Yinglin Guo ◽  
Qing Peng ◽  
Zhiqin Zhang ◽  
Shenghao Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
Cancer Cells ◽  
Flow Cytometry Analysis ◽  
Chemotherapy Drugs ◽  
The World ◽  
Hcc Cells

Abstract Hepatocellular carcinoma (HCC) was one of the most malignant cancers in the world. Cisplatin (DDP) was one of the main chemotherapy drugs for HCC, but the mechanism of DDP treatment for HCC remains unclear. In this presentation, we found that DDP inhibited the growth of HCC cells and promoted the expression of PD-1 and its ligand PD-L1 in cancer cells. Meanwhile, flow cytometry analysis revealed that DDP enhanced PD-1-CD8+ T cells expression and decreased PD-1+CD8+ T cells expression. ELISA analysis suggested that DDP decreased TGF-β expression in vivo. Therefore, the study indicated that DDP enhanced PD-1 and PD-L1 expression and inhibited the growth of HCC.

scholarly journals Potential caveats of putative microglia-specific markers for assessment of age-related cerebrovascular neuroinflammation

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Pedram Honarpisheh ◽  
Juneyoung Lee ◽  
Anik Banerjee ◽  
Maria P. Blasco-Conesa ◽  
Parisa Honarpisheh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ex Vivo ◽  
Myeloid Cells ◽  
Amyloid Angiopathy ◽  
Future Studies ◽  
Age Related ◽  
In Vivo Animal Models ◽  
Intermediate Expression ◽  
The Brain

Abstract Background The ability to distinguish resident microglia from infiltrating myeloid cells by flow cytometry-based surface phenotyping is an important technique for examining age-related neuroinflammation. The most commonly used surface markers for the identification of microglia include CD45 (low-intermediate expression), CD11b, Tmem119, and P2RY12. Methods In this study, we examined changes in expression levels of these putative microglia markers in in vivo animal models of stroke, cerebral amyloid angiopathy (CAA), and aging as well as in an ex vivo LPS-induced inflammation model. Results We demonstrate that Tmem119 and P2RY12 expression is evident within both CD45int and CD45high myeloid populations in models of stroke, CAA, and aging. Interestingly, LPS stimulation of FACS-sorted adult microglia suggested that these brain-resident myeloid cells can upregulate CD45 and downregulate Tmem119 and P2RY12, making them indistinguishable from peripherally derived myeloid populations. Importantly, our findings show that these changes in the molecular signatures of microglia can occur without a contribution from the other brain-resident or peripherally sourced immune cells. Conclusion We recommend future studies approach microglia identification by flow cytometry with caution, particularly in the absence of the use of a combination of markers validated for the specific neuroinflammation model of interest. The subpopulation of resident microglia residing within the “infiltrating myeloid” population, albeit small, may be functionally important in maintaining immune vigilance in the brain thus should not be overlooked in neuroimmunological studies.

scholarly journals Human levator veli palatini muscle: a novel source of mesenchymal stromal cells for use in the rehabilitation of patients with congenital craniofacial malformations

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniela Franco Bueno ◽  
Gerson Shigueru Kabayashi ◽  
Carla Cristina Gomes Pinheiro ◽  
Daniela Y. S. Tanikawa ◽  
Cassio Eduardo Raposo-Amaral ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cleft Palate ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Osteogenic Potential ◽  
Flow Cytometry Analysis ◽  
Non Invasive ◽  
Levator Veli Palatini

