Chromosomal stability of mesenchymal stromal cells during in vitro culture

Stultz, Brian G.; McGinnis, Kathleen A.; Thompson, Elaine E.; Surdo, Jessica Lo; Bauer, Steven R.; Hursh, Deborah A.

doi:10.1016/j.jcyt.2015.11.017

Cited by 68 publications

(59 citation statements)

References 43 publications

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“…We used three lots of previously characterized MSCs. The karyotypes, cell surface marker expression, transcriptomes, and proteomes of these lots have been examined, as well as their ability to differentiate along the adipogenic lineage [36, 37, 44–47]. These MSC lots were treated with cytokines for up to 72 hours.…”

Section: Resultsmentioning

confidence: 99%

In vitro cytokine licensing induces persistent permissive chromatin at the Indoleamine 2,3-dioxygenase promoter

et al. 2016

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Background Mesenchymal stromal cells (MSCs) are being investigated as therapies for inflammatory diseases due to their immunosuppressive capacity. IFN-γ treatment primes MSC immunosuppression partially through induction of Indoleamine 2,3-dioxygenase (IDO1), which depletes tryptophan necessary to support proliferation of activated T-cells. We investigated the role of histone modifications in the timing and maintenance of induced IDO1 expression in MSCs under clinical manufacturing conditions, such as cryopreservation. Methods We used chromatin immunoprecipitation and quantitative polymerase chain reaction (PCR) to assay levels of transcriptionally permissive acetylated H3K9 and repressive trimethylated H3K9 histone modifications surrounding the transcriptional start site for IDO1, and reverse transcriptase PCR and immunoblotting to detect mRNA and protein. Results MSCs derived from three donors approached maximum IDO1 mRNA levels following 24 hours of in vitro cytokine treatment. Induction of IDO1 expression correlated with increased acetylation of H3K9 concomitant with reduction of trimethylated H3K9 modifications at the promoter. Examination of two additional donors confirmed this result. While induced IDO1 levels declined within two days after cytokine removal and freeze thawing, the activated chromatin state was maintained. Upon re-exposure to cytokines, previously primed MSCs accumulated near-maximum IDO1 mRNA levels within four to eight hours. Discussion Our data indicate that in vitro priming of MSCs causes chromatin remodeling at the IDO1 promoter, that this alteration is maintained during processing commonly used to prepare MSCs for clinical use, and that once primed, MSCs are poised for IDO1 expression even in the absence of cytokines.

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Section: Resultsmentioning

confidence: 99%

In vitro cytokine licensing induces persistent permissive chromatin at the Indoleamine 2,3-dioxygenase promoter

et al. 2016

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“…MSC culture conditions were chosen based on well-established protocols (55). Most of the MSC lines used in this study have been extensively characterized and further information about the donors' surface marker expression, genomic, epigenetic, and proteomic profiles, as well as performance in multiple bioassays for MSC function has been published previously (12,38,47,48,(56)(57)(58). Detailed MSC culturing and expansion methods are described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

“…Nuclear morphology relates to stemness and differentiation (43,44), and there is direct coupling of the nucleus to the cytoskeleton through proteins such as Lamin A/C (44,45). Evidence suggests that nuclear morphological changes associated with a biological function may be mediated by epigenetic modifications (46)(47)(48). The effect of IFN-γ stimulation on permissive chromatin (at the IDO promoter) associated with MSC immunosuppressive function was demonstrated recently with several of the MSC lines used in this study and may be related to the change in nuclear morphology associated with IFN-γ treatment (47).…”

Section: Morphological Features Of Mscs After Ifn-γ Stimulation Corrementioning

confidence: 99%

Morphological features of IFN-γ–stimulated mesenchymal stromal cells predict overall immunosuppressive capacity

Klinker

Marklein

Surdo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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168

