Senescence Of Mesenchymal Stromal Cells Research Articles

Effects of tumor treating fields (TTFields) on human mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) within the glioblastoma microenvironment have been shown to promote tumor progression. Tumor Treating Fields (TTFields) are alternating electric fields with low intensity and intermediate frequency that exhibit anti-tumorigenic effects. While the effects of TTFields on glioblastoma cells have been studied previously, nothing is known about the influence of TTFields on MSCs. Single-cell RNA sequencing and immunofluorescence staining were employed to identify glioblastoma-associated MSCs in patient samples. Proliferation and clonogenic survival of human bone marrow-derived MSCs were assessed after TTFields in vitro. MSC' characteristic surface marker expression was determined using flow cytometry, while multi-lineage differentiation potential was examined with immunohistochemistry. Apoptosis was quantified based on caspase-3 and annexin-V/7-AAD levels in flow cytometry, and senescence was assessed with ß-galactosidase staining. MSCs' migratory potential was evaluated with Boyden chamber assays. Single-cell RNA sequencing and immunofluorescence showed the presence of glioblastoma-associated MSCs in patient samples. TTFields significantly reduced proliferation and clonogenic survival of human bone marrow-derived MSCs by up to 60% and 90%, respectively. While the characteristic surface marker expression and differentiation capacity were intact after TTFields, treatment resulted in increased apoptosis and senescence. Furthermore, TTFields significantly reduced MSCs' migratory capacity. We could demonstrate the presence of tumor-associated MSCs in glioblastoma patients, providing a rationale to study the impact of TTFields on MSCs. TTFields considerably increase apoptosis and senescence in MSCs, resulting in impaired survival and migration. The results provide a basis for further analyses on the role of MSCs in glioblastoma patients receiving TTFields.

Abstract 2957: Senescence of stromal, endothelial, and myeloid cells in the tumor microenvironment has distinct effects on cancer progression

Abstract Accumulation of senescent cells, undermining the function of vital organs, is a culprit of aging. The effects of senescence in different types of cells on tumor growth and metastasis remain incompletely characterized. Cell senescence has been shown to result from inactivation of Telomerase (TERT), an enzyme that maintains telomere length and has obscure roles in regulating gene expression and supporting mitochondrial function. As we reported previously, knockout (KO) of Tert gene in mesenchymal stromal cells (MSC) of Pdgfra+ or Pdgfrb+ lineages leads to premature replicative stromal cell senescence in adipose tissue. To assess the effect of senescence in endothelial and myeloid cells, we also created mice with Tert KO driven by the Tie2e promoter or the LysM promoter, respectively. Here, to investigate the effect of these three KOs on cancer, we used syngeneic graft models of breast, prostate, and pancreatic carcinoma. Overall, Tert KO in cells of Pdgfra+, Pdgfrb+, Tie2e+, or LysM+ lineage had a suppressive effect on tumor growth. The strongest tumor suppression effect of Tert KO was observed in female mice orthotopically grafted with E0771 breast cancer cells. Intermediate suppressive effects of Tert KO were observed in mice orthotopically grafted with KPC pancreatic ductal adenocarcinoma cells, whereas the effect on RM1 prostate cancer cells grafts in male mice was the least significant. MSC Tert KO and myeloid Tert KO also suppressed the growth of breast and prostate cells grafts. Importantly, spontaneous metastases to the liver were induced by Tie2e-Tert KO in all mice with pancreatic KPC grafts. Analysis of adipose tissue in this endothelial senescence model revealed vasculature abnormality, leakiness, and tissue hypoxia, which provides a mechanistic explanation for increased tumor dissemination. In contrast, LysM-Tert KO did not promote KPC metastases, consistent with the lack of contribution of myeloid cells to the vasculature. Pdgfrb-Tert KO in the KPC model resulted in liver metastases observed for some but not all mice, consistent with the role of MSC in supporting vascular integrity. To test an independent model of replicative MSC senescence, we exhausted their pool by several cycles of ganciclovir treatment in mice expressing thymidine kinase under the control of Pdgfrb promoter. Upon KPC grafting in this model, reduced primary pancreatic tumor growth was also observed and liver metastases were also induced. We conclude that senescence of distinct cells in the tumor microenvironment affects early and late stages of cancer progression differently. Specifically, our data indicate that vascular cell senescence suppresses tumor growth but promotes metastases. These findings are important to consider as new approaches designed to mitigate cell senescence are investigated in the context of cancer. Citation Format: Joseph Rupert, Zhanguo Gao, Yongmei Yu, Mikhail Kolonin. Senescence of stromal, endothelial, and myeloid cells in the tumor microenvironment has distinct effects on cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2957.

