scholarly journals Efficient Nonviral Transfection of Human Bone Marrow Mesenchymal Stromal Cells Shown Using Placental Growth Factor Overexpression

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Winston Y. Cheung ◽  
Owen Hovey ◽  
Jonathan M. Gobin ◽  
Gauri Muradia ◽  
Jelica Mehic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Transfection Efficiency ◽  
Transient Transfection ◽  
Placental Growth Factor ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Fluorescence Signal ◽  
Transfection Method ◽  
High Cytotoxicity

Background. Human mesenchymal stromal/stem cells (hMSCs) hold great therapeutic potential due to their immunomodulatory and tissue regenerative properties. Enhancement of biological features of hMSCs by transfection has become a focus of investigation for cell- and gene-based therapies. However, many of the current transient transfection methods result in either low transfection efficiency or high cytotoxicity. Methods. In order to find a transfection method that would address the current issues of low transfection efficiency and high cytotoxicity, 6 commercially available cationic lipid and polymer reagents were tested on human bone marrow-derived MSCs (hBM-MSCs) using GFP as a reporter gene. One transfection method using TransIT-2020 was selected and tested with an emphasis on cell quality (viability, identity, and yield), as well as efficacy with a human placental growth factor (PlGF) plasmid. Results. TransIT-2020 yielded the highest fluorescence signal per cell out of the methods that did not decrease cell recovery. Transfecting GFP to 5 hBM-MSC donors using TransIT-2020 yielded 24–36% GFP-expressing cells with a viability of 85–96%. hBM-MSC identity was unaffected as CD90, CD105, and CD73 markers were retained (>95%+) after transfection. When this method was applied to PlGF expression, there was up to a 220-fold increase in secretion. Both growth and secretion of PlGF in overexpressing hBM-MSC were sustained over 7 days, confirming the sustainability and applicability of the TransIT-2020 transfection system. Discussion. We report a simple and efficient method for transient transfection that has not been reported for hBM-MSCs, encompassing high levels of plasmid expression without significant changes to fundamental hBM-MSC characteristics.

scholarly journals Growth Factor Content in Human Sera Affects the Isolation of Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

2016 ◽  
Vol 4 ◽  
Author(s):  
Marina Montali ◽  
Serena Barachini ◽  
Francesca M. Panvini ◽  
Vittoria Carnicelli ◽  
Franca Fulceri ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Progenitor Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Factor Content ◽  
Human Sera

PLATELET DERIVED GROWTH FACTOR (PDGF) BINDS TO HUMAN BONE MARROW FIBROBLASTS AND STIMULATES THEIR PROLIFERATION.

1987 ◽  
Author(s):  
M C Bryckaert ◽  
A Wasteson ◽  
M Lindroth ◽  
G C Tobelem
Keyword(s):  
Electron Microscopy ◽  
Bone Marrow ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Human Bone ◽  
Platelet Derived Growth Factor ◽  
Human Bone Marrow ◽  
Immunofluorescent Staining ◽  
Cultured Fibroblasts ◽  
Bone Marrow Fibroblasts

A role for the Platelet Derived Growth Factor (PDGF) has been suggested in the abnormal proliferation of bone marrow fibroblasts occuring during myelofibrosis. To investigate this hypothesis, human bone marrow fibroblasts were isolated, and the cultures were characterized by immunofluorescent staining and electron microscopy. Electron microscopy eliminated the presence of endothelial cells by the absence of Weibel-Palade-Bodies. A positive intra and extra cellular antifibro-nectin staining was observed by immunofluorescent staining. The cultured cells didn’t show any labeling with specific antibodies for factor VIII von Willebrand factor, desmin or macrophage. Following the characterization of the bone marrow fibroblasts, using human pure 125I-PDGF, a specific binding of 125I-PDGF was demonstrated. The binding reached a plateau after 3 hours at 20°C, and after 4 hours at 4°C. Addition of unlabeled PDGF decreased this binding until 25 %.Saturation curve and scatchard analysis indicated two classes of sites with respectively 21,000 sites/aall and 37.000 sites/cell with an apparent Kd of 0.3 X 10-10 M and 0.5 X 10-9 M. Normal human serum at a concentration of 20 % induced a maximal DNA synthesis measured by-3H thymidine incorporation. When PDGF was added alone to the cultured fibroblasts at a concentration of 15 ng/ml, it induced a maximal DNA synthesis of 400 %.In the presence of 5 % of Platelet Poor Plasma (PPP), the same concentration of PDGF (15ng/ml) increased the incorporation of 3H thymidine up to 900%.In conclusion i) PDGF binds to human bone marrow fibroblasts, ii) PDGF stimulates their proliferation. These results are in favour of a role of PDGF in the proliferation of bone marrow fibroblasts associated with the development of myelofibrosis.

scholarly journals Characterization of Signal Transduction Pathways in Human Bone Marrow Endothelial Cells

