scholarly journals Immortalizing Mesenchymal Stromal Cells from Aged Donors While Keeping Their Essential Features

2020 ◽  
Vol 2020 ◽  
pp. 1-24 ◽  
Author(s):  
María Piñeiro-Ramil ◽  
Rocío Castro-Viñuelas ◽  
Clara Sanjurjo-Rodríguez ◽  
Silvia Rodríguez-Fernández ◽  
Tamara Hermida-Gómez ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Cell Fate ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Transgene Expression ◽  
Transduction Efficiency ◽  
Aged Patients ◽  
Differentiation Potential ◽  
Medicine Research ◽  
Age Related

Human bone marrow-derived mesenchymal stromal cells (MSCs) obtained from aged patients are prone to senesce and diminish their differentiation potential, therefore limiting their usefulness for osteochondral regenerative medicine approaches or to study age-related diseases, such as osteoarthiritis (OA). MSCs can be transduced with immortalizing genes to overcome this limitation, but transduction of primary slow-dividing cells has proven to be challenging. Methods for enhancing transduction efficiency (such as spinoculation, chemical adjuvants, or transgene expression inductors) can be used, but several parameters must be adapted for each transduction system. In order to develop a transduction method suitable for the immortalization of MSCs from aged donors, we used a spinoculation method. Incubation parameters of packaging cells, speed and time of centrifugation, and valproic acid concentration to induce transgene expression have been adjusted. In this way, four immortalized MSC lines (iMSC#6, iMSC#8, iMSC#9, and iMSC#10) were generated. These immortalized MSCs (iMSCs) were capable of bypassing senescence and proliferating at a higher rate than primary MSCs. Characterization of iMSCs showed that these cells kept the expression of mesenchymal surface markers and were able to differentiate towards osteoblasts, adipocytes, and chondrocytes. Nevertheless, alterations in the CD105 expression and a switch of cell fate-commitment towards the osteogenic lineage have been noticed. In conclusion, the developed transduction method is suitable for the immortalization of MSCs derived from aged donors. The generated iMSC lines maintain essential mesenchymal features and are expected to be useful tools for the bone and cartilage regenerative medicine research.

scholarly journals High-content analysis reveals senescence of cultured canine adipose-derived mesenchymal stromal cells to be reversible with a novel immortalization approach

2020 ◽  
Author(s):  
Ana Stojiljkovic ◽  
Veronique Gaschen ◽  
Franck Forterre ◽  
Ulrich Rytz ◽  
Michael H Stoffel ◽  
...  
Keyword(s):  
Content Analysis ◽  
Regenerative Medicine ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Replicative Senescence ◽  
Therapeutic Potential ◽  
Morphological Changes ◽  
Human Telomerase Reverse Transcriptase ◽  
Differentiation Potential ◽  
High Content Analysis

In the last decades, the scientific community spared no effort to elucidate the therapeutic potential of mesenchymal stromal cells (MSCs). Unfortunately, in vitro cellular senescence occurring along with a loss of proliferative capacity is a major drawback in view of future therapeutic applications of these cells in the field of regenerative medicine. Even though insight into the mechanisms of replicative senescence in human medicine has evolved dramatically, knowledge about replicative senescence of canine MSCs is still scarce. Thus, we developed a high-content analysis workflow to simultaneously investigate three important characteristics of senescence in canine adipose-derived MSCs (cAD-MSCs): morphological changes, activation of the cell cycle arrest machinery and increased activity of the senescence-associated beta-galactosidase. We took advantage of this tool to demonstrate that passaging of cAD-MSCs results in the appearance of a senescence phenotype and proliferation arrest. This was partially prevented upon immortalization of these cells using a newly designed PiggyBac(TM) Transposon System, which allows for the expression of the human polycomb ring finger proto-oncogene BMI1 and the human telomerase reverse transcriptase under the same promotor. Our results indicate that cAD-MSCs immortalized with this new vector maintain their proliferation capacity and differentiation potential for a longer time than untreated cAD-MSCs. This study not only offers a workflow to investigate replicative senescence in eukaryotic cells with a high-content analysis approach but also paves the way for a rapid and effective generation of immortalized MSC lines. This promotes a better understanding of these cells in view of future applications in regenerative medicine.