166

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Human mesenchymal stromal cell (MSC) lines can vary significantly in their functional characteristics, and the effectiveness of MSCbased therapeutics may be realized by finding predictive features associated with MSC function. To identify features associated with immunosuppressive capacity in MSCs, we developed a robust in vitro assay that uses principal-component analysis to integrate multidimensional flow cytometry data into a single measurement of MSC-mediated inhibition of T-cell activation. We used this assay to correlate single-cell morphological data with overall immunosuppressive capacity in a cohort of MSC lines derived from different donors and manufacturing conditions. MSC morphology after IFN-γ stimulation significantly correlated with immunosuppressive capacity and accurately predicted the immunosuppressive capacity of MSC lines in a validation cohort. IFN-γ enhanced the immunosuppressive capacity of all MSC lines, and morphology predicted the magnitude of IFN-γ-enhanced immunosuppressive activity. Together, these data identify MSC morphology as a predictive feature of MSC immunosuppressive function.H uman mesenchymal stromal cells (MSCs) can potently suppress immune responses in vitro and in animal models of human disease (1, 2), but to date MSC-based therapies have produced mixed results in clinical trials for treatment of inflammatory and autoimmune diseases (3, 4). A major challenge in the development of consistently effective MSC-based immunosuppressive therapies is that MSC lines derived from different donors and manufacturing processes (i.e., cell expansion) can possess markedly dissimilar immunosuppressive function (3,5,6). Although methods exist to assess MSC immunosuppression in vitro, they are often based on only a few measured outcomes, assay culture conditions, and donor MSC samples (5, 7-9). To improve upon these methods, we developed an experimental and analytical approach to quantify MSC-mediated immune suppression using principal-component analysis (PCA) to integrate multiple measurements of T-cell activation assessed at a range of MSC densities. This approach allowed us to determine a single value for immunosuppressive capacity for MSC lines derived from two different manufacturing processes and 13 independent donors.Another major challenge associated with MSC-based immune therapies is the lack of well-defined predictive markers to identify MSC lines with therapeutically relevant biological activities or manufacturing processes that produce more effective MSCbased products. Efforts have been made to identify MSC quality attributes associated with immunosuppression (6, 7), but the majority of clinical studies (10) rely upon the surface markers described by Dominici et al. (11). Having previously shown that morphology can predict MSC mineralization capacity (12), we hypothesized that morphological features associated with immunosuppression in MSCs could be identified and used to predict their performance in our quantitative immunosuppression assay.Using our quantitative method for as...

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“…Isolation methods influence MSC proliferation and differentiation potential Van Harmelen et al, 2004) Source of isolation effects MSC properties (Beeravolu et al, 2016;Beeravolu et al, 2017;De Ugarte et al, 2003;Im et al, 2005;Legzdina et al, 2016) Media composition influences proliferation and differentiation capacity of MSCs (Araujo et al, 2017;Beeravolu et al, 2016;Beeravolu et al, 2017;Jin et al, 2013) Microenvironment effects cell growth and differentiation of MSCs Y. Li et al, 2015;Shekaran et al, 2015) Donor age and environmental factors effect genetic stability of MSCs (D'Ippolito et al, 1999;Malgieri et al, 2010;Mattiucci et al, 2018;Stultz et al, 2016) Promotion of tumor growth by MSCs (Lacerda et al, 2015;Ridge, Sullivan, & Glynn, 2017) Abbreviation: MSC, mesenchymal stromal/stem cell.…”

Section: Description Referencementioning

confidence: 99%

Mesenchymal stem cells: Cell therapy and regeneration potential

Brown

McKee

Bakshi

et al. 2019

J Tissue Eng Regen Med

448

310

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Rapid advances in the isolation of multipotent progenitor cells, routinely called mesenchymal stromal/stem cells (MSCs), from various human tissues and organs have provided impetus to the field of cell therapy and regenerative medicine. The most widely studied sources of MSCs include bone marrow, adipose, muscle, peripheral blood, umbilical cord, placenta, fetal tissue, and amniotic fluid. According to the standard definition of MSCs, these clonal cells adhere to plastic, express cluster of differentiation (CD) markers such as CD73, CD90, and CD105 markers, and can differentiate into adipogenic, chondrogenic, and osteogenic lineages in vitro. However, isolated MSCs have been reported to vary in their potency and self‐renewal potential. As a result, the MSCs used for clinical applications often lead to variable or even conflicting results. The lack of uniform characterization methods both in vitro and in vivo also contributes to this confusion. Therefore, the name “MSCs” itself has been increasingly questioned lately. As the use of MSCs is expanding rapidly, there is an increasing need to understand the potential sources and specific potencies of MSCs. This review discusses and compares the characteristics of MSCs and suggests that the variations in their distinctive features are dependent on the source and method of isolation as well as epigenetic changes during maintenance and growth. We also discuss the potential opportunities and challenges of MSC research with the hope to stimulate their use for therapeutic and regenerative medicine.

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Chromosomal stability of mesenchymal stromal cells during in vitro culture

Cited by 68 publications

References 43 publications

In vitro cytokine licensing induces persistent permissive chromatin at the Indoleamine 2,3-dioxygenase promoter

In vitro cytokine licensing induces persistent permissive chromatin at the Indoleamine 2,3-dioxygenase promoter

Morphological features of IFN-γ–stimulated mesenchymal stromal cells predict overall immunosuppressive capacity

Mesenchymal stem cells: Cell therapy and regeneration potential

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