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2253-2259 ◽  
Author(s):  
Zhong-Ying Liu ◽  
Ramesh K. Ganju ◽  
Jian-Feng Wang ◽  
Karin Schweitzer ◽  
Babette Weksler ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Growth Factors ◽  
Growth Factor ◽  
T Antigen ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Signal Transduction Pathways ◽  
Stimulation Of

Human bone marrow endothelial cells immortalized with the T antigen of SV40 (TrHBMEC) have previously been characterized by us with regard to their properties that are similar to primary marrow endothelial cells and their utility as a model system. We now report that TrHBMEC express a recently discovered signal transduction molecule termed RAFTK (related adhesion focal tyrosine kinase), also called Pyk2 or CAK-β. RAFTK, the second member of the focal adhesion kinase (FAK) family, is known to be activated in response to calcium flux in neuronal cells and integrin stimulation in megakaryocytes and B cells. We have studied the effects of cytokines on RAFTK activation in TrHBMEC. Treatment of TrHBMEC with the vascular endothelial growth factor (VEGF ), as well as the VEGF-related protein (VRP), the recently identified ligand for the FLT-4 receptor, resulted in enhanced tyrosine phosphorylation of RAFTK. Similar changes in RAFTK phosphorylation were observed upon stimulation of TrHBMEC with basic fibroblast growth factor (bFGF ) or oncostatin M (OSM). Stimulation of these cells with growth factors also resulted in an increase in RAFTK activity and the c-Jun NH2 -terminal kinase (JNK). RAFTK coimmunoprecipitated with the cytoskeletal protein paxillin through its C-terminal proline-rich domain in TrHBMEC. These results suggest that, in marrow endothelium, activation of RAFTK by VEGF, VRP, OSM, and bFGF represents a new element in the signal transduction pathways used by these growth factors and likely acts to coordinate signaling from their surface receptors to the cytoskeleton, thereby modulating cell growth and function.

scholarly journals The Effect of Human Bone Marrow Mesenchymal Stem Cells on Epidermal Growth Factor and Epidermal Growth Factor Receptor Expression in Re-epithelialization Process in the Healing of Burns on Experimental Rats

2020 ◽  
Vol 8 (A) ◽  
pp. 508-511
Author(s):  
Gusti Revilla ◽  
Henny Mulyani
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Egfr Expression ◽  
Marrow Mesenchymal Stem Cells ◽  
Epidermal Growth

BACKGROUND: Research on human bone marrow mesenchymal stem cells (hBM-MSCs) for burns healing has been known to increase the percentage of integrin expression of α2β1, type I collagen, transforming growth factor-β, and matrix metalloproteinases-9, but research on giving hBM-MSCs to growth factor expression in the process of re-epithelialization of burn healing has not been done. AIM: This study aims to the effect of hBM-MSCs given on the expression of epidermal growth factor (EGF) and EGF receptor (EGFR) in the process re-epithelialization in the healing of burn experimental rat. MATERIALS AND METHODS: This research is experimental with the post-test only control design, using 30 Wistar rats. Rats were divided into two groups, namely, control (phosphate-buffered saline), and the treatment was given hBM-MSCs, and stem cells were given subcutaneous doses of 2 × 106 cells/ml. Before being treated rats were anesthetized using xylazine and ketamine then the rats were burned in the dorsal (spine) with full-thickness. On the 3, 7, and 14 days, skin tissue was taken to see the expression of EGF and EGFR by immunohistochemical methods. This study was approved by the Ethics Commission of the Faculty of Medicine, Andalas University, Padang. The results of the study were analyzed by the t-test. RESULTS: Immunohistochemical examination of EGF and EGFR expressions after hBM-MSCs administration has significantly increased epithelialization compared with controls. Increased EGF expression was found on days 3 and 7 with moderate positive internal revenue service (IRS) assessment and on day 14 strong positive EGF expression, as well as EGFR expression on days 3 and 7 with moderate positive IRS assessment and on day 14 robust positive EGFR expression. CONCLUSION: This study concluded that giving of hBM-MSCs can increase the expression of EGF and EGFR which enhances the process of re-epithelialization thereby accelerating the healing of burns of experimental rats.

The role of platelet α-granular proteins in the regulation of thrombopoietin messenger RNA expression in human bone marrow stromal cells

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3094-3101 ◽  
Author(s):  
Ranita Sungaran ◽  
Orin T. Chisholm ◽  
Boban Markovic ◽  
Levon M. Khachigian ◽  
Yoshihiro Tanaka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Mrna Expression ◽  
Stromal Cells ◽  
Messenger Rna ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Dependent Manner ◽  
Platelet Factor ◽  
Dose Dependent

Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet -granular proteins. We showed that platelet-derived growth factor (PDGF) BB and fibroblast growth factor (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and 20 ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet factor 4 (PF4), thrombospondin (TSP), and transforming growth factor-beta (TGF-β) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 μg/mL of both PF4 and TSP and 50 ng/mL of TGF-β, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet -granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.

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