scholarly journals mTORC1 and mTORC2 Differentially Regulate Cell Fate Programs to Coordinate Osteoblastic Differentiation in Mesenchymal Stromal Cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Theres Schaub ◽  
Dennis Gürgen ◽  
Deborah Maus ◽  
Claudia Lange ◽  
Victor Tarabykin ◽  
...  
Keyword(s):  
Cell Fate ◽  
Mesenchymal Stromal Cells ◽  
Cellular Senescence ◽  
Stromal Cells ◽  
Metabolic Diseases ◽  
Osteoblastic Differentiation ◽  
Cell Fates ◽  
Network Activation ◽  
Vascular Regeneration ◽  
Age Related

AbstractVascular regeneration depends on intact function of progenitors of vascular smooth muscle cells such as pericytes and their circulating counterparts, mesenchymal stromal cells (MSC). Deregulated MSC differentiation and maladaptive cell fate programs associated with age and metabolic diseases may exacerbate arteriosclerosis due to excessive transformation to osteoblast-like calcifying cells. Targeting mTOR, a central controller of differentiation and cell fates, could offer novel therapeutic perspectives. In a cell culture model for osteoblastic differentiation of pluripotent human MSC we found distinct roles for mTORC1 and mTORC2 in the regulation of differentiation towards calcifying osteoblasts via cell fate programs in a temporally-controlled sequence. Activation of mTORC1 with induction of cellular senescence and apoptosis were hallmarks of transition to a calcifying phenotype. Inhibition of mTORC1 with Rapamycin elicited reciprocal activation of mTORC2, enhanced autophagy and recruited anti-apoptotic signals, conferring protection from calcification. Pharmacologic and genetic negative interference with mTORC2 function or autophagy both abolished regenerative programs but induced cellular senescence, apoptosis, and calcification. Overexpression of the mTORC2 constituent rictor revealed that enhanced mTORC2 signaling without altered mTORC1 function was sufficient to inhibit calcification. Studies in mice reproduced the in vitro effects of mTOR modulation with Rapamycin on cell fates in vascular cells in vivo. Amplification of mTORC2 signaling promotes protective cell fates including autophagy to counteract osteoblast differentiation and calcification of MSC, representing a novel mTORC2 function. Regenerative approaches aimed at modulating mTOR network activation patterns hold promise for delaying age-related vascular diseases and treatment of accelerated arteriosclerosis in chronic metabolic conditions.

scholarly journals Kinship of conditionally immortalized cells derived from fetal bone to human bone-derived mesenchymal stroma cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. Marozin ◽  
B. Simon-Nobbe ◽  
S. Irausek ◽  
L. W. K. Chung ◽  
G. Lepperdinger
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Human Bone ◽  
Multilineage Differentiation ◽  
Differentiation Potential ◽  
Osteoblast Cell Line ◽  
Age Related

AbstractThe human fetal osteoblast cell line (hFOB 1.19) has been proposed as an accessible experimental model for study of osteoblast biology relating to drug development and biomaterial engineering. For their multilineage differentiation potential, hFOB has been compared to human mesenchymal progenitor cells and used to investigate bone-metabolism in vitro. Hereby, we studied whether and to what extent the conditionally immortalized cell line hFOB 1.19 can serve as a surrogate model for bone-marrow derived mesenchymal stromal cells (bmMSC). hFOB indeed exhibit specific characteristics reminiscent of bmMSC, as colony formation, migration capacity and the propensity to grow as multicellular aggregates. After prolonged culture, in contrast to the expected effect of immortalization, hFOB acquired a delayed growth rate. In close resemblance to bmMSC at increasing passages, also hFOB showed morphological abnormalities, enlargement and finally reduced proliferation rates together with enhanced expression of the cell cycle inhibitors p21 and p16. hFOB not only have the ability to undergo multilineage differentiation but portray several important aspects of human bone marrow mesenchymal stromal cells. Superior to primary MSC and osteoblasts, hFOB enabled the generation of continuous cell lines. These provide an advanced basis for investigating age-related dysfunctions of MSCs in an in vitro 3D-stem cell microenvironment.

