Co-culture Of Mesenchymal Stem Cells Research Articles

Mesenchymal Stem Cells from the Deciduous Tooth Pulp Lose their Ability to Suppress the Differentiation of Dendritic Cells during Long-Term Culturing.

A culture of cells expressing markers of mesenchymal stem cells (MSC) (CD73, CD90, CD44, CD29, and CD49b), but not hematopoietic cell markers, and capable of multilineage differentiation was isolated from the deciduous tooth pulp. Co-culturing with immature dendritic cells in the presence of LPS did not reveal an ability of the MSC to suppress the maturation of dendritic cells. On the contrary, co-culturing of MSC with monocytes in the presence of granulocyte-macrophage CSF and IL-4 led to complete suppression of monocyte differentiation into dendritic cells. However, long-term culturing of MSC from dental pulp showed that by the passage 11, they almost completely lose their suppressor ability. These results indicate that the immunological properties of MSC can change during culturing without changing their phenotypic markers. This should be taken into account when creating biomedical cell products.

An injectable anti-vascularization functionalized hydrogel for degenerative nucleus pulposus repair

Neovascularization and inflammatory cell invasion within the nucleus pulposus (NP) constitute pivotal pathological changes during the acceleration stage of intervertebral disc degeneration (IDD). Mesenchymal stem cells (MSCs), renowned for their remarkable capacity in intervertebral disc (IVD) regeneration, also exhibit the capability to secrete pro-angiogenic factors, expediting IDD progression under hypoxic conditions. Consequently, we developed a hydrogel comprised of methacrylated hyaluronic acid (HAMA), rat tail collagen I (COL), and MSCs, incorporating the vascular endothelial growth factor receptor (VEGFR) inhibitor cabozantinib (Cabo@HAMA-COL/MSCs hydrogel). This innovative construct aimed to facilitate NP regeneration while mitigating vascularization and inflammation. Our findings revealed that the hydrogel aptly mimicked the mechanical characteristics of NP tissue, exhibiting injectability, low cytotoxicity, and the preservation of the cellular phenotype of NP cells. Co-culturing of MSCs and human umbilical vein endothelial cells (HUVECs) promoted migration, tube formation, and sprouting of HUVECs, which will be inhibited by cabozantinib. In vivo experiments demonstrated that Cabo@HAMA-COL/MSCs hydrogel maintained disc height, protected NP, and alleviated vascularization and inflammation in a puncture-induced rat caudal IDD model. Consequently, our results substantiate that Cabo@HAMA-COL/MSCs hydrogel can prevent IDD degeneration by ameliorating the vascularization-inflammation pathological microenvironment, offering a promising therapeutic strategy for IDD.

Influential factors for optimizing and strengthening mesenchymal stem cells and hematopoietic stem cells co-culture.

Mesenchymal stem cells (MSCs) and Hematopoietic stem cells (HSCs) are two types of bone marrow stem cells that can proliferate and differentiate into different cell lineages. HSCs interact with MSCs under protective conditions, called niche. Numerous studies have indicated supportive effects of MSCs on HSCs proliferation and differentiation. Furthermore, HSCs have many clinical applications and could treat different hematologic and non-hematologic diseases. For this purpose, there is a need to perform in vitro studies to optimize their expansion. Therefore, various methods including co-culture with MSCs are used to address the limitations of HSCs culture. Some parameters that might be effective for improving the MSC/ HSC co-culture systems. Manipulating culture condition to enhance MSC paracrine activity, scaffolds, hypoxia, culture medium additives, and the use of various MSC sources, have been examined in different studies. In this article, we investigated the potential factors for optimizing HSCs/ MSCs co-culture. It might be helpful to apply a suitable approach for providing high-quality HSCs and improving their therapeutic applications.