Abstract Background Bone reconstruction in congenital craniofacial differences, which affect about 2–3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stromal cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent cells that can be isolated via non-invasive procedures. In this study, we analyzed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during palatoplasty in cleft palate patients, represent a novel source of MSCs with osteogenic potential. Methods We obtained levator veli palatini muscle fragments (3–5 mm3), during surgical repair of cleft palate in 5 unrelated patients. Mesenchymal stromal cells were isolated from the muscle using a pre-plating technique and other standard practices. The multipotent nature of the isolated stromal cells was demonstrated via flow cytometry analysis and by induction along osteogenic, adipogenic, and chondrogenic differentiation pathways. To demonstrate the osteogenic potential of these cells in vivo, they were used to reconstruct a critical-sized full-thickness calvarial defect model in immunocompetent rats. Results Flow cytometry analysis showed that the isolated stromal cells were positive for mesenchymal stem cell antigens (CD29, CD44, CD73, CD90, and CD105) and negative for hematopoietic (CD34 and CD45) or endothelial cell markers (CD31). The cells successfully underwent osteogenic, chondrogenic, and adipogenic cell differentiation under appropriate cell culture conditions. Calvarial defects treated with CellCeram™ scaffolds seeded with the isolated levator veli palatini muscle cells showed greater bone healing compared to defects treated with acellular scaffolds. Conclusion Cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stromal cells, both in vitro and in vivo. Our findings suggest that these cells may have clinical relevance in the surgical rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

CREB Regulates Early Myelopoiesis and Myeloid Engraftment.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1452-1452
Author(s):  
Tiffany Simms-Waldrip ◽  
Michelle Yoonha Cho ◽  
Kenneth Dorshkind ◽  
Kathleen M Sakamoto
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Research Funding ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Wild Type ◽  
In Vivo Models ◽  
Hematopoietic Stem

Abstract Abstract 1452 The cAMP-responsive element binding protein (CREB) is a nuclear transcription factor that regulates genes that control cell proliferation, differentiation, and survival. CREB overexpression leads to increased proliferation and survival of myeloid cells. Transgenic (Tg) mice overexpressing CREB under the control of the myeloid specific promoter hMRP8 develop myeloproliferative disease (MPD) but not leukemia. We hypothesized that transplantation of hematopoietic stem cells from CREB transgenic mice into lethally irradiated recipient wild type mice would lead to enhanced myelopoiesis and myeloid engraftment. The goal of our study was to determine if proliferative stress through transplantation would result in increased myeloid engraftment and progression of CREB overexpressing cells from MPD to leukemia. Steady state analyses were performed on CREB Tg mice, including flow cytometry to resolve common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), and megakaryocyte erythroid progenitors (MEP), as well as cell cycle analysis to determine baseline proliferative state. In vitro and in vivo models that exposed CREB-expressing cells to proliferative stress were used. In the former case, long-term bone marrow cultures (LTBMC) were established on an adherent layer of stromal cells prepared from wild type (WT) bone marrow (BM) with media specific for myeloid cell growth. BM cells (2 × 106) from CREB Tg mice or WT controls were seeded onto the stroma and evaluated at 4 and 8 weeks for myeloid cell proliferation. In vivo studies were conducted by transplanting (2.5 × 106) BM cells from CREB Tg mice into lethally irradiated recipients that were sacrificed at 4 weeks. Cells harvested from LTBMC or transplant recipients were analyzed by flow cytometry to evaluate cell lineage and proliferation or were plated in methylcellulose and assessed for colony formation. In addition, kinetic analyses were performed on these populations. At baseline, CREB Tg mice have an increased percentage of early progenitors (1.8% vs. 1.2%, p=0.0001) with increased absolute numbers of CMP (17,683 cells vs. 11,650 cells, p=0.0001) at 12 weeks of age compared to WT controls. CREB Tg mice also have increased number of cells in S phase at baseline (26% vs. 20%, p=0.0022) due to upregulation of cyclins A and D. LTBMCs seeded with BM cells from CREB Tg mice had greater numbers of myeloid cells at 4 weeks compared to cultures established with WT marrow (4.5 × 106 cells/mL and 1.3 × 106 cells/mL respectively, p = 0.0135). Consistent with these data, mice transplanted with CREB Tg BM had a significantly higher percentage of donor myeloid cells at 4 weeks, detected using cell surface markers Gr-1+Mac-1+ (67% vs. 40%, p=0.0061). These mice also had a higher percentage of more differentiated Mac-1+ myeloid cells (11% vs. 0%, p=0.0014) and a higher number of myeloid cells in BM colony assays compared to recipients of WT marrow (69% vs. 13%, p<0.0001). At 4 weeks post-transplant, the histology of the spleen and liver from mice transplanted with CREB Tg marrow demonstrated replacement of the lymphocytes in the white pulp with macrophages, as well as extramedullary hematopoiesis in the liver that was not observed in WT controls. Our results provide evidence that CREB overexpression enhances myelopoiesis and short-term myeloid engraftment, but is not sufficient for transformation to AML. Therefore, CREB plays a critical role in normal hematopoietic dynamics and myeloid progenitor cell kinetics. Disclosures: Sakamoto: Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.