Mesenchymal stromal cell response to intervertebral disc-like pH is tissue source dependent.

Intervertebral disc (IVD) degeneration (IVDD) has become increasingly prevalent and is a common contributing factor to low back pain. Current treatment options are limited to either symptom management or surgery. A promising treatment option being explored is intradiscal administration of mesenchymal stromal cells (MSCs). However, there remains a gap in knowledge as to whether MSCs from different tissue sources have similar responses to the low pH microenvironment of the IVD and the possible mechanisms governing these responses. To study this, MSCs from three different tissue sources: adipose (adipose-derived mesenchymal stem cell), bone marrow (bone marrow mesenchymal stem cells), and amnion (amniotic membrane mesenchymal stem cell) were cultured at low pHs representative of IVDD. MSCs were assessed for survival, senescence, apoptosis, metabolic activity, and cytokine release profile. Additionally, western blot was utilized to assess acid sensing ion channel 1 and 3 expression. The results of this study indicated that MSC viability, cell proliferation, senescence, and metabolic activity is negatively affected by low pH and alters MSC cytokine production. This study also demonstrated that MSCs behavior is dependent on tissue source. Understanding how MSC behavior is altered by pH will allow further research aimed at increasing the efficacy of MSC therapy to promote in situ IVDtissue regeneration to combat IVDD.

Senescence and Mitochondrial Transfer Ability of Bone Marrow Mesenchymal Stromal Cells Influence Patterns of Alloreactivity after Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative treatment for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). However post-transplant disease recurrence and alloreactive complications (chiefly graft versus host diseases - GvHD) are still associated with severe morbidity and mortality. Proliferative advantage of transformed cells as well as immune and microenvironmental dysregulations contribute both to promoting relapse and escape from graft versus leukemia (GvL) effect. Bone marrow (BM) mesenchymal stromal cells (MSC) are well known for their role in hematopoiesis and immune modulation. MSCs have been shown able to transfer mitochondria (MT) to different cell populations, including immune effectors, in order to (re)establish a healthy oxidative metabolism within the recipient cell. Here we hypothesize that these mechanisms are highly active in BM microenvironment after allo-HSCT and may contribute to alloreactivity, modifying the immune environment. On this basis we stipulate that those intercellular exchanges are deeply impacted by MSCs' senescence, that could be deployed as a useful parameter to predict post-transplant outcomes. To that end, we conducted a translational research, based on BM specimens prospectively collected at our Institution (NCT03357172, NCT03964922), involving patients undergoing allo-HSCT for high-risk MDS and AML and receiving fludarabine/busulfan-based conditioning regimens, anti-lymphoglobuline and cyclosporin/mycophenolate mofetil. MSC phenotype and functionality at the pre-transplant stage, MT capacity, immunomodulation towards T cells and phenotype were studied by multiparametric flow cytometry. Cell metabolism, in terms of oxygen consumption and extracellular acidification rate, was characterized with Seahorse XF technology. Percentages of senescent MSCs were determined with SA β-galactosidase test. First, we compared phenotypic and metabolic characteristics of MSC from MDS/AML (N=29) patients with those retrieved from a cohort of healthy controls (N=8). We observed that patient MSCs were characterized by higher expression of CD157 and CD146 and lower expression of CD200 as compared to healthy MSCs. Pre-transplant senescence quantification highlighted a certain degree of heterogeneity within the MSC compartment in leukemic patients (median = 37.42% sd=16.30 for AML patient and m=11.74% sd=6.4 for HC). In an attempt to link clinical phenotypes to this heterogeneity, we observed that patients with high degree of MSC senescence in pretransplant samples (mean= 51.6% sd=16.6 cells/sample), developed more frequently acute GvHD, as compared to those with low degree of pre-transplant MSC senescence (mean= 23.8% sd=14.3) who experienced instead a higher incidence of disease relapse (p=0,004). Assuming that the link between senescence and alloreactivity could rely to different metabolic states and MT capabilities in MSCs, we analyzed these biological features in our samples. Indeed, we observed that low degree of MSC senescence was associated with higher MT to T cells, in particular to CD4+ cells, shifting their metabolism and phenotype toward an immunosuppressive state The characterization of cellular metabolism showed in highly senescent MSCs the presence of markers of mitochondrial dysfunction with high oxidative power, but reduced coupling efficiency of respiratory chain. This metabolic profile involved a high mitochondrial mass characterized by a high maximal respiration capacity compared to HC MSCs. The in vitro modulation of MSC senescence with nicotinamide administration restored healthy mitochondrial metabolism and improved coupling efficacy of oxidative phosphorylation, possibly impacting on the immunomodulation capabilities of this microenvironment component. Here for the first time, we provide the evidence that MSC senescence plays an pivotal role in defining an altered immune state in transplanted AML possibly shifting the degree of alloreactivity from a high GvH/GvL response (high GvHD and low relapse) to a high immunosuppressive state (low GvHD and high relapse). These findings pave the way for implementing MSC senescence as a biomarker able to predict post-transplant outcomes and show new biological rationales to integrate donor-derived MSC products to better adjust GvH responses.

