scholarly journals Adhesive Properties of the Hyaluronan Pericellular Coat in Hyaluronan Synthases Overexpressing Mesenchymal Stem Cells

2020 ◽  
Vol 21 (11) ◽  
pp. 3827 ◽  
Author(s):  
Sebastian Reiprich ◽  
Eva Hofbauer ◽  
Stefanie Kiderlen ◽  
Hauke Clausen-Schaumann ◽  
Wolfgang Böcker ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Mesenchymal Stem Cells ◽  
Cell Adhesion ◽  
Human Mesenchymal Stem Cells ◽  
Time Lapse ◽  
Regulatory Function ◽  
Adhesive Properties ◽  
Hyaluronan Synthases ◽  
Adhesive Interactions

Hyaluronan (HA), a natural component of the extracellular matrix, is supposed to have a regulatory function in the stem cell niche. Bone marrow-derived human mesenchymal stem cells (hMSCs) are known to express all three hyaluronan synthases (HASes), which are responsible for HA production. HA is extruded into the extracellular matrix, but also stays bound to the plasma membrane forming a pericellular coat, which plays a key role during early cell adhesion. Since HAS isoenzymes, HAS1, HAS2 and HAS3, produce HA with different molecular weights, a difference in their role for cell adhesion is expected. Here, we transduced the immortalized hMSC cell line SCP1 to constitutively express eGFP-tagged HASes (SCP1-HAS-eGFP) by lentiviral gene transfer. The overexpression of the HAS-eGFP was shown on RNA and protein levels, HA was determined by ELISA and the stained HA-coat was analyzed using confocal microscopy. Time-lapse microscopy, spreading assay and single cell force spectroscopy using atomic force microscopy were applied to characterize adhesion of the different HAS transduced SCP1 cells. We showed in this study that HAS3 overexpressing cells formed the thickest pericellular coat compared with control or HAS1 and HAS2 transduced cells. Furthermore, SCP1-HAS3-eGFP displayed faster and stronger adhesion compared to cells overexpressing the other synthases or control cells. We conclude that overexpression of HASes in hMSCs differentially modulates their initial adhesive interactions with the substrate. This observation might be helpful in regenerative medicine goals.

scholarly journals Screening a chemically defined extracellular matrix mimetic substrate library to identify substrates that enhance substrate-mediated transfection

2020 ◽  
Vol 245 (7) ◽  
pp. 606-619
Author(s):  
Andrew Hamann ◽  
Alvin K Thomas ◽  
Tyler Kozisek ◽  
Eric Farris ◽  
Steffen Lück ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Mesenchymal Stem Cells ◽  
Cell Adhesion ◽  
Nucleic Acid ◽  
Gene Delivery ◽  
Human Mesenchymal Stem Cells ◽  
Sulfated Polysaccharides ◽  
A Cell ◽  
Protein Coatings

