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 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 Blowup of Accidental Images as a Passageway to Discovery: Insights into the Interaction between Hydroxyapatite Nanoparticles and Human Mesenchymal Stem Cells

2020 ◽  
Vol 10 (22) ◽  
pp. 8204
Author(s):  
Vuk Uskoković
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Adhesion ◽  
Cellular Uptake ◽  
Meta Analysis ◽  
Statistical Significance ◽  
Human Mesenchymal Stem Cells ◽  
Hydroxyapatite Nanoparticles ◽  
Nanoparticle Clusters

Despite decades of research into the interaction between cells and nanoparticles, there is a lack of consensus regarding how specific physicochemical characteristics of the nanoparticles, including chemical composition, crystallinity, size, morphology, charge, and aspect ratio, among others, govern their internalization and intracellular fate. Methodological novelties offer new perspectives on the same old problematics, and often translate into an improved understanding of the given topic. Inspired by an analogy with the theme of the movie, Lisbon Story, a conceptually unconventional method for gaining insight into the interaction between nanoparticles and cells is proposed here. It involves the random, “Take 1” capture of an atomic force micrograph showing the interaction of human mesenchymal stem cells and clusters of spherical hydroxyapatite nanoparticles with a broad distribution of sizes and shapes, the blowup of its segments, and their detailed qualitative inspection. This method led to the derivation of three illustrative hypotheses, some of which were refuted and some corroborated. Specifically, the presupposition that there is an inverse relationship between the cellular uptake efficiency and the size of nanoparticle clusters was confirmed, both empirically and through a literature meta-analysis, but the idea that the geometry of these clusters affects the uptake was refuted. The definite presence of morphological determinants of the cellular uptake at the level of elementary particles, not clusters thereof, however, was confirmed in an alternative experiment. Likewise, immunofluorescent studies demonstrated that relatively large and irregularly shaped nanoparticle clusters do get internalized and localized to the perinuclear area, where they engage in an intimate interaction with the cell nucleus. The proposed enhancement of the binding between cells and biomaterials by increasing the surface ruffling consequential to the nanoparticle uptake - in analogy with the enhanced cell adhesion achieved by introducing topographic irregularities to smooth biomaterial surfaces - was also confirmed by showing that the uptake improves the stem cell adhesion. The uptake also augmented the stem cell viability and the proliferative capacity of cells reseeded with this internal nanoparticle cargo on a fresh surface, albeit with moderate levels of statistical significance and the caveat of its presumed dependence on the cell type, the nanoparticle chemistry and dose, and the overall stage in the transition of the multipotent cells toward an osteoprogenitor lineage.

scholarly journals Chitosan/DNA Polyelectrolyte Complex Membranes for Controlling Cell Spreading and Aggregation

2008 ◽  
Vol 2 (1) ◽  
pp. 148-151 ◽  
Author(s):  
Naoki Kawazoe ◽  
Yujiro Narita ◽  
Guoping Chen ◽  
Tadaatsu Satomi ◽  
Tetsuya Tateishi
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Adhesion ◽  
Cell Viability ◽  
Dna Content ◽  
Polyelectrolyte Complex ◽  
Human Mesenchymal Stem Cells ◽  
Cell Spreading ◽  
High Ratio ◽  
Hydrophilic Surface

Three kinds of polyelectrolyte complex membranes were prepared by mixing chitosan and DNA at the ratios of 3:1, 1:1, and 1:3. The effects of the membranes on cell adhesion, spreading, and viability were investigated. The membrane with the high ratio of chitosan had a less hydrophilic surface. The surface of the polyelectrolyte complex membranes became rough as the DNA content increased. The ratio of DNA to chitosan showed some effect on cell adhesion, spreading, and viability. Human mesenchymal stem cells adhered and spread on membranes prepared at chitosan/DNA ratios of 1:1 and 3:1, while they did not on membranes prepared at a chitosan/DNA ratio of 1:3. Cells aggregated on the membrane prepared at a chitosan/DNA ratio of 1:3. Cell viability was also higher on membranes prepared at chitosan/ DNA ratios of 1:1 and 3:1 than that on the membrane prepared at a chitosan/DNA ratio of 1:3. The membrane with a high content of chitosan facilitated cell adhesion and spreading, while a high content of DNA suppressed cell adhesion and spreading.