Dnmt3a-Mutant Haematopoietic Stem and Progenitor Cells Induce Mesenchymal Stromal Cell Senescence through the Production of Alarmins S100A8/A9

Clonal haematopoiesis (CH) is an age-related condition occurring due to somatic genetic variation in haematopoietic stem and progenitor cells (HSPCs), most frequently observed in the gene DNA methyltransferase DNMT3A. CH can be a precursor condition for haematologic malignancy, but the mechanisms driving progression of CH to malignancy are incompletely understood. Given that malignant cells are understood to reprogram their bone marrow (BM) microenvironment to create a self-reinforcing niche, we hypothesized that HSPCs carrying a CH-associated mutation in DNMT3A have microenvironment-remodelling properties that promote their clonal advantage. Using single-cell RNA-seq, we comprehensively captured bone and BM cells of haematopoietic and non-haematopoietic lineages in Dnmt3a R878H/+ mice in which the Dnmt3a mutation is present in the haematopoietic but not the non-haematopoietic compartments. In the non-haematopoietic fractions, we identified enrichment of a cellular senescence program, including transcriptional signatures of cell cycle arrest and expression of the canonical senescence-associated gene Cdkn1a (p21), in BM mesenchymal stromal cells (MSCs). The MSC cellular senescence phenotype caused by Dnmt3a-mutant haematopoiesis was confirmed at the protein level by increased expression of senescence-associated beta galactosidase (SA-β-gal) and the anti-apoptotic proteins BCL-2 and BCL-xL. In ex vivo co-culture assays, primary wild-type MSCs cultured with Dnmt3a-mutant HSPCs versus control HSPCs had increased SA-β-gal, Cdkn1a (p21), and Cdkn2a (p16) expression. Moreover, transwell assays demonstrated that MSC senescence induced by Dnmt3a-mutant HSPCs is cell contact-independent. To undercover the mechanisms driving MSC senescence, we analyzed soluble factors produced by Dnmt3a-mutant HSPCs. Conditioned mediafrom Dnmt3a-mutant HSPCs had significantly elevated levels of S100A8/A9, IL-6 and TNFα, which we confirmed were also elevated in BM fluid of Dnmt3a-mutant mice. In our single cell RNA-seq data, S100a8 and S100a9 were the top significantly increased genes in Dnmt3a-mutant versus control HSCs, making S100A8/A9 our top candidates for functional studies. We tested the direct effect of recombinant S100A8/A9 on primary wild-type MSCs and found that this was sufficient to induce SA-β-gal. Ongoing studies using the S100A8/A9 inhibitor Tasquinimod are examining the dependency of Dnmt3a-mutant hematopoiesis on alarmin production ex vivo and in vivo. Given that S100A8/A9 are heavily involved in the pathogenesis of acute inflammation and myeloproliferative neoplasms, induction of senescence through the alarmin axis is a mechanistic link between aging, clonal hematopoiesis and haematologic malignancy.

