Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior

Sareen, Niketa; Sequiera, Glen Lester; Chaudhary, Rakesh; Abu‐El‐Rub, Ejlal; Chowdhury, Subir Roy; Sharma, Vikram; Surendran, Arun; Moudgil, Meenal; Fernyhough, Paul; Ravandi, Amir; Dhingra, Sanjiv

doi:10.1186/s13287-018-0867-4

Cited by 34 publications

(25 citation statements)

References 39 publications

(32 reference statements)

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“…The choice of evaluation passages was based on the scientific literature [2,3], and the experimental groups were submitted to viability tests by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), apoptosis and necrosis assays, comet test, and measurements of TBARS, nitrite, GSH, and catalase.…”

Section: Methodsmentioning

confidence: 99%

“…The in vitro senescence of multipotent cells, such as mesenchymal stem cells (MSC) and renal progenitor cells (RPC), has been commonly associated with macromolecular damage, especially to DNA induced by oxidative stress [1]. These changes are most frequently identified in cultures with prolonged incubation time, from the fourth passage, which limits the possibility of clinical application given the intrinsic need for cell expansion in vitro to obtain the minimum therapeutic concentration of 10 6 cells/mL [2,3].…”

Section: Introductionmentioning

confidence: 99%

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Renal Progenitor Cells Have Higher Genetic Stability and Lower Oxidative Stress than Mesenchymal Stem Cells during In Vitro Expansion

Silva

Neto

Bezerra

et al. 2020

Oxidative Medicine and Cellular Longevity

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In vitro senescence of multipotent cells has been commonly associated with DNA damage induced by oxidative stress. These changes may vary according to the sources of production and the studied lineages, which raises questions about the effect of growing time on genetic stability. This study is aimed at evaluating the evolution of genetic stability, viability, and oxidative stress of bone marrow mesenchymal stem cells (MSCBMsu) and renal progenitor cells of the renal cortex (RPCsu) of swine (Sus scrofa domesticus) in culture passages. P2, P5, and P9 were used for MSCBMsu and P1, P2, and P3 for RPCsu obtained by thawing. The experimental groups were submitted to MTT, apoptosis and necrosis assays, comet test, and reactive substance measurements of thiobarbituric acid (TBARS), nitrite, reduced glutathione (GSH), and catalase. The MTT test curve showed a mean viability of 1.14±0.62 and 1.12±0.54, respectively, for MSCBMsu and RPCsu. The percentages of MSCBMsu and RPCsu were presented, respectively, for apoptosis, an irregular and descending behavior, and necrosis, ascending and irregular. The DNA damage index showed higher intensity among the MSCBMsu in the P5 and P9 passages (p<0.05). In the TBARS evaluation, there was variation among the lines of RPCsu and MSCBMsu, presenting the last most significant variations (p<0.05). In the nitrite values, we identified only among the lines, in the passages P1 and P2, with the highest averages displayed by the MSCBMsu lineage (p<0.05). The measurement of antioxidant system activity showed high standards, identifying differences only for GSH values, in the RPCsu lineage, in P3 (p<0.05). This study suggests that the maintenance of cell culture in the long term induces lower regulation of oxidative stress, and RPCsu presents higher genetic stability and lower oxidative stress than MSCBMsu during in vitro expansion.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Renal Progenitor Cells Have Higher Genetic Stability and Lower Oxidative Stress than Mesenchymal Stem Cells during In Vitro Expansion

Silva

Neto

Bezerra

et al. 2020

Oxidative Medicine and Cellular Longevity

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“…To measure leukocyte-mediated cytotoxicity in rat MSCs, the leukocytes were isolated from spleen (SD rat) using HISTOPAQUE 1083 (Sigma-Aldrich) and co-cultured with allogeneic MSCs in the ratio of 10:1 as described in our previous studies 12,51 . After 72 h of co- culture, leukocyte-mediated cytotoxicity in the MSCs was assessed by measuring the LDH released from the damaged MSCs (LDH Cytotoxicity Detection Kit; Clontech).…”