scholarly journals Targeted imaging of esophageal adenocarcinoma with a near-infrared fluorescent peptide

2020 ◽  
Author(s):  
Xiaoyu Kang ◽  
Meng Li ◽  
Lei Liu ◽  
Shaopeng Liu ◽  
Hao Hu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Esophageal Carcinoma ◽  
Esophageal Adenocarcinoma ◽  
Tumor Targeting ◽  
Confocal Imaging ◽  
Block Group ◽  
Flow Cytometry Analysis ◽  
Fluorescent Peptide

Abstract Background: Targeted optical imaging offers a noninvasive and accurate method for the early detection of gastrointestinal tumors, especially for flat appearances. In our previous study, a sequence of SNFYMPL (SNF) was identified as a specific peptide to bind to esophageal carcinoma using phage-display technology. This study aimed to evaluate the tumor-targeting efficacy of Cy5.5-conjugated SNF probe for imaging of esophageal carcinoma in vitro and in vivo.Methods: The SNF-Cy5.5 probe was synthesized and then identified using High Performance Liquid Chromatography (HPLC) and mass spectrometry (MS). Confocal fluorescence imaging and Flow cytometry analysis were performed to evaluate the binding specificity and the receptor binding affinity of SNF-Cy5.5 to OE33. In vivo imaging was performed to evaluate the targeting ability of SNF-Cy5.5 to esophageal carcinoma.Results：The confocal imaging and flow cytometry analysis showed that SNF-Cy5.5 bound specifically to the plasma membrane of OE33 cells with a high affinity. In vivo, for non-block group, SNF-Cy5.5 probe exhibited rapid OE33 tumor targeting during 24 h p.i. and excellent tumor-to-background contrast at 2 h p.i. For the block group, SNF-Cy5.5 was not observed in the mice after 4h p.i. Ex vivo imaging also revealed that a higher fluorescent signal intensity value of the tumors was clearly observed in the non-block group than that in the block group (2.6 ± 0.32× 109 vs 0.8 ± 0.08× 109, p < 0.05).Conclusions: SNF -Cy5.5 was synthesized and characterized with a high efficiency and purity. The higher affinity, specificity, and tumor targeting efficacy of SNF-Cy5.5 were confirmed by in vitro and in vivo tests. SNF-Cy5.5 is a promising optical probe for the imaging of esophageal adenocarcinoma.

scholarly journals Successful transplantation of spermatogonial stem cells into the seminiferous tubules of busulfan-treated mice

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Hossein Azizi ◽  
Amirreza Niazi Tabar ◽  
Thomas Skutella
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Spermatogonial Stem Cells ◽  
Pcr Analysis ◽  
Seminiferous Tubules ◽  
Flow Cytometry Analysis ◽  
Rt Pcr ◽  
Expression Levels ◽  
Successful Transplantation

Abstract Background Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. Methods In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology—immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer. Results ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P < 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. Conclusions In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

scholarly journals Successful Transplantation of Spermatogonial Stem Cells Into the Seminiferous Tubules of Busulfan-treated Mice

2020 ◽  
Author(s):  
Hossein Azizi ◽  
Amirreza Niazi Tabar ◽  
Thomas Skutella
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Spermatogonial Stem Cells ◽  
Pcr Analysis ◽  
Seminiferous Tubules ◽  
Flow Cytometry Analysis ◽  
Rt Pcr ◽  
Expression Levels ◽  
Successful Transplantation

