Normal Mesenchymal Stem Cells Research Articles

610 The Effectiveness of Adipose Tissue Derived Mesenchymal Stem Cells Mixed with Platelet Rich Plasma in the Healing of Inflammatory Bowel Anastomoses: A Pre-Clinical Study in Rats

Abstract Aim Anastomotic leak is a detrimental complication following bowel surgery leading to prolonged hospital stay, increased healthcare costs and significant increase in morbidity and mortality. The aim of this study is to investigate the effect of adipose derived mesenchymal stem cells (MSCs) mixed with platelet rich plasma (PRP) in the healing of bowel anastomoses, in an inflammatory environment . Method Thirty-five male Wistar rats were divided into five groups of seven animals: normal controls, colitis controls, PRP, MSCs, and PRP + MSCs. All groups underwent laparotomy, segmental colectomy and anastomosis in situ. Colitis was previously established by transrectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) except for the normal controls. Post-mortem histopathological, tissue hydroxyproline and anastomotic bursting pressure (ABP) assessments were performed. Results Tissue hydroxyproline and ABP were significantly increased in the group of PRP+MSCs compared to colitis controls (p=0.0151 and p=0.0104, respectively). Inflammatory cell infiltration was lower and fibroblast activity higher in PRP + MSCs group, but not statistically significant. Neoangiogenesis (p=0.0073) and anastomotic area epithelialization (p=0.0182) were significantly higher in PRP + MSCs group compared to colitis controls. Conclusions The synergic effect of the PRP and MSCs is shown to significantly improve healing in bowel anastomoses even on a background of inflammation. These results give hope for primary anastomosis and potentially avoiding stoma and its complications, especially in immunocompromised or malnourished patients undergoing bowel resection. Clinical studies should follow in order to support the clinical application of PRP and MSCs combination in gastrointestinal anastomoses.

Single-cell transcriptomics reveals the intra-tumoral heterogeneity and SQSTM1/P62 and Wnt/β-catenin mediated epithelial to mesenchymal transition and stemness of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is characterized by the complex tumor microenvironment (TME) consisting of an abundance of mesenchymal stem cells (MSCs), which is known to facilitate epithelial-to-mesenchymal transition (EMT). The development of single-cell genomics is a powerful method for defining the intricate genetic landscapes of malignancies. In this study, we have employed single-cell RNA sequencing (scRNA-seq) to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare consequential cell subpopulations of significance. The scRNA-seq analysis of TNBC and Normal patient derived samples revealed that EMT markers and transcription factors were most upregulated in MSC population. Further, exploration of gene expression analysis among TNBC and Normal patient-derived MSCs ascertained the role of SQSTM1/P62 and Wnt/β-catenin in TNBC progression. Wnt/β-catenin and Wnt/PCP signaling pathways are prominent contributors of EMT, stemness, and cancer stem cell (CSC) properties of TNBC. SQSTM1/P62 cooperates with the components of the Wnt/PCP signaling pathway and is critically involved at the interface of autophagy and EMT.Moreover, siRNA targeting SQSTM1/P62 and inhibitor of Wnt/β-catenin (FH535) in conjunction was used to explore molecular modification of EMT and stemness markers. Although SQSTM1/P62 is not crucial for cell survival, cytotoxicity assay revealed synergistic interaction between the siRNA/inhibitor. Modulation of these important pathways helped in reduction of expression of genes and proteins contributing to CSC properties. Gene and protein expression analysis revealed the induction of EMT to MET. Moreover, co-treatment resulted in inactivation of non-canonical Wnt VANGL2-JNK signaling axis. The synergistic impact of inhibition of SQSTM1/P62 and Wnt/β-catenin signaling facilitates the development of a potential therapeutic regimen for TNBC.

Tumor-derived mesenchymal progenitor cell-related genes in the regulation of breast cancer proliferation.