Immunological effects of human decidual mesenchymal stem cells in spontaneous and recurrent abortions

The aim of this study was to evaluate the immunological activities of human decidual mesenchymal stem cells (MSCs) on proliferation, apoptosis and percentage of regulatory T cells (Treg) in abortions and to investigate whether these activities differ in spontaneous abortions (SA) and recurrent abortions (RA). This prospective cohort study included women who had a first-trimester abortion between 2019 and 2022. Women with uterine anomaly, endocrinological disease, known autoimmune or thrombophilic disease, and fetal chromosomal abnormality in abortion material were excluded. Decidual MSCs isolated from abortion materials were classified as spontaneous abortion-MSCs (SA-MSCs) and recurrent abortion-MSCs (RA-MSCs). Peripheral blood mononuclear cells were isolated from venous blood and co-cultured with SA-MSCs and RA-MSCs. The effects of MSCs on proliferation and apoptosis of lymphocytes, and Tregs levels were compared between SA-MSCs and RA-MSCs groups. Thirty cases (15 SA-MSCs and 15 RA-MSCs) were included in the study. The presence of MSC in co-cultures increased percentage of Treg cells while reducing proliferation and apoptosis compared to those without MSCs (p < 0.0001, p < 0.0001 and p < 0.0001). The increase in percentage of Treg cells and the reduction in apoptosis were significantly lower in the RA-MSCs group compared to the SA-MSCs group (p < 0.0001 and p < 0.001, respectively). Although the proliferation reducing effect of the presence of MSCs was lower in the RA-MSCs group compared to the SA-MSCs group, the difference was not significant (p = 0.07). MSCs contribute to maternal immunotolerance to semi-allogeneic fetus by suppressing proliferation and apoptosis, and increasing percentage of Treg cells. However, the immunoregulatory effects of MSCs are lower in RA compared to SA.

Layer by layer assembly of strontium substituted nanowire hydroxyapatite and hybrid of carbon derivatives as potential biomaterial for bone metastasis treatment

To devise a highly effective implant for addressing bone metastasis with the unique capability of self-oxygen generation, a multifunctional film was engineered using a Layer-by-Layer technique. The film, comprised of strontium-substituted hydroxyapatite nanowires (SrwHAP) and a graphitic carbon nitride/graphene oxide (g-CN/GO) hybrid, was meticulously designed. To optimize the film's interaction with mesenchymal stem cells (MSCs), the number of layers was adjusted. Subsequent surface coating involved extracellular matrix (ECM) derived from osteogenic cells. The final film, ((CN/GO). SrwHAP)2. ECM, exhibited pronounced MSC attachment and an accelerated proliferation rate. Key osteogenic markers such as alkaline phosphatase activity, mineralization, RUNX2, and osteocalcin expression remarkably increased when compared to the glass substrate. Notably, the presence of LED irradiation yielded further enhancements in proliferation and osteogenic markers, attributed to the photocatalytic properties of the (CN/GO) hybrid alongside O2 generation. Direct co-culturing of MSCs and breast cancer cells (MCF7) on its surface, coupled with LED irradiation not only bolstered the osteogenic potential of MSCs but also reduced tumor colonies over time. Impressively, ((CN/GO). SrwHAP)2. ECM also demonstrated potent antibacterial properties against Gram-positive bacteria (Staphylococcus aureus). Based on the results obtained, ((CN/GO). SrwHAP)2. ECM emerges as a promising candidate for addressing bone defects in patients afflicted by breast cancer bone metastasis.

Overexpression of Nrf2 in Mesenchymal Stem Cells Promotes Migration and Invasion of B- Acute Lymphoblastic Leukemia Cells

