scholarly journals Effects of Donor Age, Long-Term Passage Culture, and Cryopreservation on Tonsil-Derived Mesenchymal Stem Cells

2015 ◽  
Vol 36 (1) ◽  
pp. 85-99 ◽  
Author(s):  
Jin-Sik Choi ◽  
Byung-Joo Lee ◽  
Hee-Young Park ◽  
Ji-Sun Song ◽  
Sung-Chan Shin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Surface Markers ◽  
Donor Age ◽  
Human Tonsil ◽  
Differentiation Potential ◽  
Morphological Differences ◽  
Proliferation Potential

Objectives: Human mesenchymal stem cells (MSCs) are efficacious in various cellular therapeutic applications and have been isolated from several tissues. Recent studies have reported that human tonsil tissue contains a new source of progenitor cells, potentially applicable for cell-based therapies. Information about the effects of donor age, long-term passage and cryopreservation are essential for clinical applications and cell-based therapies. Therefore, the authors investigated how the morphology, cell-surface markers, proliferation potential and differentiation capacity of tonsil-derived MSCs (T-MSCs) were affected by donor age, long-term passage, and cryopreservation. Materials and Methods: T-MSCs were isolated from tonsillar tissue of 20 patients undergoing tonsillectomy. Authors evaluated the effects of donor-age, long-term passage, and cryopreservation on the morphology, surface markers, proliferation potential and differentiation capacities of T-MSCs. Results: T-MSCs exhibited a fibroblast-like, spindle-shaped appearance. There were no significant morphological differences according to donor age, long-term passage or cryopreservation. T-MSCs isolated from donors of various ages were positive for markers CD90, CD44, and CD73, but negative for CD45, CD31, and HLA-DR. There were no significant differences in the expression of positive and negative surface markers as a function of donor age, long-term passage and cryopreservation. T-MSCs from different donor age groups showed similar proliferation potentials after passage 2. After long-term passage and cryopreservation, there were no significant morphological differences. Cryopreservation did not affect the proliferation potential of T-MSCs, but there was a significant decrease in the proliferation potential in long-term passage T-MSCs (passage 15). The effect of donor age, long-term passage and cryopreservation on the in vitro adipogenic, osteogenic, and chondrogenic differentiation potential of T-MSCs was not significant. Conclusion: The effect of donor age, long-term passage culture, and cryopreservation on T-MSC properties are negligible, except for the proliferation capacity of long-term cultured T-MSCs. Therefore, T-MSCs are considered to be promising MSCs that can be used as future alternative sources for autologous or allogenic MSCs.

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 Magnetic Nanoparticles and Magnetic Field Exposure Enhances Chondrogenesis of Human Adipose Derived Mesenchymal Stem Cells But Not of Wharton Jelly Mesenchymal Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Luminita Labusca ◽  
Dumitru-Daniel Herea ◽  
Anca Emanuela Minuti ◽  
Cristina Stavila ◽  
Camelia Danceanu ◽  
...  
Keyword(s):  
Magnetic Field ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Magnetic Nanoparticles ◽  
Cellular Senescence ◽  
Field Exposure ◽  
Differentiation Potential ◽  
Cartilage Engineering