Breast cancer (BC) is one of the most common malignancies worldwide, and its development is affected in various ways by the tumor microenvironment (TME). Tumor-derived mesenchymal progenitor cells (MPCs), as the most important components of the TME, participate in the proliferation and metastasis of BC in several ways. In this study, we aimed to characterize the genes associated with tumor-derived MPCs and determine their effects on BC cells. Tumor-derived MPCs and normal breast tissue-derived mesenchymal stem cells (MSCs) were isolated from tissues specimens of patients with BC. We conducted culture and passage, phenotype identification, proliferation and migration detection, inflammatory factor release detection, and other experiments on isolated MPCs from tumors and MSCs from normal breast tissues. Three paired tumor-derived MPCs and normal breast tissue-derived MSCs were then subjected to transcriptome analysis to determine the expression profiles of the relevant genes, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to further confirm gene expression. Subsequently, the overexpression plasmids were transfected into tumor-derived MPCs, and the expression of various inflammatory factors of tumor-derived MPCs and their proliferation were characterized with a cell viability test reagent (Cell Counting Kit 8). Subsequently, the transfected tumor-derived MPCs were cocultured with BC cells using a conditioned medium coculture method to clarify the role of tumor-derived MSCs in BC. Tumor-derived MPCs expressed stem cell characteristics including CD105, CD90, and CD73 and exhibited adipogenic and osteogenic differentiation in vitro. The proliferation of tumor-derived MPCs was significantly lower than that of normal breast tissue-derived MSCs, and the invasive metastatic ability was comparable; however, MPCs were found to release inflammatory factors such as interleukin 6 (IL-6) and transforming growth factor β (TGF-β). Transcriptome analysis showed that stomatin (STOM), collagen and calcium binding EGF domains 1 (CCBE1), and laminin subunit alpha 5 (LAMA5) were significantly upregulated in tumor-derived MPCs. Among them, STOM was highly expressed in tumor-derived MPCs, which mediated the slow proliferation of MPCs and promoted the proliferation of BC cells. STOM, CCBE1, and LAMA5 were highly expressed in tumor-derived MPCs, with STOM being found to retard the proliferation of MPCs but promote the proliferation of BC cells. There findings present new possibilities in targeted microenvironmental therapy for BC.

Selective cargo sorting in stem cell-derived small extracellular vesicles: impact on therapeutic efficacy for intervertebral disc degeneration.

Growing evidence has suggested the role of stem cell-derived small extracellular vesicles (sEVs) in intervertebral disc degeneration (IVDD). The cargo sorting of sEVs, particularly miRNAs, may be influenced when the donor cell is subjected to oxidative stress. Here, we discovered that miRNAs containing specific motifs are selectively sorted into intraluminal vesicles within mesenchymal stem cells (MSCs) in response to oxidative stress. Analysis of miRNA cargoes in sEVs derived from normal MSCs (C-sEVs) or stressed MSCs (T-sEVs) was conducted using miRNA sequencing. Differential expressed miRNAs in sEVs and the identification of motifs were evaluated through bioinformatics analysis. Protein binding was assessed using immunofluorescent staining and immunoprecipitation analysis. Additionally, RNA pull down and RNA immunoprecipitation (RIP) immunoprecipitation were employed to determine the binding between miRNAs and proteins. The effects of C-sEVs and T-sEVs on IVDD were compared by detecting the expression levels of phenotypic genes in vitro or histological evaluation in vivo. The sorting process of miRNAs is mediated by the nucleocytoplasmic transport of heterogeneous nuclear ribonucleoproteins, which in turn facilitates the phosphorylation of SNAP25 and promotes the transport and secretion of sEVs. Additionally, CHMP1B plays a role in membrane repair and protects against cell ferroptosis upon oxidative stress, concurrently affecting the release of sEVs. Notably, stem cell-derived sEVs associated with ferroptosis impair the therapeutic efficacy for IVDD. However, the application of engineered sEVs containing a specific miRNA inhibitor exhibits the potential to reinstate the therapeutic efficacy for IVDD both in vitro and in vivo. Taken together, our findings shed light on the mechanism of miRNAs sorting into sEVs and offer new insights for the optimization of sEV-based treatments during intervertebral disc regeneration. regeneration.

IGF2BP2-meidated m6A modification of CSF2 reprograms MSC to promote gastric cancer progression

The interaction between tumor cells and stromal cells within the tumor microenvironment plays a critical role in cancer progression. Mesenchymal stem cells (MSCs) are important tumor stromal cells that exhibit pro-oncogenic activities when reprogrammed by the tumor. However, the precise mechanisms underlying MSC reprogramming in gastric cancer remain not well understood. QRT-PCR, western blot, and immunohistochemistry were used to examine gene and protein expression levels. In vitro and in vivo experiments were conducted to assess the biological functions of gastric cancer cells. RNA-sequencing, RNA immunoprecipitation (RIP), and meRIP assays were performed to investigate underlying molecular mechanisms. We found a significant increase in the expression and N6-methyladenosine (m6A) modification levels of colony-stimulating factor 2 (CSF2) in gastric cancer MSCs. CSF2 gene overexpression induced the reprogramming of normal MSCs into cancer-promoting MSCs, thereby enhancing the proliferation, migration, and drug resistance of gastric cancer cells through the secretion of various pro-inflammatory factors. Additionally, we demonstrated that the m6A reader IGF2BP2 bound to and stabilized CSF2 mRNA in gastric cancer MSCs. Notably, overexpression of IGF2BP2 mimicked the effect of CSF2 on MSCs, promoting gastric cancer progression. Finally, we unveiled that CSF2 induced the ubiquitination of Notch1 to reprogram MSCs. Our study highlights a critical role of IGF2BP2-mediated m6A modification of CSF2 in reprogramming MSCs, which presents a promising therapeutic target for gastric cancer.