CHRONOLOGICAL AGE AFFECTS MSC SENESCENCE IN VITRO: A SYSTEMATIC REVIEW

The use of mesenchymal stromal cells (MSCs) in regenerative medicine and tissue engineering is well established, given their properties of self-renewal and differentiation. However, several studies have shown that these properties diminish with age, and understanding the pathways involved are important to provide regenerative therapies in an ageing population.In this PRISMA systematic review, we investigated the effects of chronological donor ageing on the senescence of MSCs. We identified 3023 studies after searching four databases including PubMed, Web of Science, Cochrane, and Medline. Nine studies met the inclusion and exclusion criteria and were included in the final analyses.These studies showed an increase in the expression of p21, p53, p16, ROS, and NF- B with chronological age. This implies an activated DNA damage response (DDR), as well as increased levels of stress and inflammation in the MSCs of older donors. Additionally, highlighting the effects of an activated DDR in cells from older donors, a decrease in the expression of proliferative markers including Ki67, MAPK pathway elements, and Wnt/ -catenin pathway elements was observed. Furthermore, we found an increase in the levels of SA- -galactosidase, a specific marker of cellular senescence.Together, these findings support an association between chronological age and MSC senescence. The precise threshold for chronological age where the reported changes become significant is yet to be defined and should form the basis for further scientific investigations. The outcomes of this review should direct further investigations into reversing the biological effects of chronological age on the MSC senescence phenotype.

Phenotyping senescent mesenchymal stromal cells using AI image translation.

Mesenchymal stromal cells (MSCs) offer promising potential in biomedical research, clinical therapeutics, and immunomodulatory therapies due to their ease of isolation and multipotent, immunoprivileged, and immunosuppersive properties. Extensive efforts have focused on optimizing the cell isolation and culture methods to generate scalable, therapeutically-relevant MSCs for clinical applications. However, MSC-based therapies are often hindered by cell heterogeneity and inconsistency of therapeutic function caused, in part, by MSC senescence. As such, noninvasive and molecular-based MSC characterizations play an essential role in assuring the consistency of MSC functions. Here, we demonstrated that AI image translation algorithms can effectively predict immunofluorescence images of MSC senescence markers from phase contrast images. We showed that the expression level of senescence markers including senescence-associated beta-galactosidase (SABG), p16, p21, and p38 are accurately predicted by deep-learning models for Doxorubicin-induced MSC senescence, irradiation-induced MSC senescence, and replicative MSC senescence. Our AI model distinguished the non-senescent and senescent MSC populations and simultaneously captured the cell-to-cell variability within a population. Our microscopy-based phenotyping platform can be integrated with cell culture routines making it an easily accessible tool for MSC engineering and manufacturing.

Paracrine Senescence of Mesenchymal Stromal Cells Involves Inflammatory Cytokines and the NF-κB Pathway.

It has been known that senescence-associated secretory phenotype (SASP) triggers senescence of the surrounding normal cells. However, SASP signaling regarding mesenchymal stromal cell aging remains to be fully elucidated. Therefore, the present study aimed to clarify the molecular mechanism of late (passage) MSC-induced paracrine SASP-mediated senescence of early (passage) MSCs during ex vivo expansion. Here, we conducted an extensive characterization of senescence features in bone-marrow (BM)-derived MSCs from healthy human donors. Late MSCs displayed an enlarged senescent-like morphology, induced SASP-related proinflammatory cytokines (IL-1α and IL-8), and reduced clonogenic capacity and osteogenic differentiation when compared to early MSCs. Of note, paracrine effects of SASP-related IL-1α and IL-8 from late MSCs induced cellular senescence of early MSCs via an NF-κB-dependent manner. Moreover, cellular senescence of early MSCs was promoted by the synergistic action of IL-1α and IL-8. However, inhibition of NF-κB by shRNA transfection or using inhibitors in early MSCs blocked early MSCs cellular senescence caused by paracrine SASP of late MSCs. In conclusion, these findings reveal that late MSCs display features of senescence and that, during ex vivo expansion, SASP-related proinflammatory cytokines contribute to activate a cellular senescence program in early MSCs that may ultimately impair their functionality.

