Correlation between in vitro expansion-related cell stiffening and differentiation potential of human mesenchymal stem cells

2015 ◽  
Vol 90 (1-3) ◽  
pp. 1-15 ◽  
Author(s):  
Courtney E. LeBlon ◽  
Meghan E. Casey ◽  
Caitlin R. Fodor ◽  
Tony Zhang ◽  
Xiaohui Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cells ◽  
Differentiation Potential ◽  
Related Cell ◽  
In Vitro Expansion

Effect of Substrate Elasticity on In Vitro Aging of Human Mesenchymal Stem Cells

2012 ◽  
Vol 1498 ◽  
pp. 39-45
Author(s):  
Courtney E. LeBlon ◽  
Caitlin R. Fodor ◽  
Tony Zhang ◽  
Xiaohui Zhang ◽  
Sabrina S. Jedlicka
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Elastic Modulus ◽  
Myogenic Differentiation ◽  
Human Mesenchymal Stem Cells ◽  
Immunofluorescent Staining ◽  
Differentiation Potential ◽  
Gene And Protein Expression ◽  
The Impact

ABSTRACTHuman mesenchymal stem cells (hMSCs) were routinely cultured on tissue-culture polystyrene (TCPS) to investigate the in vitro aging and cell stiffening. hMSCs were also cultured on thermoplastic polyurethane (TPU), which is a biocompatible polymer with an elastic modulus of approximately 12.9MPa, to investigate the impact of substrate elastic modulus on cell stiffening and differentiation potential. Cells were passaged over several generations on each material. At each passage, cells were subjected to osteogenic and myogenic differentiation. Local cell elastic modulus was measured at every passage using atomic force microscopy (AFM) indentation. Gene and protein expression was examined using qRT-PCR and immunofluorescent staining, respectively, for osteogenic and myogenic markers. Results show that the success of myogenic differentiation is highly reliant on the elastic modulus of the undifferentiated cells. The success of osteogenic differentiations is most likely somewhat dependent on the cell elastic modulus, as differentiations were more successful in earlier passages, when cells were softer.

scholarly journals Soft Substrate Maintains Proliferative and Adipogenic Differentiation Potential of human Mesenchymal Stem Cells on Long Term Expansion by Delaying Senescence

2018 ◽  
Author(s):  
Sanjay K. Kureel ◽  
Pankaj Mogha ◽  
Akshada Khadpekar ◽  
Vardhman Kumar ◽  
Rohit Joshi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture System ◽  
Human Mesenchymal Stem Cells ◽  
Population Doubling ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Poly Acrylamide

AbstractHuman mesenchymal stem cells (hMSCs), when cultured on tissue culture plate (TCP) for in vitro expansion, they spontaneously lose their proliferative capacity and multi-lineage differentiation potential. They also lose their distinct spindle morphology and become large and flat. After a certain number of population doubling, they enter into permanent cell cycle arrest, called senescence. This is a major roadblock for clinical use of hMSCs which demands large number of cells. A cell culture system is needed which can maintain the stemness of hMSCs over long term passages yet simple to use. In this study, we explore the role of substrate rigidity in maintaining stemness. hMSCs were serially passaged on TCP and 5 kPa poly-acrylamide gel for 20 population doubling. It was found that while on TCP, cell growth reached a plateau at cumulative population doubling (CPD) = 12.5, on 5 kPa gel, they continue to proliferate linearly till we monitored (CPD = 20). We also found that while on TCP, late passage MSCs lost their adipogenic potential, the same was maintained on soft gel. Cell surface markers related to MSCs were also unaltered. We demonstrated that this maintenance of stemness was correlated with delay in onset of senescence, which was confirmed by β-gal assay and by differential expression of vimentin, Lamin A and Lamin B. As preparation of poly-acrylamide gel is a simple, well established, and well standardized protocol, we believe that this system of cell expansion will be useful in therapeutic and research applications of hMSCs.One Sentence SummaryhMSCs retain their stemness when expanded in vitro on soft polyacrylamide gel coated with collagen by delaying senescence.Significance StatementFor clinical applications, mesenchymal stem cells (MSCs) are required in large numbers. As MSCs are available only in scarcity in vivo, to fulfill the need, extensive in vitro expansion is unavoidable. However, on expansion, they lose their replicative and multi-lineage differentiation potential and become senescent. A culture system that can maintain MSC stemness on long-term expansion, without compromising the stemness, is need of the hour. In this paper, we identified polyacrylamide (PAA) hydrogel of optimum stiffness that can be used to maintain stemness of MSCs during in vitro long term culture. Large quantity of MSCs thus grown can be used in regenerative medicine, cell therapy, and in treatment of inflammatory diseases.

scholarly journals Human Mesenchymal Stem Cells from Chorionic Villi and Amniotic Fluid are not Susceptible to Transformation after Extensive in Vitro Expansion

2011 ◽  
Vol 20 (5) ◽  
pp. 643-654 ◽  
Author(s):  
Antonella Poloni ◽  
Giulia Maurizi ◽  
Lucia Babini ◽  
Federica Serrani ◽  
Eleonora Berardinelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Amniotic Fluid ◽  
Human Mesenchymal Stem Cells ◽  
Chorionic Villi ◽  
In Vitro Expansion

scholarly journals Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Yueh-Hsun Kevin Yang ◽  
Courtney R. Ogando ◽  
Carmine Wang See ◽  
Tsui-Yun Chang ◽  
Gilda A. Barabino
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cells ◽  
Differentiation Potential

scholarly journals An Improved Harvest andin VitroExpansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Song Xu ◽  
Ann De Becker ◽  
Ben Van Camp ◽  
Karin Vanderkerken ◽  
Ivan Van Riet
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Surface Markers ◽  
Differentiation Potential ◽  
Murine Bone Marrow ◽  
Expansion Time ◽  
Expansion Protocol ◽  
In Vitro Expansion

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time.

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