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 Display Reduced Expression of CD105 after Culture in Serum-Free Medium

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Peter Mark ◽  
Mandy Kleinsorge ◽  
Ralf Gaebel ◽  
Cornelia A. Lux ◽  
Anita Toelk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Growth Media ◽  
Clinical Use ◽  
Differentiation Potential ◽  
Serum Free ◽  
The Impact

Human Mesenchymal Stem Cells (hMSCs) present a promising tool for regenerative medicine. However,ex vivoexpansion is necessary to obtain sufficient cells for clinical therapy. Conventional growth media usually contain the critical component fetal bovine serum. For clinical use, chemically defined media will be required. In this study, the capability of two commercial, chemically defined, serum-free hMSC growth media (MSCGM-CD and PowerStem) for hMSC proliferation was examined and compared to serum-containing medium (MSCGM). Immunophenotyping of hMSCs was performed using flow cytometry, and they were tested for their ability to differentiate into a variety of cell types. Although the morphology of hMSCs cultured in the different media differed, immunophenotyping displayed similar marker patterns (high expression of CD29, CD44, CD73, and CD90 cell surface markers and absence of CD45). Interestingly, the expression of CD105 was significantly lower for hMSCs cultured in MSCGM-CD compared to MSCGM. Both groups maintained mesenchymal multilineage differentiation potential. In conclusion, the serum-free growth medium is suitable for hMSC culture and comparable to its serum-containing counterpart. As the expression of CD105 has been shown to positively influence hMSC cardiac regenerative potential, the impact of CD105 expression onto clinical use after expansion in MSCGM-CD will have to be tested.

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

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 In Vitro Uptake of Hydroxyapatite Nanoparticles and Their Effect on Osteogenic Differentiation of Human Mesenchymal Stem Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Xing Yang ◽  
Yuanyuan Li ◽  
Xujie Liu ◽  
Ranran Zhang ◽  
Qingling Feng
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Human Mesenchymal Stem Cells ◽  
Exposure Dose ◽  
Immunofluorescent Staining ◽  
Activity Measurement ◽  
Hydroxyapatite Nanoparticles ◽  
Dose Dependent

There have been many applications in biomedical fields based on hydroxyapatite nanoparticles (HA NPs) over the past decades. However, the biocompatibility of HANPs is affected by exposure dose, particle size, and the way of contact with cells. The objective of this study is to investigate the effect of HA NPs with different sizes on osteogenesis using human mesenchymal stem cells (hMSCs). Three different-sized HA NPs (~50, ~100, and ~150 nm, resp.) were synthesized to study the cytotoxicity, cellular uptake, and effect on osteogenic differentiation of hMSCs. The results clearly showed that each size of HA NPs had dose-dependent cytotoxicity on hMSCs. It was found that HA NPs could be uptaken into hMSCs. The osteogenic differentiation of hMSCs was evaluated through alkaline phosphatase (ALP) activity measurement, ALP staining, immunofluorescent staining for osteopontin (OPN), and real-time polymerase chain reaction (RT-PCR) examination. As expected, HA NPs of all sizes could promote the differentiation of hMSCs towards osteoblast lineage. Among the three sizes, smaller-sized HA NPs (~50 and ~100 nm) appeared to be more effective in stimulating osteogenic differentiation of hMSCs.

scholarly journals Tsc1 Regulates the Proliferation Capacity of Bone-Marrow Derived Mesenchymal Stem Cells

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2072 ◽  
Author(s):  
Maria V. Guijarro ◽  
Laura S. Danielson ◽  
Marta Cañamero ◽  
Akbar Nawab ◽  
Carolina Abrahan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Myogenic Differentiation ◽  
Negative Regulator ◽  
Benign Tumors ◽  
Hematopoietic Stem ◽  
The Impact

TSC1 is a tumor suppressor that inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). TSC1 mutations are associated with Tuberous Sclerosis Complex (TSC), characterized by multiple benign tumors of mesenchymal and epithelial origin. TSC1 modulates self-renewal and differentiation in hematopoietic stem cells; however, its effects on mesenchymal stem cells (MSCs) are unknown. We investigated the impact of Tsc1 inactivation in murine bone marrow (BM)-MSCs, using tissue-specific, transgelin (Tagln)-mediated cre-recombination, targeting both BM-MSCs and smooth muscle cells. Tsc1 mutants were viable, but homozygous inactivation led to a dwarfed appearance with TSC-like pathologies in multiple organs and reduced survival. In young (28 day old) mice, Tsc1 deficiency-induced significant cell expansion of non-hematopoietic BM in vivo, and MSC colony-forming potential in vitro, that was normalized upon treatment with the mTOR inhibitor, everolimus. The hyperproliferative BM-MSC phenotype was lost in aged (1.5 yr) mice, and Tsc1 inactivation was also accompanied by elevated ROS and increased senescence. ShRNA-mediated knockdown of Tsc1 in BM-MSCs replicated the hyperproliferative BM-MSC phenotype and led to impaired adipogenic and myogenic differentiation. Our data show that Tsc1 is a negative regulator of BM-MSC proliferation and support a pivotal role for the Tsc1-mTOR axis in the maintenance of the mesenchymal progenitor pool.

scholarly journals The subpopulation of CD105 negative mesenchymal stem cells show strong immunomodulation capacity compared to CD105 positive mesenchymal stem cells

2019 ◽  
Vol 6 (4) ◽  
pp. 3131-3140 ◽  
Author(s):  
Liem Hieu Pham ◽  
Ngoc Bich Vu ◽  
Phuc Van Pham
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Umbilical Cord ◽  
Immune Modulation ◽  
Human Mesenchymal Stem Cells ◽  
Human Adipose Tissue ◽  
Human Umbilical Cord ◽  
Differentiation Potential

Introduction: Human mesenchymal stem cells (MSCs) are the most popular stem cells applied in disease treatment. MSCs can be isolated and in vitro expanded from various sources such as bone marrow, peripheral blood, umbilical cord blood, umbilical cord tissue, and adipose tissue. According to Dominici et al. (2006), MSCs should express CD105, an essential marker used to confirm MSCs. However, some recent studies have show that MSCs contained a subpopulation that is negative for CD105. This study aimed to compare the immune modulation capacity of 2 populations of CD105 positive (CD105+) and negative (CD105-) MSCs derived from 2 sources: human adipose tissue (AT) and human umbilical cord (UC). Methods: MSCs were isolated from human adipose tissues (adipose tissue-derived mesenchymal stem cells – AT-MSCs) and human umbilical cord (umbilical cord-derived mesenchymal stem cells – UC-MSCs) according to previously published protocols. The two populations of CD105- and CD105+ MSCs were sorted based on the expression of CD105 from AT-MSCs and UC-MSCs. Four populations of CD105 (AT-MSCs, CD105+ AT-MSCs, CD105- UC-MSCs, and CD105+ UC-MSCs) were used to compare the phenotype as well as in vitro differentiation potential; then they were used to evaluate the immune modulation capacity by allogeneic T cell suppression and cytokine release. Results: The results showed that CD105- MSCs from AT and UC exhibited an immune modulation capacity that was much stronger than CD105+ MSCs from the same source of AT and UC. The strong immunomodulation of CD105- MSCs may relate to autocrine production of TGF-beta 1 by MSCs. Conclusion: The results suggested that CD105- MSCs are promising MSCs for application in regenerative medicine, especially for the treatment of diseases related to inflammation.  

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