CD26 is a senescence marker associated with reduced immunopotency of human adipose tissue-derived multipotent mesenchymal stromal cells

IntroductionHuman mesenchymal stromal cells (MSCs) have immunomodulatory, anti-inflammatory, and tolerogenic effects. Long-term in vitro expansion of MSCs to generate clinical grade products results in the accumulation of senescent-functionally impaired MSCs. Markers to assess the ‘senescent load’ of MSC products are needed.MethodsEarly and late passage human adipose tissue (AT) MSCs from pediatric and adult donors were characterized using established senescent markers [i.e., MSC size, granularity, and autofluorescence by flow cytometry; β-galactosidase staining (SA-β-gal); CDKN2A and CDKN1A by qRT-PCR]. In gene set enrichment analysis, DPP4 (also known as adenosine deaminase complexing protein 2 or CD26) was found as a prominent dysregulated transcript that was increased in late passage MSC(AT). This was confirmed in a larger number of MSC samples by PCR, flow cytometry, Western blotting, and immunofluorescence. In vitro immunopotency assays compared the function of CD26high and CD26low MSC(AT). The effect of senolytics on the CD26high subpopulation was evaluated in senescent MSC(AT).ResultsLate passage MSC(AT) had a senescence transcriptome signature. DPP4 was the most differentially enriched gene in senescent MSCs. Late passage senescent MSC(AT) had higher CD26 surface levels and total protein abundance. Moreover, CD26 surface levels were higher in early passage MSC(AT) from adults compared to pediatric donors. CD26 abundance correlated with established senescence markers. CD26high MSC(AT) had reduced immunopotency compared to CD26low MSC(AT). Senolytic treatment induced MSC apoptosis, which decreased the frequencies of CD26high MSC(AT).ConclusionsDPP4 gene expression and DPP4/CD26 protein abundance are markers of replicative senescence in MSC(AT). Samples enriched in CD26high MSC(AT) have reduced immunopotency and CD26high MSCs are reduced with senolytics.

Novel immortalization approach defers senescence of cultured canine adipose-derived mesenchymal stromal cells.

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 β-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™ 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.

Biomolecular Evaluation of Piceatannol's Effects in Counteracting the Senescence of Mesenchymal Stromal Cells: A New Candidate for Senotherapeutics?

Several investigations on senescence and its causative role in aging have underscored the importance of developing senotherapeutics, a field focused on killing senescent cells and/or preventing their accumulation within tissues. Using polyphenols in counteracting senescence may facilitate the development of senotherapeutics given their presence in the human diet, their confirmed tolerability and absence of severe side effects, and their role in preventing senescence and inducing the death of senescent cells. Against that background, we evaluated the effect of piceatannol, a natural polyphenol, on the senescence of mesenchymal stromal cells (MSCs), which play a key role in the body’s homeostasis. Among our results, piceatannol reduced the number of senescent cells both after genotoxic stress that induced acute senescence and in senescent replicative cultures. Such senotherapeutics activity, moreover, promoted the recovery of cell proliferation and the stemness properties of MSCs. Altogether, our findings demonstrate piceatannol’s effectiveness in counteracting senescence by targeting its associated pathways and detecting and affecting P53-dependent and P53-independent senescence. Our study thus suggests that, given piceatannol’s various mechanisms to accomplish its pleiotropic activities, it may be able to counteract any senescent phenotypes.

Chronological Age Affects MSC Senescence In Vitro—A Systematic Review

The use of mesenchymal stromal cells (MSCs) in regenerative medicine and tissue engineering is well established, given their properties of self-renewal and differentiation. However, several studies have shown that these properties diminish with age, and understanding the pathways involved are important to provide regenerative therapies in an ageing population. In this PRISMA systematic review, we investigated the effects of chronological donor ageing on the senescence of MSCs. We identified 3023 studies after searching four databases including PubMed, Web of Science, Cochrane, and Medline. Nine studies met the inclusion and exclusion criteria and were included in the final analyses. These studies showed an increase in the expression of p21, p53, p16, ROS, and NF-κB with chronological age. This implies an activated DNA damage response (DDR), as well as increased levels of stress and inflammation in the MSCs of older donors. Additionally, highlighting the effects of an activated DDR in cells from older donors, a decrease in the expression of proliferative markers including Ki67, MAPK pathway elements, and Wnt/β-catenin pathway elements was observed. Furthermore, we found an increase in the levels of SA-β-galactosidase, a specific marker of cellular senescence. Together, these findings support an association between chronological age and MSC senescence. The precise threshold for chronological age where the reported changes become significant is yet to be defined and should form the basis for further scientific investigations. The outcomes of this review should direct further investigations into reversing the biological effects of chronological age on the MSC senescence phenotype.