Background and objective:B -Acute lymphoblastic leukemia (B-ALL) is one of the most difficult cancers to treat in adults, especially recurrence and drug resistance are still the biggest challenges for B-ALL. In recent years, a new concept has been proposed, that is, the interaction between bone marrow microenvironment and leukemic cells can reduce the sensitivity of leukemic cells to chemotherapy. The components of bone marrow microenvironment (BMM) have been shown to mediate the occurrence and development of leukemia. There is growing evidence that leukemic cells teach the bone marrow microenvironment, change its function and establish leukemia protective interactions with stromal cells and immune cells. Bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BM-MSCs) are stromal cells in leukemia microenvironment, which can promote the progression of leukemia under certain conditions. Nuclear factor erythroid related factor 2 (Nrf2) is an important transcription factor and a regulator of cellular antioxidant response, which regulates the expression of genes encoding antioxidant enzymes to prevent various oxidative changes. It has been confirmed that the high expression of Nrf2 in leukemic cells is related to drug resistance and poor prognosis of leukemia. However, the interaction mechanism of Nrf2 overexpression in MSCs on B-cell acute lymphoblastic leukemia (B-ALL) cells is not clear. We will for the first time propose the mechanism of Nrf2 overexpression in MSCs on the migration and invasion of B-ALL leukemic cells. Methods :Bone marrow nucleated cells were detected in 16 patients with complete remission (B-ALL/CR), 12 patients with relapsed/refractory (B-ALL/R/R) ALL and 10 normal controls. The expression of Nrf2 was detected byreal-time PCR and Western Blot. In vitro cell experiments, real-time PCR and Western Blot were used to detect the expression of Nrf2 in mesenchymal stem cells (MSCs) alone and in the co-culture of MSCs and cell lines (Nalm-6/RS4:11) express the situation Mouse model experiment in vivo, transwell chamber experiment in vitro, three-dimensional (3D) cell culture model, lentivirus transfection, WB and PCR were used to explore the possible mechanism of Nrf2 overexpression in mesenchymal stem cells on the migration and invasion of B-ALL cells. Results: In clinical samples, the expression of Nrf2 in B-ALL-R/R group was significantly higher than that in ALL-CR group and normal control group, suggesting that the expression of Nrf2 was closely related to the recurrence and drug resistance of ALL. In vitro cell experiment, the expression level of Nrf2 in MSCs co-cultured with cell line (NALM-6/RS4:11) was higher than that in MSC cultured alone(P &amp;lt; 0.05). By regulating the overexpression of Nrf2 in MSCs, it can significantly promote the growth, proliferation, migration and invasion of leukemia cells, and promote the expression of CXCR4 in tumor tissues. We further regulated the expression of Nrf2 in MSCs. The results showed that it significantly promoted the migration and invasion of NALM-6/RS4:11 cells. We concluded that the overexpression of Nrf2 in MSCs promotes the migration and invasion of leukemic cells by activating the activity of SDF-1-CXCR4 axis. In vivo experiments, we used tail vein injection of MSCs and B-ALL leukemia cells to explore the protective effect of Nrf2 overexpression in mesenchymal stem cells on leukemia cells and promote extramedullary infiltration of leukemia. The results showed that the infiltration degree of extramedullary organs of mice after combined infusion of two kinds of cells was significantly higher than that of leukemia cells alone, and the survival time of mice was significantly shortened after combined infusion. After statistical treatment, the difference was statistically significant (P &amp;lt; 0.05). Conclusion:The expression of Nrf2 in MSCs may be a key component in mediating the interaction between MSCs and ALL cells. This result may provide a new entry point for deepening all disease progression mechanisms and finding more promising targeted therapies.On the other hand, activation of PI3K/AKTERK1/2 signaling pathway in B-ALL cells further promoted the evolution of B-ALL. This may provide new ideas for findingpotential therapeutic targets for B-ALL extramedullary infi ltration, and help to further solve the current problem of B-ALL relapse and refractory.

Mesenchymal stem cells of the bone marrow raise infectivity of Plasmodium falciparum gametocytes.

Plasmodium falciparum is a parasite that causes the deadly human disease, malaria, and exhibits a complex life cycle in human and mosquito hosts. In the sexual stages of the parasite, gametocytes mature in the human body and propagate malaria when they are picked up by mosquitoes to infect new hosts. Previous research has shown that gametocytes home to the bone marrow of the host, where they complete their maturation and alter the behavior of resident mesenchymal stem cells (MSCs). In this study, we investigated the alternate side of this host-pathogen interaction, whether MSCs could alter the behavior of gametocytes. Gametocytes were co-cultured with MSCs until maturity and subsequently fed to mosquitoes to measure the oocysts produced. Here, we report, for the first time, that MSCs co-culture significantly elevated oocyst numbers in the infected mosquito compared to conventional culture medium. This enhancement appeared to be most effective during the early stages of gametocyte development and was not replicated by other cell types. MSC co-culture also increased the infectivity of field isolated P. falciparum parasites. This effect was partially mediated by soluble factor(s) as conditioned medium harvested from MSCs could also partially raise infectivity of gametocytes to nearly half compared to MSC co-culture. Together, this study reveals novel host-pathogen interactions, where the human MSCs are elevating the infectivity of malaria gametocytes. IMPORTANCE While prior research has established that Plasmodium gametocytes sequester in the bone marrow and can influence resident stem cells, the question of why they would choose this compartment and these cells remained a mystery. This study, for the first time, shows that being in the presence of mesenchymal stem cells (MSCs) alters the biology of the P. falciparum parasite and makes it more infectious to mosquitoes, hinting at novel mechanisms in its life cycle. This method also facilitates mosquito infections with field isolated parasites, affording research teams new infection models with parasites, which are challenging to infect into mosquitos using conventional culture methods. Finally, our findings that MSC-conditioned medium can also raise infectivity open avenues of investigation into mechanisms involved but can also serve as a practical tool for researchers hoping to increase oocyst yields.