The effect of bovine milk lactoferrin-loaded exosomes (exoLF) on human MDA-MB-231 breast cancer cell line

BackgroundCancer is still the most challenging disease and is responsible for many deaths worldwide. Considerable research now focuses on targeted therapy in cancer using natural components to improve anti-tumor efficacy and reduce unfavorable effects. Lactoferrin is an iron-binding glycoprotein found in body fluids. Increasing evidence suggests that lactoferrin is a safe agent capable of inducing anti-cancer effects. Therefore, we conducted a study to evaluate the effects of the exosomal form of bovine milk lactoferrin on a human MDA-MB-231 breast cancer cell line.MethodsThe exosomes were isolated from cancer cells by ultracentrifugation and incorporated with bovine milk lactoferrin through the incubation method. The average size of the purified exosome was determined using SEM imaging and DLS analysis. The maximum percentage of lactoferrin-loaded exosomes (exoLF) was achieved by incubating 1 mg/ml of lactoferrin with 30 µg/ml of MDA-MB-231 cells-derived exosomes. Following treatment of MDA-MB-231 cancer cells and normal cells with 1 mg/ml exoLF MTT assay applied to evaluate the cytotoxicity, PI/ annexin V analysis was carried out to illustrate the apoptotic phenotype, and the real-time PCR was performed to assess the pro-apoptotic protein, Bid, and anti-apoptotic protein, Bcl-2.ResultsThe average size of the purified exosome was about 100 nm. The maximum lactoferrin loading efficiency of exoLF was 29.72%. MTT assay showed that although the 1 mg/ml exoLF treatment of MDA-MB-231 cancer cells induced 50% cell growth inhibition, normal mesenchymal stem cells remained viable. PI/ annexin V analysis revealed that 34% of cancer cells had late apoptotic phenotype after treatment. The real-time PCR showed an elevated expression of pro-apoptotic protein Bid and diminished anti-apoptotic protein Bcl-2 following exoLF treatment.ConclusionThese results suggested that exoLF could induce selective cytotoxicity against cancer cells compared to normal cells. Incorporating lactoferrin into the exosome seems an effective agent for cancer therapy. However, further studies are required to evaluate anti-tumor efficacy and the underlying mechanism of exoLF in various cancer cell lines and animal models.

302 Super Mitochondria, the Next Super Therapy to Reverse Ischemic Damage in Stroke?

INTRODUCTION: Stroke represents a massive healthcare emergency, ranking in the top 5 causes of death and disability and impacting nearly 800,000 Americans annually. Current therapies are limited. A deeper analysis of the mechanisms of cell death after ischemic brain insult may usher novel safe and effective treatments. Mitochondrial damage serves as a key modulator of ischemic cell death, which may be reversible with transfer of healthy mitochondria from mesenchymal stem cells (MSCs) to ischemic neurons. METHODS: MSCs were grown under ambient condition (normal MSCs) (nMSCs) or metabolic switching paradigm involving exchanging galactose for glucose in the cell culture medium (switched MSCs) (sMSCs) then assayed for oxygen consumption rates using the Seahorse technology. Subsequently, primary neurons were subjected to OGD then co-cultured with either nMSCs or sMSCs, and assessed for cell viability, cell metabolism, mitochondrial reactive oxidative species mRNA, and mitochondrial ATP mRNA. RESULTS: Compared to nMSCs, sMSCs displayed robust mitochondrial respiration as evidenced by higher basal energy production, larger spare respiratory capacity, greater ATP production, and decreased proton (H+) leak. Co-culture of OGD-exposed neurons with sMSCs conferred increased cell viability and enhanced cell metabolism, coupled with reduced mitochondrial reactive oxidative species mRNA and elevated mitochondrial ATP mRNA, which were significantly better than those afforded by nMSCs. CONCLUSIONS: These findings suggest sMSCs generated “super” mitochondria. Metabolic switching produces healthy mitochondria that may enhance the mitochondrial transfer between MSCs and ischemic cells warranting consideration as a stroke therapeutic.

Mesenchymal stem cells protect against sepsis-associated acute kidney injury by inducing Gal-9/Tim-3 to remodel immune homeostasis

Objective The present study investigated the specific mechanism by which mesenchymal stem cells (MSCs) protect against sepsis-associated acute kidney injury (SA-AKI). Methods Male C57BL/6 mice underwent cecal ligation and puncture surgery to induce sepsis and then received either normal IgG or MSCs (1 × 106 cells, intravenously) plus Gal-9 or soluble Tim-3 3 h after surgery. Results After cecal ligation and puncture surgery, the mice injected with Gal-9 or MSCs plus Gal-9 had a higher survival rate than the mice in the IgG treatment group. Treatment with MSCs plus Gal-9 decreased serum creatinine and blood urea nitrogen levels, improved tubular function recovery, reduced IL-17 and RORγt levels and induced IL-10 and FOXP3 expression. Additionally, the Th17/Treg cell balance was altered. However, when soluble Tim-3 was used to block the Gal-9/Tim-3 pathway, the septic mice developed kidney injury and exhibited increased mortality. Treatment with MSCs plus soluble Tim-3 blunted the therapeutic effect of MSCs, inhibited the induction of Tregs, and suppressed the inhibition of differentiation into Th17 cells. Conclusion Treatment with MSCs significantly reversed the Th1/Th2 balance. Thus, the Gal-9/Tim-3 pathway may be an important mechanism of MSC-mediated protection against SA-AKI.

