scholarly journals Senescence-associated metabolomic phenotype in primary and iPSC-derived mesenchymal stromal cells

2019 ◽  
Author(s):  
Eduardo Fernandez-Rebollo ◽  
Julia Franzen ◽  
Jonathan Hollmann ◽  
Alina Ostrowska ◽  
Matteo Oliverio ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Replicative Senescence ◽  
Cell Types ◽  
Cellular Aging ◽  
Metabolic Switch ◽  
Differentiation Potential ◽  
Long Term Culture ◽  
Functional Changes

Long-term culture of primary cells is reflected by functional and secretory changes, which ultimately result in replicative senescence. In contrast, induced pluripotent stem cells (iPSCs) do not reveal any signs of cellular aging while in the pluripotency state, whereas little is known how they senesce upon differentiation. Furthermore, it is largely unclear how the metabolome of cells changes during replicative senescence and if such changes are consistent across different cell types. In this study, we have directly compared culture expansion of primary mesenchymal stromal cells (MSCs) and iPSC-derived MSCs (iMSCs) until they reached growth arrest after a mean of 21 and 17 cumulative population doublings, respectively. Both cell types acquired similar changes in morphology, in vitro differentiation potential, up-regulation of senescence-associated beta-galactosidase, and senescence-associated DNA methylation changes. Furthermore, MSCs and iMSCs revealed overlapping gene expression changes during culture expansion, particularly in functional categories related to metabolic processes. We subsequently compared the metabolome of MSCs and iMSCs at early and senescent passages and observed various significant and overlapping senescence-associated changes in both cell types, including down-regulation of nicotinamide ribonucleotide and up-regulation of orotic acid. Replicative senescence of both cell types was consistently reflected by the metabolic switch from oxidative to glycolytic pathways. Taken together, long-term culture of iPSC-derived MSCs evokes very similar molecular and functional changes as observed in primary MSCs. Replicative senescence is associated with a highly reproducible senescence-associated metabolomics phenotype, which may be used to monitor the state of cellular aging.

scholarly journals Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term Culture Associated Alterations of Cellular Properties

2021 ◽  
Author(s):  
Chenghai Li
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mhc Class Ii ◽  
Stromal Cells ◽  
Replicative Senescence ◽  
Cell Types ◽  
Long Term Culture ◽  
Medicine Research

Mesenchymal stem cell/stromal cells (MSCs) can differentiate into a variety of cell types, including osteocytes, adipocytes and chondrocytes. MSCs are present in the multiple types of adult tissue, such as bone marrow, adipose tissue, and various neonatal birth-associated tissues. Given their self-renewal and differentiation potential, immunomodulatory and paracrine properties, and lacking major histocompatibility complex (MHC) class II molecules, MSCs have attracted much attention for stem cell-based translational medicine research. Due to a very low frequency in different types of tissue, MSCs can be isolated and expanded in vitro to derive sufficient cell numbers prior to the clinical applications. In this chapter, the methodology to obtain primary bone marrow-derived MSCs as well as their in vitro culture expansion will be described. To assess the functional properties, differentiation assays, including osteogenesis, chondrogenesis and adipogenesis, 3-D culture of MSCs and co-culture of MSCs and tumor cells are also provided. Finally, the long-term culture associated alterations of MSCs, such as replicative senescence and spontaneous transformation, will be discussed for better understanding of the use of MSCs at the early stages for safe and effective cell-based therapy.

scholarly journals Human adipose-derived mesenchymal stromal cells from face and abdomen undergo replicative senescence and loss of genetic integrity after long-term culture

2021 ◽  
Author(s):  
Priscilla B Delben ◽  
Helena D Zomer ◽  
Camila A Silva ◽  
Rogerio S Gomes ◽  
Fernanda R Melo ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Replicative Senescence ◽  
Genetic Integrity ◽  
Cell Therapies ◽  
Long Term Culture ◽  
Morphological Alterations ◽  
Fat Depots