Nonviral gene delivery, though limited by inefficiency, has extensive utility in cell therapy, tissue engineering, and diagnostics. Substrate-mediated gene delivery (SMD) increases efficiency and allows transfection at a cell-biomaterial interface, by immobilizing and concentrating nucleic acid complexes on a surface. Efficient SMD generally requires substrates to be coated with serum or other protein coatings to mediate nucleic acid complex immobilization, as well as cell adhesion and growth; however, this strategy limits reproducibility and may be difficult to translate for clinical applications. As an alternative, we screened a chemically defined combinatorial library of 20 different extracellular matrix mimetic substrates containing combinations of (1) different sulfated polysaccharides that are essential extracellular matrix glycosaminoglycans (GAGs), with (2) mimetic peptides derived from adhesion proteins, growth factors, and cell-penetrating domains, for use as SMD coatings. We identified optimal substrates for DNA lipoplex and polyplex SMD transfection of fibroblasts and human mesenchymal stem cells. Optimal extracellular matrix mimetic substrates varied between cell type, donor source, and transfection reagent, but typically contained Heparin GAG and an adhesion peptide. Multiple substrates significantly increased transgene expression (i.e. 2- to 20-fold) over standard protein coatings. Considering previous research of similar ligands, we hypothesize extracellular matrix mimetic substrates modulate cell adhesion, proliferation, and survival, as well as plasmid internalization and trafficking. Our results demonstrate the utility of screening combinatorial extracellular matrix mimetic substrates for optimal SMD transfection towards application- and patient-specific technologies. Impact statement Substrate-mediated gene delivery (SMD) approaches have potential for modification of cells in applications where a cell-material interface exists. Conventional SMD uses ill-defined serum or protein coatings to facilitate immobilization of nucleic acid complexes, cell attachment, and subsequent transfection, which limits reproducibility and clinical utility. As an alternative, we screened a defined library of extracellular matrix mimetic substrates containing combinations of different glycosaminoglycans and bioactive peptides to identify optimal substrates for SMD transfection of fibroblasts and human mesenchymal stem cells. This strategy could be utilized to develop substrates for specific SMD applications in which variability exists between different cell types and patient samples.

Evaluation of Stemness Maintenance Properties of the Recombinant Human Laminin α2 LG1-3 Domains in Human Mesenchymal Stem Cells

2019 ◽  
Vol 26 (10) ◽  
pp. 785-791
Author(s):  
Ji-Eun Kim ◽  
Hye-Jin Seo ◽  
SuJin Lee ◽  
Jun-Hyeog Jang
Keyword(s):  
Escherichia Coli ◽  
Stem Cells ◽  
Molecular Weight ◽  
Mesenchymal Stem Cells ◽  
Cell Adhesion ◽  
Human Mesenchymal Stem Cells ◽  
Adhesion Assay ◽  
Proliferation Assay ◽  
Undifferentiated State ◽  
Laminin Α2

Background: Laminin, a member of the Extracellular Matrix (ECM), is a glycoprotein that is used as a factor that affects cell adhesion, proliferation, survival, and differentiation. Of these, five globular domains (LG domains) of the alpha chain play an important role in influencing the cell by binding to the integrin. Objective: This study aimed to evaluate the ability of globular domains 1-3 of laminin alpha2 (rhLAMA2LG1-3) in maintaining the pluripotency of human Mesenchymal Stem Cells (hMSCs), which are widely used in regenerative medicine. Methods: hMSCs were grown in the medium supplemented with rhLAMA2LG1-3, then the effect of the protein on hMSCs were confirmed through cell adhesion assay, proliferation assay and RTPCR. Results: rhLAMA2LG1-3 expressed in Escherichia coli has a molecular weight of 70 kDa, at 1 µg/ml concentration of rhLAMA2LG1-3, the attachment and proliferation of hMSCs were approximately 3.18-fold and 1.67-fold, respectively, more efficient than those of untreated controls. In addition, the undifferentiated state and degree of stemness of hMSCs were measured, on the basis of CD90 and CD105 levels. In the rhLAMA2LG1-3-treated hMSCs, the expression levels of CD90 and CD105 increased by 2.83-fold and 1.62-fold, respectively, compared to those in untreated controls. Conclusion: rhLAMA2LG1-3 can be potentially used in stem cell therapy to improve the viability and maintain the undifferentiated state of hMSCs.

scholarly journals Laminin-5 activates extracellular matrix production and osteogenic gene focusing in human mesenchymal stem cells

2007 ◽  
Vol 26 (2) ◽  
pp. 106-114 ◽  
Author(s):  
Robert F. Klees ◽  
Roman M. Salasznyk ◽  
Scott Vandenberg ◽  
Kristin Bennett ◽  
George E. Plopper
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cells ◽  
Laminin 5 ◽  
Extracellular Matrix Production ◽  
Matrix Production ◽  
Osteogenic Gene

scholarly journals Tumor Immunotherapy Using Gene-Modified Human Mesenchymal Stem Cells Loaded into Synthetic Extracellular Matrix Scaffolds