The Antioxidant and Proapoptotic Effects of Sternbergia clusiana Bulb Ethanolic Extract on Triple-Negative and Estrogen-Dependent Breast Cancer Cells In Vitro.

Sternbergia clusiana belongs to the Amaryllidaceae family and is recognized for the valuable biological activity of its major bioactive compounds. The aim of the current is to evaluate the anticancer effects of the ethanolic bulb extract of Sternbergia clusiana (ScBEE) on breast cancer cells in vitro and to further reveal the underlying cellular mechanism. An MTS cell viability assay was performed on MDA-MB-231 and MCF-7 cells, along with cell cycle analysis, cell death ELISA, Western blot analysis and an ROS production assay to decipher the mechanism of death. LC-MS/MS was also performed to identify the chemical composition of this ethanolic extract. The results show a selective antiproliferative effect on both cell lines with no effect on normal mesenchymal stem cells. Further analysis suggested the activation of the apoptotic pathway as reflected by the increase in cellular and DNA fragmentation and alterations in apoptotic proteins such as Bax, Bcl-2 and c-PARP. ScBEE was also found to exhibit antioxidant effect, as shown by a decrease in ROS production. The underlying mechanism of action was explained by the presence of several bioactive compounds identified by LC-MS/MS, including alkaloids, terpenoids and phenols, which are elaborated in the manuscript. This study highlights the antioxidant and anticancerous properties of S.clusiana for breast cancer treatment.

MiR-30a inhibits the osteogenic differentiation of the tibia-derived MSCs in congenital pseudarthrosis via targeting HOXD8.

BackgroundCongenital pseudarthrosis of the tibia (CPT) is an uncommon congenital deformity and a special subtype of bone nonunion. The lower ability of osteogenic differentiation in CPT-derived mesenchymal stem cells (MSCs) could result in progression of CPT, and miR-30a could inhibit osteogenic differentiation. However, the role of miR-30a in CPT-derived MSCs remains unclear.MethodsThe osteogenic differentiation of CPT-derived MSCs treated with the miR-30a inhibitor was tested by Alizarin Red S staining and alkaline phosphatase (ALP) activity. The expression levels of protein and mRNA were assessed by Western blot or quantitative reverse transcription-polymerase chain reaction (RT-qPCR), respectively. The interplay between miR-30a and HOXD8 was investigated by a dual-luciferase reporter assay. Chromatin immunoprecipitation (ChIP) was conducted to assess the binding relationship between HOXD8 and RUNX2 promoter.ResultsCPT-derived MSCs showed a lower ability of osteogenic differentiation than normal MSCs. miR-30a increased in CPT-derived MSCs, and miR-30a downregulation promoted the osteogenic differentiation of CPT-derived MSCs. Meanwhile, HOXD8 is a direct target for miR-30a, and HOXD8 could transcriptionally activate RUNX2. In addition, miR-30a could inhibit the osteogenic differentiation of CPT-derived MSCs by negatively regulating HOXD8.ConclusionmiR-30a inhibits the osteogenic differentiation of CPT-derived MSCs by targeting HOXD8. Thus, this study might supply a novel strategy against CPT.

The effects of Twinlight laser treatment on the titanium surface proliferation and osteogenic differentiation of mesenchymal stem cells

BackgroundThe study aimed to observe the effects of a Twinlight laser on the titanium surface proliferation of inflammatory Mesenchymal stem cells (MSCs), inflammatory cytokine expression, and osteogenic differentiation.MethodsThe MSCs were collected from bone tissue of healthy individuals.The cellular inflammatory model was established with 1 μg/mL lipopolysaccharide (LPS).Under the cellular inflammatory model,divided into five groups: the normal control group (C); the inflammatory control group (L); Er:YAG laser group (L + E); Nd:YAG laser group (L + N); Er:YAG laser and Nd:YAG laser group (L + E + N). The treated cells were inoculated onto titanium disks.The normal and inflammatory MSCs on the surface of titanium surface were examined by CCK-8, scanning election microscopy (SEM), quantitative real-time polymerase chain reaction (qRT‑PCR) and other methods for their proliferation, growth pattern, expression of inflammatory factors Interleukin-6 (IL-6), Interleukin-8 (IL-8) and osteogenic genes Runx2 (Runt-related transcription factor 2) and alkaline phosphatase (ALP), providing the theoretical basis and experimental data for the Twinlight laser-assisted treatment of peri-implantitis. Statistical analyses were performed using a Student's t test with SPSS 17.0 software.ResultsThrough observation using SEM, the cell densities of the L + E + N, L + E, and L + N groups were similar, but cell bodies in the L + E + N group were fuller and each had more than two pseudopodia. The expression level of IL-6 mRNA in the L, L + N, L + E, and L + E + N groups was higher than in group C (P < 0.05), and the expression level of IL-8 mRNA in the L + E + N group was significantly lower than in group L (P < 0.0001). On day 7, the expression level of ALP mRNA in the L, L + N, L + E, and L + E + N groups was lower than in group C (P < 0.05). On day 14, there was no significant difference in the expression level of ALP mRNA among the L + N, L + E + N, and C groups (P > 0.05). On day 7, the expression level of RUNX2 mRNA in the L + E + N group was higher than in group L (P < 0.001). On day 14, the expression level of RUNX2 mRNA in the L + E + N group was higher than in group L (P < 0.01).ConclusionTwinlight laser treatment promoted cell proliferation, inhibited the expression of inflammatory cytokines, and effectively enhanced the osteogenic differentiation of cells on a titanium surface.