Abstract Background: Spermatogonial stem cells (SSCs) in the testis are crucial for transferring genetic information to the next generation. Successful transplantation of SSCs to infertile men is an advanced therapeutic application in reproductive biology research. Methods: In this experimental research, both in vitro and in vivo characterization of undifferentiated and differentiated SSCs were performed by morphology - immunocytochemistry (ICC), immunohistochemistry (IMH), Fluidigm Real-Time polymerase chain reaction (RT-PCR) and flow cytometry analysis. The isolated SSCs were finally microinjected into the rete testis of busulfan-treated mice. The compact undifferentiated and more loosely connected round differentiated SSCs were isolated during testicular cell expansion from their specific feeder layer.Results: ICC analysis indicated high and low expression levels of Zbtb16 in undifferentiated and differentiated germ cells. Also, IMH analysis showed different expression levels of Zbtb16 in the two different germ stem cell populations of the testicular tissue. While Fluidigm RT-PCR analysis indicated overexpression of the TAF4B germ cell gene, the expression of DAZL, VASA, and Zbtb16 were down-regulated during the differentiation of SSCs (P< 0.05). Also, flow cytometry analysis confirmed the significant downregulation of Itgb1 and Itga4 during differentiation. By transplantation of SSCs into busulfan-treated NOD/SCID mice, GFP-labeled sperm cells developed. Conclusions: In the current study, we performed a transplantation technique that could be useful for the future microinjection of SSCs during infertility treatment and for studying in vivo differentiation of SSCs into sperm.

scholarly journals Human levator veli palatini muscle: A novel source of mesenchymal stem cells for use in the rehabilitation of patients with congenital craniofacial malformations

2020 ◽  
Author(s):  
Daniela Franco Bueno ◽  
Gerson Shigueru Kabayashi ◽  
Carla Cristina Gomes Pinheiro ◽  
Daniela Y S Tanikawa ◽  
Cassio Eduardo Raposo-Amaral ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Osteogenic Potential ◽  
Flow Cytometry Analysis ◽  
Non Invasive ◽  
Levator Veli Palatini

Abstract Background. Bone reconstruction in congenital craniofacial differences, which affect about 2-3% of newborns, has long been the focus of intensive research in the field of bone tissue engineering. The possibility of using mesenchymal stem cells in regenerative medicine protocols has opened a new field of investigation aimed at finding optimal sources of multipotent stem cells that can be isolated via non-invasive procedures. Here we analysed whether levator veli palatini muscle fragments, which can be readily obtained in non-invasive manner during surgical rehabilitation of cleft p­­atients during palatoplasty, represent a novel source of MSCs with osteogenic potential. Methods. We obtained levator veli palatini muscle fragments, in non-invasive procedure during surgical rehabilitation of 5 unrelated cleft palate patients (palatoplasty surgery). The levator veli palatini muscle fragments was used to obtain the mesenchymal cells using pre-plating technique in a clean rooms infrastructure and all procedures were performed at good practices of manipulation conditions. To prove that levator veli palatini muscle are mesenchymal stem cells they were induced to flow cytometry analysis and to differentiation into bone, cartilage, fat and muscle. To demonstrate the osteogenic potential of these cells in vivo a bilateral full thickness calvarial defect model was made in immunocompentent rats.Results. Flow cytometry analysis showed that the cells were positive for mesenchymal stem cell antigens (CD29, CD73, CD90), while negative for hematopoietic (CD45) or endothelial cell markers (CD31). Moreover, these cells were capable of undergoing chondrogenic, adipogenic, osteogenic and skeletal muscle cell differentiation under appropriate cell culture conditions characterizing them as mesenchymal stem cell. Defects treated with CellCeramTM scaffolds seeded with levator veli palatini muscle cells showed significantly greater bone healing compared to defects treated with acellular scaffolds. Conclusion. We have demonstrated that cells derived from levator veli palatini muscle have phenotypic characteristics similar to other mesenchymal stem cells, both in vitro and in vivo. Our findings suggest that these cells may have clinical relevance in the rehabilitation of patients with cleft palate and other craniofacial anomalies characterized by significant bone deficit.

Sign in / Sign up

Export Citation Format

Share Document