scholarly journals K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

2021 ◽  
Vol 22 (2) ◽  
pp. 598
Author(s):  
Madalina Dumitrescu ◽  
Ana Maria Vacaru ◽  
Violeta Georgeta Trusca ◽  
Ioana Madalina Fenyo ◽  
Radu Ionita ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Mammalian Cells ◽  
Receptor Expression ◽  
Transduction Efficiency ◽  
Mouse Strains ◽  
Differentiation Potential ◽  
Adenoviral Transduction ◽  
Foreign Dna ◽  
Different Levels

Adenoviral vectors are important vehicles for delivering therapeutic genes into mammalian cells. However, the yield of the adenoviral transduction of murine mesenchymal stromal cells (MSC) is low. Here, we aimed to improve the adenoviral transduction efficiency of bone marrow-derived MSC. Our data showed that among all the potential transduction boosters that we tested, the K2 Transfection System (K2TS) greatly increased the transduction efficiency. After optimization of both K2TS components, the yield of the adenoviral transduction increased from 18% to 96% for non-obese diabetic (NOD)-derived MSC, from 30% to 86% for C57BL/6-derived MSC, and from 0.6% to 63% for BALB/c-derived MSC, when 250 transduction units/cell were used. We found that MSC derived from these mouse strains expressed different levels of the coxsackievirus and adenovirus receptors (MSC from C57BL/6≥NOD>>>BALB/c). K2TS did not increase the level of the receptor expression, but desensitized the cells to foreign DNA and facilitated the virus entry into the cell. The expression of Stem cells antigen-1 (Sca-1) and 5′-nucleotidase (CD73) MSC markers, the adipogenic and osteogenic differentiation potential, and the immunosuppressive capacity were preserved after the adenoviral transduction of MSC in the presence of the K2TS. In conclusion, K2TS significantly enhanced the adenoviral transduction of MSC, without interfering with their main characteristics and properties.

scholarly journals The Identification of Marker Genes for Predicting the Osteogenic Differentiation Potential of Mesenchymal Stromal Cells

2021 ◽  
Vol 43 (3) ◽  
pp. 2157-2166
Author(s):  
Masami Kanawa ◽  
Akira Igarashi ◽  
Katsumi Fujimoto ◽  
Tania Saskianti ◽  
Ayumu Nakashima ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Marker Genes ◽  
Mrna Levels ◽  
Differentiation Potential ◽  
Alp Activity ◽  
Osteogenic Induction ◽  
Promising Source

Mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of mature cell types and are a promising source of regenerative medicine. The success of regenerative medicine using MSCs strongly depends on their differentiation potential. In this study, we sought to identify marker genes for predicting the osteogenic differentiation potential by comparing ilium MSC and fibroblast samples. We measured the mRNA levels of 95 candidate genes in nine ilium MSC and four fibroblast samples before osteogenic induction, and compared them with alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation after induction. We identified 17 genes whose mRNA expression levels positively correlated with ALP activity. The chondrogenic and adipogenic differentiation potentials of jaw MSCs are much lower than those of ilium MSCs, although the osteogenic differentiation potential of jaw MSCs is comparable with that of ilium MSCs. To select markers suitable for predicting the osteogenic differentiation potential, we compared the mRNA levels of the 17 genes in ilium MSCs with those in jaw MSCs. The levels of 7 out of the 17 genes were not substantially different between the jaw and ilium MSCs, while the remaining 10 genes were expressed at significantly lower levels in jaw MSCs than in ilium MSCs. The mRNA levels of the seven similarly expressed genes were also compared with those in fibroblasts, which have little or no osteogenic differentiation potential. Among the seven genes, the mRNA levels of IGF1 and SRGN in all MSCs examined were higher than those in any of the fibroblasts. These results suggest that measuring the mRNA levels of IGF1 and SRGN before osteogenic induction will provide useful information for selecting competent MSCs for regenerative medicine, although the effectiveness of the markers is needed to be confirmed using a large number of MSCs, which have various levels of osteogenic differentiation potential.