Enhancing oxidative phosphorylation over glycolysis for energy production in cultured mesenchymal stem cells.

ObjectiveStrokes represent as one of the leading causes of death and disability in the USA, however, there is no optimal treatment to reduce the occurrence or improve prognosis. Preconditioning of tissues triggers ischemic tolerance, a physiological state that may involve a metabolic switch (i.e. from glycolysis to oxidative phosphorylation or OxPhos) to preserve tissue viability under an ischemic insult. Here, we hypothesized that metabolic switching of energy source from glucose to galactose in cultured mesenchymal stem cells (MSCs) stands as an effective OxPhos-enhancing strategy.MethodsMSCs were grown under ambient condition (normal MSCs) or metabolic switching paradigm (switched MSCs) and then assayed for oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) using the Seahorse technology to assess mitochondrial respiration.ResultsNormal MSCs showed a lower OCR/ECAR ratio than switched MSCs at baseline (P < 0.0001), signifying that there were greater levels of OxPhos compared to glycolysis in switched MSCs. By modulating the mitochondrial metabolism with oligomycin (time points 4–6), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (7–9), and rotenone and antimycin (time points 10–12), switched MSCs greater reliance on OxPhos was further elucidated (time points 5–12; P < 0.0001; time point 4; P < 0.001).ConclusionThe metabolic switch from glycolytic to oxidative metabolism amplifies the OxPhos potential of MSCs, which may allow these cells to afford more robust therapeutic effects against neurological disorders that benefit from ischemic tolerance.

Normal mesenchymal stem cells can improve the abnormal function of T cells in psoriasis via upregulating transforming growth factor-β receptor.

Psoriasis, a chronic inflammatory skin disease, is a refractory disorder. Previous studies have shown that the imbalance of the T-helper (Th)17/regulatory T cells (Treg) results in the immune imbalance of T cells in psoriatic patients, and that mesenchymal stem cells display an immunosuppressive role by promoting the differentiation of T cells into Treg, leading to a reduction in the proportion of Th17/Treg. Utility of mesenchymal stem cells is becoming a new approach for the treatment of immune disorders. Following co-culture of dermal mesenchymal stromal cells (DMSC) and CD3+ T cells with or without transforming growth factor (TGF)-β receptor inhibitor, the biological function and relative signal pathway of CD3+ T cells were assessed by flow cytometry, transwell, real-time polymerase chain reaction and western blotting, respectively. Normal DMSC were more potent than psoriatic DMSC in inhibition of CD3+ T-cell proliferation, and stimulation of CD3+ T-cell apoptosis than psoriasis DMSC. Moreover, normal DMSC decreased the ratio of Th17/Treg, while enhancing the immunosuppressive effect of Tregs on effector T cells. However, TGF-β receptor (TGF-βR)inhibitor attenuated the effect of normal DMSC on CD3+ T cells and Th17/Treg ratio. Additionally, the normal DMSC were more potent than the psoriatic DMSC in increasing TGF-β receptors and activation of TGF-β/SMAD pathway in psoriatic CD3+ T cells. In conclusion, normal DMSC can partially improve the biological function and immunosuppressive ability of psoriatic CD3+ T cells, possibly via upregulating the TGF-β receptors.

Therapeutic Effects and Repair Mechanism of HGF Gene-Transfected Mesenchymal Stem Cells on Injured Endometrium.

There are many studies on the advantages of using mesenchymal stem cells (MSCs) that secrete various paracrine factors for repairing endometrial injury. However, the stability and effectiveness of MSCs require improvement to become a viable therapy. Hepatocyte growth factor (HGF), one of the cytokines secreted by MSCs, promotes vascular repair and mesenchymal to epithelial transformation (MET). Therefore, HGF likely promotes the repair process of the endometrium. We prepared MSCs transfected with the HGF gene to explore its repair effects and mechanism using a damaged endometrium mouse model. HGF gene-transfected MSCs were prepared by electroporation. The transfected MSCs retained their cellular characteristics and significantly increased the expression of HGF (P < 0.01). HGF gene-transfected MSCs helped damaged endometrium to recover its morphological characteristics, improved proliferation and decreased apoptosis of endometrial cells, increased the expression of endometrial vascular growth-related factors, and activated phosphorylated c-Met and AKT in the mouse endometrial damage model (P < 0.05). Compared with normal MSCs, HGF gene-transfected MSCs produced a more significant effect on damaged endometrial epithelium repair by activating the HGF/c-Met and downstream signaling pathways. Our results indicate that HGF gene-transfected MSCs provide an effective and promising tool for injured endometrium therapy.