Section: Methodsmentioning

confidence: 99%

Hypoxia-induced 26S proteasome dysfunction increases immunogenicity of mesenchymal stem cells

et al. 2019

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Bone marrow-derived allogeneic (donor derived) mesenchymal stem cells (MSCs) are immunoprivileged and are considered to be prominent candidates for regenerative therapy for numerous degenerative diseases. Even though the outcome of initial allogeneic MSCs based clinical trials was encouraging, the overall enthusiasm, of late, has dimmed down. This is due to failure of long-term survival of transplanted cells in the recipient. In fact, recent analyses of allogeneic MSC-based studies demonstrated that cells after transplantation turned immunogenic and were subsequently rejected by host immune system. The current study reveals a novel mechanism of immune switch in MSCs. We demonstrate that hypoxia, a common denominator of ischemic tissues, induces an immune shift in MSCs from immunoprivileged to immunogenic state. The immunoprivilege of MSCs is preserved by downregulation or the absence of major histocompatibility complex class II (MHC-II) molecules. We found that 26S proteasome-mediated intracellular degradation of MHC-II helps maintain the absence of MHC-II expression on cell surface in normoxic MSCs and preserves their immunoprivilege. The exposure to hypoxia leads to dissociation of 19S and 20S subunits, and inactivation of 26S proteasome. This prevented the degradation of MHC-II and, as a result, the MSCs became immunogenic. Furthermore, we found that hypoxia-induced decrease in the levels of a chaperon protein HSP90α is responsible for inactivation of 26S proteasome. Maintaining HSP90α levels in hypoxic MSCs preserved the immunoprivilege of MSCs. Therefore, hypoxia-induced inactivation of 26S proteasome assembly instigates loss of immunoprivilege of allogeneic mesenchymal stem cells. Maintaining 26S proteasome activity in mesenchymal stem cells preserves their immunoprivilege.

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“…The cells expressing CD44, CD29 (Santa Cruz Inc), and negative for CD45, CD34 (Santa Cruz Inc) as assessed by flow cytometry, were used for subsequent experiments. 13,24,25 data suggest that the new strategies that target CSN5-COX2 signaling may improve survival and utility of transplanted allogeneic MSCs in the ischemic heart.…”

Section: Isolation and Characterization Of Rat Bone Marrow Mscsmentioning

confidence: 99%

Hypoxia‐induced downregulation of cyclooxygenase 2 leads to the loss of immunoprivilege of allogeneic mesenchymal stem cells

et al. 2020

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Allogeneic mesenchymal stem cells (MSCs) from young and healthy donors are reported to hold the potential to treat several immunological and degenerative disorders. However, recent data from animal studies and clinical trials demonstrate that immunogenicity and poor survival of transplanted MSCs impaired the efficacy of cells for regenerative applications. It is reported that initially immunoprivileged under in vitro conditions, MSCs are targeted by the host immune system after transplantation in the ischemic tissues in vivo. We performed in vitro (in MSCs) and in vivo (in the rat model of myocardial infarction [MI]) studies to elucidate the mechanisms responsible for the change in the immunophenotype of MSCs from immunoprivileged to immunogenic under ischemic conditions. We have recently reported that a soluble factor prostaglandin E2 (PGE2) preserves the immunoprivilege of allogeneic MSCs. In the current study, we found that PGE2 levels, which were elevated during normoxia, decreased in MSCs following exposure to hypoxia. Further, we found that proteasome-mediated degradation of cyclooxygenase-2 (COX2, rate-limiting enzyme in PGE2 biosynthesis) in hypoxic MSCs is responsible for PGE2 decrease and loss of immunoprivilege of MSCs. While investigating the mechanisms of COX2 degradation in hypoxic MSCs, we found that in normoxic MSCs, COP9 signalosome subunit 5 (CSN5) binds to COX2 and prevents its degradation by the proteasome. However, exposure to hypoxia leads to a decrease in CSN5 levels and its binding to COX2, rendering COX2 protein susceptible to proteasome-mediated degradation. This subsequently causes PGE2 downregulation and loss of immunoprivilege of MSCs. Maintaining COX2 levels in MSCs preserves immunoprivilege in vitro and improves the survival of transplanted MSCs in a rat model of MI. These data provide novel mechanistic evidence that PGE2 is downregulated in hypoxic MSCs which is responsible for the post-transplantation rejection of allogeneic MSCs. Therefore, our

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Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior

Cited by 34 publications

References 39 publications

Renal Progenitor Cells Have Higher Genetic Stability and Lower Oxidative Stress than Mesenchymal Stem Cells during In Vitro Expansion

Renal Progenitor Cells Have Higher Genetic Stability and Lower Oxidative Stress than Mesenchymal Stem Cells during In Vitro Expansion

Hypoxia-induced 26S proteasome dysfunction increases immunogenicity of mesenchymal stem cells

Hypoxia‐induced downregulation of cyclooxygenase 2 leads to the loss of immunoprivilege of allogeneic mesenchymal stem cells

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