scholarly journals Enhancer-priming in ageing human bone marrow mesenchymal stromal cells contributes to immune traits

2021 ◽  
Author(s):  
Mang Ching Lai ◽  
Mariana Ruiz-Velasco ◽  
Christian Arnold ◽  
Olga Sigalova ◽  
Daria Bunina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Genome Wide Association Study ◽  
Study Data ◽  
Proteomics Data ◽  
Differentiation Potential ◽  
Complex Information ◽  
Age Related ◽  
Gene Regulatory

AbstractBone marrow mesenchymal stromal cells (BMSCs) can differentiate into adipocytes and osteoblasts, and are important regulators of the haematopoietic system. Ageing associates with an increased ratio of bone marrow adipocytes to osteoblasts and immune dysregulation. Here, we carried out an integrative multiomics analysis of ATAC-Seq, RNA-Seq and proteomics data from primary human BMSCs in a healthy cohort age between 20 - 60. We identified age-sensitive elements uniquely affecting each molecular level where transcription is mostly spared, and characterised the underlying biological pathways, revealing the interplay of age-related gene expression mechanism changes spanning multiple gene regulatory layers. Through data integration with enhancer-mediated gene regulatory network analysis, we discovered that enhancers and transcription factors influence cell differentiation potential in the ageing BMSCs. By combining our results with genome-wide association study data, we found that age-specific changes could contribute to common traits related to BMSC-derived tissues such as bone and adipose tissue, and to immune-related traits on a systemic level such as asthma. We demonstrate here that a multiomics approach is crucial for unravelling complex information, providing new insights on how ageing contributes to bone marrow- and immune-related disorders.

scholarly journals Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Weichao Zhai ◽  
Jerome Tan ◽  
Tobias Russell ◽  
Sixun Chen ◽  
Dennis McGonagle ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Preclinical Model ◽  
Label Free ◽  
Current Standard ◽  
Differentiation Potential ◽  
Human Mesenchymal Stromal Cells ◽  
Endogenous Fluorophores

AbstractHuman mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical $$\upbeta $$ β -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-$$\upbeta $$ β -galactosidase activity using the fluorogenic substrate, C$${_{12}}$$ 12 FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in $$\upbeta $$ β -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p $$\le $$ ≤ 0.001 for the fluorogenic C$${_{12}}$$ 12 FDG method, and r = 0.72, p $$\le $$ ≤ 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

scholarly journals Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marie-Theresa Weickert ◽  
Judith S. Hecker ◽  
Michèle C. Buck ◽  
Christina Schreck ◽  
Jennifer Rivière ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Fate ◽  
Stromal Cells ◽  
Adipogenic Differentiation ◽  
Cell Types ◽  
Bone Marrow Niche ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Bm Niche

AbstractMyelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.

scholarly journals Expansion and characterization of bone marrow derived human mesenchymal stromal cells in serum-free conditions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samatha Bhat ◽  
Pachaiyappan Viswanathan ◽  
Shashank Chandanala ◽  
S. Jyothi Prasanna ◽  
Raviraja N. Seetharam
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Population Doubling ◽  
Seeding Density ◽  
Population Doubling Time ◽  
Differentiation Potential ◽  
Serum Free ◽  
Safety Issues ◽  
Free Media

AbstractBone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

scholarly journals Metabolic programming of mesenchymal stromal cells by oxygen tension directs chondrogenic cell fate

2014 ◽  
Vol 111 (38) ◽  
pp. 13954-13959 ◽  
Author(s):  
J. Leijten ◽  
N. Georgi ◽  
L. Moreira Teixeira ◽  
C. A. van Blitterswijk ◽  
J. N. Post ◽  
...  
Keyword(s):  
Cell Fate ◽  
Oxygen Tension ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Metabolic Programming
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