Body fat depots are heterogeneous concerning their embryonic origin, structure, exposure to environmental stressors, and availability. Thus, investigating adipose-derived mesenchymal stromal cells (ASCs) from different sources is essential to standardization for future therapies. In vitro amplification is also critical because it may predispose cell senescence and mutations, reducing regenerative properties and safety. Here, we evaluated long-term culture of human facial ASCs (fASCs) and abdominal ASCs (aASCs) and showed that both met the criteria for MSCs characterization, but presented differences in their immunophenotypic profile, and differentiation and clonogenic potentials. The abdominal tissue yielded more ASCs, but facial cells displayed fewer mitotic errors at higher passages. However, both cell types reduced clonal efficiency over time and entered replicative senescence around P12, as evaluated by progressive morphological alterations, reduced proliferative capacity, and SA-β-galactosidase expression. Loss of genetic integrity was detected by a higher proportion of cells showing nuclear alterations and γH2AX expression. Our findings indicate that the source of ASCs can substantially influence their phenotype and therefore should be carefully considered in future cell therapies, avoiding, however, long-term culture to ensure genetic stability.

scholarly journals Human adipose-derived mesenchymal stromal cells from face and abdomen undergo replicative senescence and loss of genetic integrity after long-term culture

2021 ◽  
pp. 112740
Author(s):  
Priscilla Barros Delben ◽  
Helena Debiazi Zomer ◽  
Camila Acordi da Silva ◽  
Rogério Schutzler Gomes ◽  
Fernanda Rosene Melo ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Replicative Senescence ◽  
Genetic Integrity ◽  
Long Term Culture

scholarly journals Amniotic Mesenchymal Stromal Cells Exhibit Preferential Osteogenic and Chondrogenic Differentiation and Enhanced Matrix Production Compared With Adipose Mesenchymal Stromal Cells

2017 ◽  
Vol 45 (11) ◽  
pp. 2637-2646 ◽  
Author(s):  
Natasha Topoluk ◽  
Richard Hawkins ◽  
John Tokish ◽  
Jeremy Mercuri
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Cell Types ◽  
Cell Counting ◽  
Gene Transcript ◽  
Differentiation Potential ◽  
Musculoskeletal Tissue ◽  
Cell Groups ◽  
And Cartilage

Background: Therapeutic efficacy of various mesenchymal stromal cell (MSC) types for orthopaedic applications is currently being investigated. While the concept of MSC therapy is well grounded in the basic science of healing and regeneration, little is known about individual MSC populations in terms of their propensity to promote the repair and/or regeneration of specific musculoskeletal tissues. Two promising MSC sources, adipose and amnion, have each demonstrated differentiation and extracellular matrix (ECM) production in the setting of musculoskeletal tissue regeneration. However, no study to date has directly compared the differentiation potential of these 2 MSC populations. Purpose: To compare the ability of human adipose- and amnion-derived MSCs to undergo osteogenic and chondrogenic differentiation. Study Design: Controlled laboratory study. Methods: MSC populations from the human term amnion were quantified and characterized via cell counting, histologic assessment, and flow cytometry. Differentiation of these cells in comparison to commercially purchased human adipose-derived mesenchymal stromal cells (hADSCs) in the presence and absence of differentiation media was evaluated via reverse transcription polymerase chain reaction (PCR) for bone and cartilage gene transcript markers and histology/immunohistochemistry to examine ECM production. Analysis of variance and paired t tests were performed to compare results across all cell groups investigated. Results: The authors confirmed that the human term amnion contains 2 primary cell types demonstrating MSC characteristics—(1) human amniotic epithelial cells (hAECs) and (2) human amniotic mesenchymal stromal cells (hAMSCs)—and each exhibited more than 90% staining for MSC surface markers (CD90, CD105, CD73). Average viable hAEC and hAMSC yields at harvest were 2.3 × 106 ± 3.7 × 105 and 1.6 × 106 ± 4.7 × 105 per milliliter of amnion, respectively. As well, hAECs and hAMSCs demonstrated significantly greater osteocalcin ( P = .025), aggrecan ( P < .0001), and collagen type 2 ( P = .044) gene expression compared with hADSCs, respectively, after culture in differentiation medium. Moreover, both hAECs and hAMSCs produced significantly greater quantities of mineralized ( P < .0001) and cartilaginous ( P = .0004) matrix at earlier time points compared with hADSCs when cultured under identical osteogenic and chondrogenic differentiation conditions, respectively. Conclusion: Amnion-derived MSCs demonstrate a greater differentiation potential toward bone and cartilage compared with hADSCs. Clinical Relevance: Amniotic MSCs may be the source of choice in the regenerative treatment of bone or osteochondral musculoskeletal disease. They show significantly higher yields and better differentiation toward these tissues than MSCs derived from adipose.