Purpose: Iron oxide based magnetic nanoparticles (MNP) are versatile tools in biology and medicine. Adipose derived mesenchymal stem cells (ADSC) and Wharton Jelly mesenchymal stem cells (WJMSC) are currently tested in different strategies for regenerative regenerative medicine (RM) purposes. Their superiority compared to other mesenchymal stem cell consists in larger availability, and superior proliferative and differentiation potential. Magnetic field (MF) exposure of MNP-loaded ADSC has been proposed as a method to deliver mechanical stimulation for increasing conversion to musculoskeletal lineages. In this study, we investigated comparatively chondrogenic conversion of ADSC-MNP and WJMSC with or without MF exposure in order to identify the most appropriate cell source and differentiation protocol for future cartilage engineering strategies.Methods: Human primary ADSC and WJMSC from various donors were loaded with proprietary uncoated MNP. The in vitro effect on proliferation and cellular senescence (beta galactosidase assay) in long term culture was assessed. In vitro chondrogenic differentiation in pellet culture system, with or without MF exposure, was assessed using pellet histology (Safranin O staining) as well as quantitative evaluation of glycosaminoglycan (GAG) deposition per cell.Results: ADSC-MNP complexes displayed superior proliferative capability and decreased senescence after long term (28 days) culture in vitro compared to non-loaded ADSC and to WJMSC-MNP. Significant increase in chondrogenesis conversion in terms of GAG/cell ratio could be observed in ADSC-MNP. MF exposure increased glycosaminoglycan deposition in MNP-loaded ADSC, but not in WJMSC.Conclusion: ADSC-MNP display decreased cellular senescence and superior chondrogenic capability in vitro compared to non-loaded cells as well as to WJMSC-MNP. MF exposure further increases ADSC-MNP chondrogenesis in ADSC, but not in WJMSC. Loading ADSC with MNP can derive a successful procedure for obtaining improved chondrogenesis in ADSC. Further in vivo studies are needed to confirm the utility of ADSC-MNP complexes for cartilage engineering.

scholarly journals Hepatocyte growth factor (HGF) and stem cell factor (SCF) maintained the stemness of human bone marrow mesenchymal stem cells (hBMSCs) during long-term expansion by preserving mitochondrial function via the PI3K/AKT, ERK1/2, and STAT3 signaling pathways

2020 ◽  
Author(s):  
Zeyuan Cao ◽  
Yunyi Xie ◽  
Le Yu ◽  
Yi Li ◽  
Yan Wang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Signaling Pathways ◽  
Mitochondrial Function ◽  
Key Factors ◽  
Differentiation Potential ◽  
Stat3 Signaling ◽  
Marrow Mesenchymal Stem Cells

Abstract Background: Mesenchymal stem cells (MSCs) have a limited self-renewal ability, impaired multi-differentiation potential, and undetermined cell senescence during in vitro series expansion. To address this concern, we investigated the effects of the microenvironment provided by stem cells from human exfoliated deciduous teeth (SHED) in maintaining the stemness of human bone marrow mesenchymal stem cells (hBMSCs) and identified the key factors and possible mechanisms responsible for maintaining the stemness of MSCs during long-term expansion in vitro.Methods: The passage 3 (P3) to passage 8 (P8) hBMSCs were cultured in the conditioned medium from SHED (SHED-CM). The percentage of senescent cells was evaluated by β-galactosidase staining. In addition, the osteogenic differentiation potential was analyzed by reverse transcription quantitative PCR (RT-qPCR), Western blot, alizarin red and alkaline phosphatase (ALP) staining. Furthermore, RT-qPCR results identified hepatocyte growth factor (HGF) and stem cell factor (SCF) as key factors. Thus, the effects of HGF and SCF on mitochondrial function were assessed by measuring the ROS and mitochondrial membrane potential levels. Finally, selected mitochondrial-related proteins associated with the PI3K/AKT, ERK1/2, and STAT3 signaling pathways were investigated to determine the effects of HGF and SCF in preserving the mitochondrial function of hBMSCs during long-term expansion.Results: SHED-CM had significantly enhanced the cell viability, reduced the senescent cells, and maintained the osteogenesis and pro-angiogenic capacity in P8 hBMSCs during long-term expansion. In addition, hBMSCs treated with 100 ng/ml HGF and 10 ng/ml SCF had reduced ROS levels, and preserved mitochondrial membrane potential compared with P8 hBMSCs during long-term expansion. Furthermore, HGF and SCF upregulated the expression of mitochondrial-related proteins associated with the PI3K/AKT, ERK1/2, and STAT3 signaling pathways, possibly contributing to the maintenance of hBMSCs stemness by preserving mitochondrial function.Conclusion: Both HGF and SCF are key factors in maintaining the stemness of hBMSCs by preserving mitochondrial function through the expression of proteins associated with the PI3K/AKT, ERK1/2, and STAT3 signaling pathways. This study provides new insights into the anti-senescence capability of HGF and SCF, as well as new evidence for their potential application in optimizing the long-term culture of MSCs.