Stem Cells ◽  
2009 ◽  
Vol 27 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Marta Compte ◽  
Ángel M. Cuesta ◽  
David Sánchez-Martín ◽  
Vanesa Alonso-Camino ◽  
José Luís Vicario ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cells ◽  
Tumor Immunotherapy ◽  
Extracellular Matrix Scaffolds

Vessel graft fabricated by the on-site differentiation of human mesenchymal stem cells towards vascular cells on vascular extracellular matrix scaffold under mechanical stimulation in a rotary bioreactor

2019 ◽  
Vol 7 (16) ◽  
pp. 2703-2713 ◽  
Author(s):  
Na Li ◽  
Alex P. Rickel ◽  
Hanna J. Sanyour ◽  
Zhongkui Hong
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Extracellular Matrix ◽  
Mesenchymal Stem Cells ◽  
Mechanical Stimulation ◽  
Vascular Tissue ◽  
Human Mesenchymal Stem Cells ◽  
Stem Cell Differentiation ◽  
Vascular Tissue Engineering ◽  
Extracellular Matrix Scaffold

Stem cell differentiation on a decellularized native blood vessel scaffold under mechanical stimulation for vascular tissue engineering.

Annexin II Mediates Plasminogen-Dependant Matrix Invasion by Adult Human Mesenchymal Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2326-2326
Author(s):  
Paul B. Bolno ◽  
Doris A. Morgan ◽  
Mahesh Sharma ◽  
Martin Lazorik ◽  
Andrew S. Wechsler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Western Blot ◽  
Human Mesenchymal Stem Cells ◽  
Angiogenic Factors ◽  
Annexin Ii ◽  
Adult Human

Abstract Background: Annexin II (ANX2) is a fibrinolytic receptor that serves as a binding site for plasminogen and tissue plasminogen activator, facilitating the generation of plasmin. ANX2 is present on a wide variety of cells including vascular endothelial cells as well as macrophages. ANX2 has been shown to play a key role in extracellular matrix degradation, cellular migration, and invasion. This degradation of extracellular matrix may also cause the release of matrix-bound angiogenic factors such as VEGF and FGF. We hypothesized that adult human mesenchymal stem cells (hMSCs) express ANX2 and utilize this receptor for plasmin generation to facilitate basement membrane invasion. Methods: Primary hMSCs were isolated from the sternal bone marrow of patients undergoing median sternotomy. Stem cell surface markers were characterized via immuno-fluorescence. The presence of ANX2 protein by hMSCs was established via western blot. ANX2 mediated plasminogen activation and plasmin generation was quantified using chromozyme-P as a colorimetric substrate. Invasion assays were performed in dual-chamber culture wells containing matrigel inserts. hMSCs were plated into upper chambers containing: serum-free medium (SFM), SFM + Plasminogen, or SFM + Plasminogen + epsilon-aminocaproic acid (e-ACA inhibits binding of plasminogen to ANX2). After 24 hours, invasive cells were isolated and counted. Results: Sternal bone marrow derived hMSCs expressed the membrane phenotype CD34 (−), CD14 (−), CD44 (+), CD105 (+), CD106 (+). The presence of ANX2 was confirmed by western blot analysis. hMSCs generated 1.95 units of plasmin per milligram of protein. There was a 20% (p 0 .004) increase in hMSC invasion in the wells containing plasminogen as compared to SFM alone. When e-ACA was introduced there was a decrease in hMSC invasion back to control values. Conclusion: Our observations establish for the first time the presence and functional activity of ANX2 in hMSCs. These data suggest that mesenchymal stem cell expression of ANX2 facilitates plasminogen-mediated hMSC trans-endothelial invasion, migration and the release of pro-angiogenic factors from within the extracellular matrix, promoting stem cell directed repair and angiogenesis.

Sign in / Sign up

Export Citation Format

Share Document