scholarly journals Interrupted reprogramming into induced pluripotent stem cells does not rejuvenate human mesenchymal stromal cells

2018 ◽  
Author(s):  
Carolin Göbel ◽  
Roman Goetzke ◽  
Thomas Eggermann ◽  
Wolfgang Wagner
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Replicative Senescence ◽  
Chromosome 1 ◽  
Differentiation Potential ◽  
Pluripotency Factors ◽  
Induced Pluripotent

AbstractReplicative senescence hampers application of mesenchymal stromal cells (MSCs) because it limits culture expansion, impairs differentiation potential, and hinders reliable standardization of cell products. MSCs can be rejuvenated by reprogramming into induced pluripotent stem cells (iPSCs), which is associated with complete erasure of age- and senescence-associated DNA methylation (DNAm) patterns. However, this process is also associated with erasure of cell-type and tissue-specific epigenetic characteristics that are not recapitulated upon re-differentiation towards MSCs. In this study, we therefore followed the hypothesis that overexpression of pluripotency factors under culture conditions that do not allow full reprogramming might reset senescence-associated changes without entering a pluripotent state. MSCs were transfected with episomal plasmids and either successfully reprogrammed into iPSCs or cultured in different media with continuous passaging every week. Overexpression of pluripotency factors without reprogramming did neither prolong culture expansion nor ameliorate molecular and epigenetic hallmarks of senescence. Notably, transfection resulted in immortalization of one cell preparation with gain of large parts of the long arm of chromosome 1. Taken together, premature termination of reprogramming does not result in rejuvenation of MSCs and harbours the risk of transformation. This approach is therefore not suitable to rejuvenate cells for cellular therapy.

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 Clonal chromosomal and genomic instability during human multipotent mesenchymal stromal cells long-term culture

PLoS ONE ◽  
2018 ◽  
Vol 13 (2) ◽  
pp. e0192445 ◽  
Author(s):  
Victoria Nikitina ◽  
Tatiana Astrelina ◽  
Vladimir Nugis ◽  
Aleksandr Ostashkin ◽  
Tatiana Karaseva ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Long Term Culture

scholarly journals DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells

Aging Cell ◽  
2010 ◽  
Vol 9 (1) ◽  
pp. 54-63 ◽  
Author(s):  
Simone Bork ◽  
Stefan Pfister ◽  
Hendrik Witt ◽  
Patrick Horn ◽  
Bernhard Korn ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Methylation Pattern ◽  
Long Term Culture ◽  
Human Mesenchymal Stromal Cells ◽  
Dna Methylation Pattern

scholarly journals Mesenchymal stromal cells of osteosarcoma patients do not show evidence of neoplastic changes during long-term culture

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Emilie P Buddingh ◽  
S Eriaty N Ruslan ◽  
Christianne M A Reijnders ◽  
Karoly Szuhai ◽  
Marieke L Kuijjer ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Long Term Culture ◽  
Show Evidence

GENETIC INTEGRITY OF HUMAN ADIPOSE DERIVED MESENCHYMAL STROMAL CELLS FROM FACE AND ABDOMEN IN LONG-TERM CULTURE AND UVB RADIATION.

Cytotherapy ◽  
2021 ◽  
Vol 23 (4) ◽  
pp. 16-17
Author(s):  
C Acordi-Silva ◽  
P Barros-Delben ◽  
HD Zomer ◽  
RS Gomes ◽  
AG Trentin
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Genetic Integrity ◽  
Uvb Radiation ◽  
Long Term Culture
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