AML-108: Exogenous Mitochondrial Transfer and Endogenous Mitochondrial Fission Facilitate Resistance to OxPhos Inhibition in AML

<h3>Context:</h3> Oxidative phosphorylation (OxPhos) inhibition induces mitochondrial trafficking from BM stromal cells to AML and is accompanied by mitochondrial fission and mitophagy. The mitochondrial coupling and crosstalk with BM stromal cells facilitate the development of resistance to OxPhos inhibition in AML cells. <h3>Objective:</h3> Acute myeloid leukemia (AML) cells are highly dependent on OxPhos for survival and continually adapt to fluctuations in nutrient and oxygen availability in the environment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. <h3>Design:</h3> Primary AML cells from 26 patients, primary normal MSCs, and AML cell lines were utilized and treated with the OxPhos inhibitor IACS-0107598 and/or cytarabine. Cap analysis of gene expression transcriptome analyses for AML PDXs, primary samples, and AML cell lines has been performed. We performed an extracellular flux assay to measure the oxygen consumption rate. To visualize mitochondria, AML cell lines and MSCs were stably transfected with mitochondria-targeted PDHA1-GFP and -dsRed, respectively. Electron microscopy and immunoelectron microscopy analysis have been performed. <h3>Results:</h3> Cellular adhesion, growth, and apoptosis assays, along with measurements of mtDNA expression and mitochondrial reactive oxygen species generation, indicated that direct interactions with BM stromal cells triggered the compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, OxPhos inhibition induced (1) transfer of mesenchymal stem cell (MSC)-derived mitochondria to AML cells via tunneling nanotubes under direct-contact co-culture conditions and (2) mitochondrial fission with an increase in functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, and we observed mitochondrial transport to the leading edge of protrusions of migrating AML cells toward MSCs by electron microscopy analysis. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased IACS-010759-triggered mitochondrial transfer from MSCs to AML cells. <h3>Conclusions:</h3> Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells and of endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.

Leukemia-Induced Cellular Senescence and Stemness Alterations in Mesenchymal Stem Cells Are Reversible upon Withdrawal of B-Cell Acute Lymphoblastic Leukemia Cells.

Leukemic cell growth in the bone marrow (BM) induces a very stressful condition. Mesenchymal stem cells (MSC), a key component of this BM niche, are affected in several ways with unfavorable consequences on hematopoietic stem cells favoring leukemic cells. These alterations in MSC during B-cell acute lymphoblastic leukemia (B-ALL) have not been fully studied. In this work, we have compared the modifications that occur in an in vitro leukemic niche (LN) with those observed in MSC isolated from B-ALL patients. MSC in this LN niche showed features of a senescence process, i.e., altered morphology, increased senescence-associated β-Galactosidase (SA-βGAL) activity, and upregulation of p53 and p21 (without p16 expression), cell-cycle arrest, reduced clonogenicity, and some moderated changes in stemness properties. Importantly, almost all of these features were found in MSC isolated from B-ALL patients. These alterations rendered B-ALL cells susceptible to the chemotherapeutic agent dexamethasone. The senescent process seems to be transient since when leukemic cells are removed, normal MSC morphology is re-established, SA-βGAL expression is diminished, and MSC are capable of re-entering cell cycle. In addition, few cells showed low γH2AX phosphorylation that was reduced to basal levels upon cultivation. The reversibility of the senescent process in MSC must impinge important biological and clinical significance depending on cell interactions in the bone marrow at different stages of disease progression in B-ALL.