Aging of Bone Marrow Mesenchymal Stromal Cells: Hematopoiesis Disturbances and Potential Role in the Development of Hematologic Cancers.

Simple SummaryAs for many other cancers, the risk of developing hematologic malignancies increases considerably as people age. In recent years, a growing number of studies have highlighted the influence of the aging microenvironment on hematopoiesis and tumor progression. Mesenchymal stromal cells are a major player in intercellular communication inside the bone marrow microenvironment involved in hematopoiesis support. With aging, their functions may be altered, leading to hematopoiesis disturbances which can lead to hematologic cancers. A good understanding of the mechanisms involved in mesenchymal stem cell aging and the consequences on hematopoiesis and tumor progression is therefore necessary for a better comprehension of hematologic malignancies and for the development of therapeutic approaches.Aging of bone marrow is a complex process that is involved in the development of many diseases, including hematologic cancers. The results obtained in this field of research, year after year, underline the important role of cross-talk between hematopoietic stem cells and their close environment. In bone marrow, mesenchymal stromal cells (MSCs) are a major player in cell-to-cell communication, presenting a wide range of functionalities, sometimes opposite, depending on the environmental conditions. Although these cells are actively studied for their therapeutic properties, their role in tumor progression remains unclear. One of the reasons for this is that the aging of MSCs has a direct impact on their behavior and on hematopoiesis. In addition, tumor progression is accompanied by dynamic remodeling of the bone marrow niche that may interfere with MSC functions. The present review presents the main features of MSC senescence in bone marrow and their implications in hematologic cancer progression.

Replicative Senescence and Expression of Autophagy Genes in Mesenchymal Stromal Cells.

Cell senescence leads to a number of changes in the properties of mesenchymal stromal cells (MSCs). In particular, the number of damaged structures is increased producing negative effect on intracellular processes. Elimination of the damaged molecules and organelles occurs via autophagy that can be important in the context of aging. Cultivation under low oxygen level can be used as an approach for enhancement of MSC therapeutic properties and "slowing down" cell senescence. The goal of this work was to study some morphological and functional characteristics and expression of autophagy-associated genes during replicative senescence of MSCs under different oxygen concentration. The study revealed changes in the regulation of autophagy at the transcriptional level. Upregulation of the expression of autophagosome membrane growth genes ATG9A and ULK1, of the autophagosome maturation genes CTSD, CLN3, GAA, and GABARAPL1, of the autophagy regulation genes TP53, TGFB1, BCL2L1, FADD, and HTT was shown. These changes were accompanied by downregulation of IGF1 and TGM2 expression. Increase of the lysosomal compartment volume was observed in the senescent MSCs that also indicated increase of their degradation activity. The number of lysosomes was decreased following prolonged cultivation under low oxygen concentration (5%). The replicative senescence of MSCs under conditions of different oxygen levels led to the similar modifications in the expression of the autophagy-associated genes.

Obesity is associated with senescence of mesenchymal stromal cells derived from bone marrow, subcutaneous and visceral fat of young mice.

White adipose tissue (WAT) is distributed in several depots with distinct metabolic and inflammatory functions. In our body there are subcutaneous (sWAT), visceral (vWAT) and bone marrow (bWAT) fat depots. Obesity affects the size, function and inflammatory state of WATs. In particular, obesity may affect the activity of mesenchymal stromal cells (MSCs) present in WAT. MSCs are a heterogeneous population containing stromal cells, progenitor cells, fibroblasts and stem cells that are able to differentiate among adipocytes, chondrocytes, osteocytes and other mesodermal derivatives.In the first study of this kind, we performed a comparison of the effects of obesity on MSCs obtained from sWAT, vWAT and bWAT. Our study showed that obesity affects mainly the biological functions of MSCs obtained from bone marrow and vWAT by decreasing the proliferation rate, reducing the percentage of cells in S phase and triggering senescence. The onset of senescence was confirmed by expression of genes belonging to RB and P53 pathways.Our study revealed that the negative consequences of obesity on body physiology may also be related to impairment in the functions of the stromal compartment present in the several adipose tissues. This finding provides new insights as to the targets that should be considered for an effective treatment of obesity-related diseases.