scholarly journals The Effect of Culture on Human Bone Marrow Mesenchymal Stem Cells: Focus on DNA Methylation Profiles

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Angela Bentivegna ◽  
Gaia Roversi ◽  
Gabriele Riva ◽  
Laura Paoletta ◽  
Serena Redaelli ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Differentiation Potential ◽  
Marrow Mesenchymal Stem Cells

Human bone marrow mesenchymal stem cells (hBM-MSCs) are the best characterized multipotent adult stem cells. Their self-renewal capacity, multilineage differentiation potential, and immunomodulatory properties have indicated that they can be used in many clinical therapies. In a previous work we studied the DNA methylation levels of hBM-MSC genomic DNA in order to delineate a kind of methylation signature specific for early and late passages of culture. In the present work we focused on the modification of the methylation profiles of the X chromosome and imprinted loci, as sites expected to be more stable than whole genome. We propose a model where cultured hBM-MSCs undergo random modifications at the methylation level of most CGIs, nevertheless reflecting the original methylation status. We also pointed out global genome-wide demethylation connected to the long-term culture and senescence. Modification at CGIs promoters of specific genes could be related to the decrease in adipogenic differentiation potential. In conclusion, we showed important changes in CGIs methylation due to long-termin vitroculture that may affect the differentiation potential of hBM-MSCs. Therefore it is necessary to optimize the experimental conditions forin vitroexpansion in order to minimize these epigenetic changes and to standardize safer procedures.

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.

scholarly journals Hepatocyte growth factor (HGF) and stem cell factor (SCF) maintained the stemness of human bone marrow mesenchymal stem cells (hBMSCs) during long-term expansion by preserving mitochondrial function via the PI3K/AKT, ERK1/2, and STAT3 signaling pathways

2020 ◽  
Author(s):  
Zeyuan Cao ◽  
Yunyi Xie ◽  
Le Yu ◽  
Yi Li ◽  
Yan Wang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Signaling Pathways ◽  
Mitochondrial Function ◽  
Key Factors ◽  
Differentiation Potential ◽  
Stat3 Signaling ◽  
Marrow Mesenchymal Stem Cells

Abstract Background: Mesenchymal stem cells (MSCs) have a limited self-renewal ability, impaired multi-differentiation potential, and undetermined cell senescence during in vitro series expansion. To address this concern, we investigated the effects of the microenvironment provided by stem cells from human exfoliated deciduous teeth (SHED) in maintaining the stemness of human bone marrow mesenchymal stem cells (hBMSCs) and identified the key factors and possible mechanisms responsible for maintaining the stemness of MSCs during long-term expansion in vitro.Methods: The passage 3 (P3) to passage 8 (P8) hBMSCs were cultured in the conditioned medium from SHED (SHED-CM). The percentage of senescent cells was evaluated by β-galactosidase staining. In addition, the osteogenic differentiation potential was analyzed by reverse transcription quantitative PCR (RT-qPCR), Western blot, alizarin red and alkaline phosphatase (ALP) staining. Furthermore, RT-qPCR results identified hepatocyte growth factor (HGF) and stem cell factor (SCF) as key factors. Thus, the effects of HGF and SCF on mitochondrial function were assessed by measuring the ROS and mitochondrial membrane potential levels. Finally, selected mitochondrial-related proteins associated with the PI3K/AKT, ERK1/2, and STAT3 signaling pathways were investigated to determine the effects of HGF and SCF in preserving the mitochondrial function of hBMSCs during long-term expansion. Results: SHED-CM had significantly enhanced the cell proliferation, reduced the senescent cells, and maintained the osteogenesis and pro-angiogenic capacity in P8 hBMSCs during long-term expansion. In addition, hBMSCs treated with 100 ng/ml HGF and 10 ng/ml SCF had reduced ROS levels, and preserved mitochondrial membrane potential compared with P8 hBMSCs during long-term expansion. Furthermore, HGF and SCF upregulated the expression of mitochondrial-related proteins associated with the PI3K/AKT, ERK1/2, and STAT3 signaling pathways, possibly contributing to the maintenance of hBMSCs stemness by preserving mitochondrial function. Conclusion: Both HGF and SCF are key factors in maintaining the stemness of hBMSCs by preserving mitochondrial function through the expression of proteins associated with the PI3K/AKT, ERK1/2, and STAT3 signaling pathways. This study provides new insights into the anti-senescence capability of HGF and SCF, as well as new evidence for their potential application in optimizing the long-term culture of MSCs.