Abstract 1016: A new biomarker in hematological malignancies

Abstract Background: Extracellular vesicles (EVs) are biologically active nanoparticles. EVs from explant prostate cancer induce a neoplastic phenotype in normal prostate cell lines. We have also shown EVs from mesenchymal stem cells (MSC) can have a healing effect, reversing the malignant phenotype in prostate and colorectal cancer; as well as mitigating radiation damage to marrow. The role of EVs in leukemia and its microenvironment remains to be studied, and may provide insight for therapeutic advances. We hypothesize that EVs derived from normal MSC can have a healing effect, inhibiting the growth of myelogenous leukemia. Methods: Kasumi AML cells lines were seeded in a 96 well plate with various concentrations of MSC-derived EVs. Vesicles were isolated usingan established differential centrifugation technique, and were co-cultured with Kasumi. To study cellular proliferation we measured fluorescence after 60 min. Fluorescence intensities were normalized to control wells containing non-EV treated cells alone. Results: Proliferation of AML cells after one day of co-culture with MSC-EVs respectively was inhibited in a dose dependent manner. Three days of co-culture with similar doses resulted in ~40% and ~80%reduction in proliferation when normalized to control. At day 6 of co-culture growth was inhibited by ~80% at both EV concentrations when normalized to control. Summary/Conclusion: MSC-derived EVs inhibits the growth of the AMLcell line in vitro. This effect is seen as early as 1 day of co-culture andpersists out to 3, and 6 days implicating an miRNA-mediated mechanismthat has been discussed in previous works. We feel this is perhaps a modelof how a normal marrow works to suppress early cancer.As leukemia develops the cross-talk between AML and its microenvironmentalters the MSCs to promote a survival signal favouring AMLgrowth. Future work involves the capacity of AML-derived EVs to alterthe phenotype of normal marrow towards a pro-leuekmic phenotype.Employing mathematical models to quantify and ultimately predict thesechanges allows for precise therapeutic intervention. Citation Format: Theo Borgovan. A new biomarker in hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1016.

Single-Cell Analysis of Mesenchymal Stem Cells Reveals Their Molecular and Functional Heterogeneity in Myeloma

IntroductionBone marrow (BM) mesenchymal stem cells (MSCs) are rare but heterogeneous cell populations, and both MSCs and their lineages mediate BM niches, hematopoiesis, and myeloma (MM) growth.The aim of the study was to assess variation in gene expression of unexpanded single MSCs generated from the same source, and study the heterogeneous effect on MM cell growth of MSCs lines generated from single MSCs or normal MSCs primed by MM cells. We also studied changes in MSCs during MM progression and identified MSC factors that mediate MM growth.MethodsCells collected from bone pieces following enzymatic digestion were cultured for 2-4 days. The adherent cells were trypsinized, replated to separate MSCs from macrophages and allowed to adhere overnight. Cells were then trypsinized and single MSCs were sorted into 96-well plates. Validation of single cell sorting and viability was assessed microscopically by staining for CFSE.Single MSCs were generated from two normal samples and two MM patients. Sorted single cells were subjected to qRT-PCR using primers of 34 genes implicated in MSC biology (e.g. CD73), cell cycle (e.g. KI67, CDKN2A), differentiation (e.g. RUNX2, PPARG, CYR61), transcription (e.g. FOXC1), and specific MSC genes we identified to be top overexpressed (e.g. POSTN) or underexpressed (e.g. IGFBP2, LEPR) in bone biopsy samples from high-risk MM patients. Also included were CD45 and CD34 to exclude hematopoietic cells, and GAPDH and ACTB as housekeeping genes. After excluding CD45+ cells and cells with low expression of housekeeping genes a total of 111 and 104 single MSCs from normal and MM subjects, respectively, were analyzed. Single MSCs expressed typical MSC markers such as CD73, CD90, FN1 and FAP.For generating MSC lines from the same source, single MSCs from each source were subjected to long-term culture supplemented with CM from whole BM and MSC cultures.For preparing primed MSCs, normal MSCs generated by standard method were cultured alones (unprimed MSCs) or cocultured with MM cells for 5 days (primed MSCs).The cultured and co-cultured cells were trypsinized, replated for 40 min followed by serial washing that attain >95% purified adherent MSCs. Primed and unprimed MSCs were subjected to gene expression profiling (GEP) and their CM used for proteomics and growth assays.ResultsSingle MSCs from the same source (i.e. normal and MM bones) heterogeneously expressed genes related to cell cycle, differentiation, and extracellular matrix. MM single MSCs had lower expression of markers of proliferation (e.g. KI67, p<0.005) and differentiation (e.g. RUNX2, p<0.03), and higher expression of the senescence marker CDKN2A (p<0.001).To further capture MSC heterogeneity, MSCs lines were generated from single MSCs and tested for their ability to support MM cells. Growth of individual MSCs varied in culture, most had a limited proliferation rate, and few were capable of generating MSC lines. MSC lines generated from the same source (4-10 lines from normal or MM subjects) had variable effects on growth of MM cells in co-cultures, or when their CM used on MM cell grown alone (p<0.0008 between lowest to highest growth stimulating lines).CM from primed MSCs stimulated the growth of primary MM cells compared to CM from unprimed MSCs in short-term culture (n=10, p<0.007) or 10-days co-couture with normal MSCs (n=6, p<0.002).Primed MM cells had reduced expression and secretion of factors related to the IGF1 pathways including IGFBP2, IGFBP3 and IGF2, whereas IGF1 was not impacted. Recombinant IGF1 promoted growth of BM-dependent MM cells in serum-free conditions, an effect that was significantly blocked by recombinant IGFBP2 (p<0.0003).IGFBP2 expression by GEP was lower in biopsy samples from MM patients (n=531) than normal subjects (n=68), lower in samples from high-risk than low-risk patients, and lower in focal lesion than random BM biopsies from the same patients (n=77). Immunohistochemistry showed high IGFBP2 protein expression by BM mesenchymal cells, particularly, small adipocytes, and changes in the proportion of IGFBP2+ cells correlated with the reduced gene expression in MM patients' biopsies, and with disease stage.ConclusionsThese findings suggest that MSCs from normal subjects and MM patients are heterogeneous population of cells, are deregulated as part of MM disease progression, and that production of factors such as IGFBP2 by these cells impacts bioavailability of IGF1. DisclosuresDavies:Bristol-Myers: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers: Consultancy, Honoraria.