Hydrogel Culture Surface Stiffness Modulates Mesenchymal Stromal Cell Secretome and Alters Senescence.

Current cell culture surfaces used for the expansion and production of mesenchymal stromal cells (MSCs) are not optimized for the production of highly secretory and nonsenescent cells. In this study, we used poly (ethylene glycol) hydrogel substrates with tunable mechanical and biochemical properties to screen the effect of culture surfaces on pro-regenerative secretome by multiplex enzyme-linked immunosorbent assay, proliferation by PicoGreen DNA analysis, and senescence by senescence-associated β-galactosidase activity. We demonstrate that MSCs cultured on 30 kPa hydrogels, regardless of biochemical functionalization, broadly enhanced the secretion of immunomodulatory and regenerative factors versus stiffer 100 kPa or tissue culture plastic surfaces, but did not support robust proliferation. In contrast, culture on 100 kPa hydrogel surfaces promoted proliferation at a similar level and did not substantially alter the amount of secreted factors as compared with tissue culture plastic. Culture on integrin-engaging, cadherin-engaging, and hyaluronic acid-containing 30 kPa substrates enhanced MSC-conditioned media (CM) angiogenic activity in a human umbilical vein endothelial cell tube formation assay and human THP-1 monocyte chemoattraction in a transwell assay. However, 30 kPa substrate culture did not impact the myogenic activity of MSC CM in a C2C12 myoblast tube formation assay. Culture on selected 100 kPa surfaces enhanced CM angiogenic activity and monocyte chemotaxis, but not myogenic activity. Serial culture on 100 kPa RGD hydrogel surfaces significantly reduced senescence in MSCs versus tissue culture plastic, while maintaining the capacity of the cells to enhance their secretome in response to 30 kPa surfaces. Thus, hydrogel substrates that exhibit stiffness orders of magnitude lower than standard tissue culture plastic can serve as novel surfaces for the production of MSCs with an improved therapeutic secretory capacity and reduced senescence. Impact statement The success of mesenchymal stromal cell (MSC)-based therapies is dependent on the manufacture of a large number of cells with high therapeutic potency. Among the culture surfaces tested in this study, we demonstrate that substrate stiffness rather than biochemical functionalization predominantly guides changes in MSC proliferation and secretory capacity. We have identified substrate parameters to support MSC proliferation, enhance secretion of paracrine factors, and to reduce replicative senescence. By maximizing secretory capacity and reducing senescence through the choice of hydrogel culture materials, these findings have great potential to improve the large-scale production of therapeutic MSCs.

Cellular senescence induced by S100A9 in mesenchymal stromal cells through NLRP3 inflammasome activation.

Bone marrow stromal cells from patients with myelodysplastic syndrome (MDS) display a senescence phenotype, but the underlying mechanism has not been elucidated. Pro-inflammatory signaling within the malignant clone and the bone marrow microenvironment has been identified as a key pathogenetic driver of MDS. Our study revealed that S100A9 is highly-expressed in lower-risk MDS. Moreover, normal primary mesenchymal stromal cells (MSCs) and the human stromal cell line HS-27a co-cultured with lower-risk MDS bone marrow mononuclear cells acquired a senescence phenotype. Exogenous supplemented S100A9 also induced cellular senescence in MSCs and HS-27a cells. Importantly, Toll-like receptor 4 (TLR4) inhibition or knockdown attenuated the cellular senescence induced by S100A9. Furthermore, we showed that S100A9 induces NLRP3 inflammasome formation, and IL-1β secretion; findings in samples from MDS patients further confirmed these thoughts. Moreover, ROS and IL-1β inhibition suppressed the cellular senescence induced by S100A9, whereas NLRP3 overexpression and exogenous IL-1β supplementation induces cellular senescence. Our study demonstrated that S100A9 promotes cellular senescence of bone marrow stromal cells via TLR4, NLRP3 inflammasome formation, and IL-1β secretion for its effects. Our findings deepen the understanding of the molecular mechanisms involved in MDS reprogramming of MSCs and indicated the essential role of S100A9 in tumor-environment interactions in bone marrow.

KDM3A and KDM4C Regulate Mesenchymal Stromal Cell Senescence and Bone Aging via Condensin-mediated Heterochromatin Reorganization.