Adhesion of human mesenchymal stem cells can be controlled by electron beam-microstructured titanium alloy surfaces during osteogenic differentiation

2015 ◽  
Vol 60 (3) ◽  
Author(s):  
Sabine Neuss ◽  
Claudia Panfil ◽  
Daniela Filipa Duarte Campos ◽  
Michael Weber ◽  
Christian Otten ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Titanium Alloy ◽  
Electron Beam ◽  
Cell Adhesion ◽  
Bone Tissue Engineering ◽  
Osteogenic Differentiation ◽  
Human Mesenchymal Stem Cells ◽  
Implant Surface

AbstractSeveral studies focusing on bone tissue engineering demonstrated that given microstructuring of an implant surface has a strong effect on its interaction with cells, and their adhesion and differentiation. In the present study, geometrically structured titanium alloy surfaces are shown to be able to guide cell adhesion during differentiation

Silver-Doped Apatite as a Bioactive and an Antimicrobial Bone Material

2011 ◽  
Vol 493-494 ◽  
pp. 27-30 ◽  
Author(s):  
Eng San Thian ◽  
P.N. Lim ◽  
Z. Shi ◽  
B.Y. Tay ◽  
K.G. Neoh
Keyword(s):  
Escherichia Coli ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cells ◽  
Bone Material ◽  
Considerable Potential ◽  
E Coli ◽  
Time Points ◽  
Phase Pure

Phase-pure nanostructured silver-doped hydroxyapatite (nAgHA) of various Ag contents was synthesised. These nAgHA were then evaluated in-vitro using human mesenchymal stem cells (hMSCs) and Escherichia coli (E. coli). Results revealed that hMSCs grew generally well on all nAgHA at all time points. In addition, fewer E. coli were seen attaching on the surface of all nAgHA. Hence, this work demonstrated that nAgHA offers considerable potential as a biomaterial.

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.

Construction and Evaluation of Recombinant Chimeric Fibrillin and Elastin Fragment in Human Mesenchymal Stem Cells

2021 ◽  
Vol 28 ◽  
Author(s):  
Eui-Seung Jeong ◽  
Bo-Hyun Park ◽  
Sujin Lee ◽  
Jun-Hyeog Jang
Keyword(s):  
Stem Cells ◽  
Molecular Weight ◽  
Mesenchymal Stem Cells ◽  
Smooth Muscle ◽  
Stem Cell ◽  
Cell Adhesion ◽  
Cell Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Control Group

Background: Diverse extracellular matrix (ECM) proteins physically interact with stem cells and regulate stem cell function. However, the large molecular weight of the natural ECM renders large-scale fabrication of a similar functional structure challenging. Objective: The objective of this study was to construct a low molecular weight and multifunctional chimeric form of recombinant ECM to stimulate mesenchymal stem cell (MSC) for tissue repair. We engineered Fibrillin-1PF14 fused to an elastin-like polypeptide to develop a new biomimetic ECM for stem cell differentiation and investigated whether this recombinant chimeric Fibrillin-Elastin fragment (rcFE) was effective on human nasal inferior turbinate-derived mesenchymal stem cells (hTMSCs). Methods: hTMSCs were grown in the medium supplemented with rcFE, then the effect of the protein was confirmed through cell adhesion assay, proliferation assay, and real-time PCR. Results: rcFE enhanced the adhesion activity of hTMSCs by 2.7-fold at the optimal concentration, and the proliferation activity was 2.6-fold higher than that of the control group (non-treatment rcFE). In addition, when smooth muscle cell differentiation markers were identified by real-time PCR, Calponin increased about 6-fold, α-actin about 9-fold, and MYH11 about 10-fold compared to the control group. Conclusion: Chimeric rcFE enhanced cellular functions such as cell adhesion, proliferation, and smooth muscle differentiation of hTMSCs, suggesting that the rcFE can facilitate the induction of tissue regeneration.

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