Geranylgeraniol prevents zoledronic acid-mediated reduction of viable mesenchymal stem cells via induction of Rho-dependent YAP activation.

Long-term use of zoledronic acid (ZA) increases the risk of medication-related osteonecrosis of the jaw (MRONJ). This may be attributed to ZA-mediated reduction of viable mesenchymal stem cells (MSCs). ZA inhibits protein geranylgeranylation, thus suppressing cell viability and proliferation. Geranylgeraniol (GGOH), which is a naturally found intermediate compound in the mevalonate pathway, has positive effects against ZA. However, precise mechanisms by which GGOH may help preserve stem cell viability against ZA are not fully understood. The objective of this study was to investigate the cytoprotective mechanisms of GGOH against ZA. The results showed that while ZA dramatically decreased the number of viable MSCs, GGOH prevented this negative effect. GGOH-rescued ZA-exposed MSCs formed mineralization comparable to that produced by normal MSCs. Mechanistically, GGOH preserved the number of viable MSCs by its reversal of ZA-mediated Ki67+ MSC number reduction, cell cycle arrest and apoptosis. Moreover, GGOH prevented ZA-suppressed RhoA activity and YAP activation. The results also established the involvement of Rho-dependent YAP and YAP-mediated CDK6 in the cytoprotective ability of GGOH against ZA. In conclusion, GGOH preserves a pool of viable MSCs with osteogenic potency against ZA by rescuing the activity of Rho-dependent YAP activation, suggesting GGOH as a promising agent and YAP as a potential therapeutic target for MRONJ.

Senescence‐Associated Exosome Exchange Activates Myofibroblast Phenotype in Mesenchymal Stem Cells

Introduction Bone marrow derived mesenchymal stem cells (MSCs) are recruited and accumulate in primary tumors due to their natural tropism for inflammatory tissues. MSCs can undergo senescence as a result of ionizing radiation therapy. Senescent cells are often resistant to apoptosis and rapidly develop a senescence-associated secretory phenotype (SASP), which is linked to increased secretion of pro-inflammatory cytokines and matrix degradation enzymes. The potency of inflammatory SASP factors is widely accepted in many tumor models. Senescence also affects the number and content of secreted exosomes. Exosomes are nanovesicles that can transfer important protein, RNA, and miRNA during cell-cell communication. Previous studies have also shown that senescence-associated exosomes (SA-EXOs) can serve as key mediators of disease progression. The objective of this study is to understand how radiation-induced senescent cells and SA-EXOs induce myofibroblast phenotypes in neighboring stromal cells. We hypothesize that SA-EXOs play critical roles in cell-cell communication to promote persistent fibroblast activation that drives local matrix remodeling for subsequent tumor metastasis. Methods MSCs from female donors were treated with gamma-irradiation of 15Gy and senescence was confirmed 10 days later based on positive staining for β-Galactosidase and increased gene expression of senescent markers (Fig 1a-b). All exosome populations were harvested using standard ultracentrifugation methods. miRNA microarrays were carried out according to the manufacturer's published protocol. Results Our proteomics data showed that proteins crucial in exosome secretion and biogenesis pathways were elevated in senescent MSCs compared to normal MSCs. This was further validated using a gene expression panel that probed for markers of the same pathways (Fig 2a-b). Dynamic Light Scattering techniques revealed that senescent MSCs secreted 2-fold more exosomes than normal MSCs (Fig 3). Exosome miRNA microarrays showed 27 differentially expressed miRNAs (non-SA-EXOs vs. SA-EXOs)—22 upregulated and 5 downregulated. Many of the miRNAs were involved in mechanosensitive pathways. Finally, we treated normal MSCs with SA-EXOs and stained for alpha smooth muscle actin (α-SMA) and ki-67. α-SMA is a protein that is elevated in senescence and myofibroblasts; ki-67 is a proliferative marker. SA-EXOs increased α-SMA and ki-67 expression in normal MSCs (Fig 4) compared to control groups. Discussion/Conclusions We showed that radiation-induced senescent MSCs secreted elevated amounts of SA-EXOs that possess unique miRNA content and that SA-EXO exchange with neighboring MSCs increased α-SMA. This highlights that these exosomes are important SASP factors that can induce myofibroblast phenotypes, which can play a large role in promoting tumor progression.