SummaryEpigenomic changes and stem cell deterioration are two hallmarks of aging. Accumulating evidence suggest that senescence of mesenchymal stromal cells (MSCs) perpetuates aging or age-related diseases. Here we report that two H3K9 demethylases, KDM3A and KDM4C, regulate heterochromatin reorganization via transcriptionally activating condensin components NCAPD2 and NCAPG2 during MSC senescence. Suppression of KDM3A or KDM4C by either genetic or biochemical approach leads to robust DNA damage response and aggravates cellular senescence, whereas overexpression of KDM3A/KDM4C or NCAPD2 promotes heterochromatin reorganization and blunts DNA damage response. Moreover, MSCs derived from Kdm3a−/− mice exhibit defective chromosome organization and exacerbated DNA damage response, which are associated with accelerated bone aging. Consistently, analysis of human bone marrow MSCs and transcriptome database reveals inverse correlation of KDM3A/KDM4C and/or NCAPD2/NCAPG2 with aging. Taken together, the present finding unveils that H3K9 demethylases function as a surveillance mechanism to restrain DNA damage accumulation in stem cells during aging.

3170 – ABERRANT BONE MARROW MICROENVIRONMENT IN THERAPY RELATED MYELOID NEOPLASM

Introduction Therapy related myeloid neoplasm (t-MN) is a lethal second hematological malignancy following chemotherapy (CT) and radiotherapy (RT) for primary cancers. It accounts for 15-20% of myelodysplastic syndromes (MDS) and acute myeloid leukemia. Prognosis of t-MN is poor with no effective therapies. Although CT/RT can damage the bone marrow microenvironment, very limited studies assessed it in t-MN. Aim To compare bone marrow microenvironment milieu and mesenchymal stromal cells (MSC) from primary MDS (pMDS), t-MN and double cancers (DC, primary cancer and MDS but without a history of CT/RT for primary cancer). Methods Bone Marrow plasma cytokines/chemokines (CCK) and bone marrow MSC proliferation, senescence, morphology and differentiation potential of pMDS, t-MN and DC was compared. Results Proliferation and clonogenic potential of MSC from t-MN were significantly lower than pMDS and DC (Fig.1A). MSC from t-MN could not be maintained in culture and had higher senescence rates, assessed by β-galactosidase positive cells, compared to pMDS and DC (p Conclusions Our data demonstrate that MSC from t-MN patients have significantly altered morphology, a lower proliferation rate, and higher senescence rate compared to pMDS and DC. Osteogenic differentiation of t-MN MSC was higher at the cost of reduced adipogenic differentiation. This data suggests that RT/CT leads to long term damages to bone marrow microenvironment which may play a role in t-MN pathogenesis.

The Upregulation of Toll-Like Receptor 3 via Autocrine IFN-β Signaling Drives the Senescence of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Through JAK1.

Although mesenchymal stromal cells (MSCs) are among the most promising cell sources for cell-based therapies and regenerative medicine, the decline in their function with age due to cellular senescence limits their therapeutic applications. Unveiling the underlying mechanism of MSC senescence is therefore of substantial interest with regard to advancing MSC-based cell therapies. We here show that the induction of human umbilical cord blood-derived MSC (UCB-MSC) senescence causes the predominant upregulation of Toll-like receptor 3 (TLR3). Subsequent TLR3 activation by polyinosinic-polycytidylic acid triggers the prominent features of senescence. Using a clustered regularly interspaced short palindromic repeats/Cas9 library screening system, we identified Janus kinase 1 (JAK1) as the candidate regulatory factor for TLR3-mediated MSC senescence. A JAK1 deficiency blocked the MSC senescence phenotype upon TLR3 activation and TLR3 induction. Targeting the JAK1 pathway using chemical JAK1 inhibitors also significantly suppressed TLR3-mediated MSC senescence. Importantly, we further observed that UCB-MSC senescence is driven by a senescence-associated secretory phenotype (SASP) and that interferon-β (IFN-β) is a component of TLR3-dependent SASP, whereby its autocrine actions upregulate TLR3 and suppress cell proliferation. A JAK1 depletion significantly interrupted these effects of IFN-β, indicating that JAK1 is a signaling mediator linking IFN-β activity to TLR3 expression and the process of MSC senescence. Collectively, our findings provide new mechanistic insights into UCB-MSC senescence by revealing the role of an autocrine regulatory loop of SASP evoked by TLR3 activation.