scholarly journals Manufacturing and Banking Canine Adipose-Derived Mesenchymal Stem Cells for Veterinary Clinical Application

2020 ◽  
Author(s):  
Huina Luo ◽  
Dongsheng Li ◽  
Zhisheng Chen ◽  
Bingyun Wang ◽  
Shengfeng Chen
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Clinical Applications ◽  
Surface Markers ◽  
Clinical Use ◽  
Therapeutic Application ◽  
Differentiation Potential ◽  
Safety And Efficacy

Abstract BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. RESULTS: After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. No adverse events were noted in two cases treated with intravenously AD-MSCs. CONCLUSION: Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

scholarly journals Isolation and Localization of Mesenchymal Stem Cells in Human Palatine Tonsil by W5C5 (SUSD2)

2016 ◽  
Vol 38 (1) ◽  
pp. 83-93 ◽  
Author(s):  
Byung-Joo Lee ◽  
Dae-Woon Kang ◽  
Hee-Young Park ◽  
Ji-Sun Song ◽  
Ji-Min Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stromal Cells ◽  
Human Tonsil ◽  
Differentiation Potential ◽  
Surface Marker Expression ◽  
Single Marker ◽  
Forming Efficiency ◽  
Perivascular Area ◽  
Proliferation Potential

Background/Aims: Although tonsil-mesenchymal stem cells (T-MSCs) have been studied as a new autologous or homologous source of MSCs, research on specific markers of MSCs and localization for purified T-MSC isolation has not yet been reported. This study investigates the expression of W5C5 (SUSD2) in tonsil stromal cells and the colony-forming ability and differentiation potential of W5C5+ cells to determine the usefulness of W5C5+ MSCs as a marker that can be used for the purification of T-MSCs. In addition, the location of W5C5+ cells expressed in the tonsil tissues is examined. Methods: T-MSCs were isolated from the tonsillar tissues of 12 patients undergoing tonsillectomy. The colony-forming ability, surface markers, proliferation potential, and differentiation capacities of purified W5C5+ MSCs, W5C5- MSCs, and unselected T-MSCs were evaluated. The location of the W5C5+ cells in the tonsillar tissues was also investigated by immunohistochemistry. Results: W5C5 was expressed in 2.5±0.4% of fresh human tonsil stromal cells. W5C5+ cells formed many colonies, but W5C5- cells did not form any colonies. The colony-forming number of W5C5+ cells (74.4 ± 9.8) was significantly higher than that of unselected tonsil stromal cells (23.6 ± 3.7). However, the differences in proliferation potential, surface marker expression, and differentiation potential between W5C5+ T-MSCs and unselected T-MSCs were not significant. W5C5+ cells were identified in the perivascular area around the blood vessels. Conclusion: W5C5+ T-MSCs possessed typical MSC properties with high colony-forming efficiency, and niches of W5C5+ T-MSCs were located in the perivascular area of tonsil tissues. These findings suggest that W5C5 is a useful single marker for the isolation of purified T-MSCs.

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