Integrin beta1 mediates the effect of telocytes on mesenchymal stem cell proliferation and migration in the treatment of acute lung injury.

Co-transplantation of mesenchymal stem cells (MSCs) with telocytes (TCs) was found to have therapeutic effects, although the mechanism of intercellular communication is still unknown. Our current studies aim at exploring the potential molecular mechanisms of TCs interaction and communication with MSCs with a focus on integrin beta1 (ITGB1) in TCs. We found that the co-culture of MSCs with ITGB1-deleted TCs (TCITGB1-ko ) changed the proliferation, differentiation and growth dynamics ability of MSC in responses to LPS or PI3K inhibitor. Changes of MSC proliferation and apoptosis were accompanied with the dysregulation of cytokine mRNA expression in MSCs co-cultured with TCITGB1-ko during the exposure of PI3Kα/δ/β inhibitor, of which IL-1β, IL-6 and TNF-α increased, while IFN-γ, IL-4 and IL-10 decreased. The responses of PI3K p85, PI3K p110 and pAKT of MSCs co-cultured with TCITGB1-ko to LPS or PI3K inhibitor were opposite to those with ITGB1-presented TCs. The intraperitoneal injection of TCITGB1-ko , TCvector or MSCs alone, as well as the combination of MSCs with TCITGB1-ko or TCvector exhibited therapeutic effects on LPS-induced acute lung injury. Thus, our data indicate that telocyte ITGB1 contributes to the interaction and intercellular communication between MSCs and TCs, responsible for influencing other cell phenomes and functions.

Mesenchymal Stem Cells Inhibit Epithelial-to-Mesenchymal Transition by Modulating the IRE1α Branch of the Endoplasmic Reticulum Stress Response.

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial lung disease, and it carries a poor prognosis due to a lack of efficient diagnosis methods and treatments. Epithelial-mesenchymal transition (EMT) plays a key role in IPF pathogenesis. Endoplasmic reticulum (ER) stress contributes to fibrosis via EMT-mediated pathways. Mesenchymal stem cell (MSC) transplantation is a promising treatment strategy for pulmonary fibrosis and ameliorates lung fibrosis in animal models via paracrine effects. However, the specific mechanisms underlying the effect of transplanted MSCs are not known. We previously reported that MSCs attenuate endothelial injury by modulating ER stress and endothelial-to-mesenchymal transition. The present study investigated whether modulation of ER stress- and EMT-related pathways plays essential roles in MSC-mediated alleviation of IPF. We constructed a A549 cell model of transforming growth factor-β1 (TGF-β1)-induced fibrosis. TGF-β1 was used to induce EMT in A549 cells, and MSC coculture decreased EMT, as indicated by increased E-cadherin levels and decreased vimentin levels. ER stress participated in TGF-β1-induced EMT in A549 cells, and MSCs inhibited the expression of XBP-1s, XBP-1u, and BiP, which was upregulated by TGF-β1. Inhibition of ER stress contributed to MSC-mediated amelioration of EMT in A549 cells, and modulation of the IRE1α-XBP1 branch of the ER stress pathway may have played an important role in this effect. MSC transplantation alleviated bleomycin (BLM)-induced pulmonary fibrosis in mice. MSC treatment decreased the expression of ER stress- and EMT-related genes and proteins, and the most obvious effect of MSC treatment was inhibition of the IRE1α/XBP1 pathway. The present study demonstrated that MSCs decrease EMT by modulating ER stress and that blockade of the IRE1α-XBP1 pathway may play a critical role in this effect. The current study provides novel insight for the application of MSCs for IPF treatment and elucidates the mechanism underlying the preventive effects of MSCs against pulmonary fibrosis.

Contribution of peripheral blood mononuclear cells isolated by advanced filtration system to myogenesis of human bone marrow mesenchymal stem cells co-cultured with myoblasts

BackgroundContribution of peripheral blood mononuclear cells (PBMCs) in myogenesis is still under debate, even though blood filtration systems are commonly used in clinical practice for successfully management of critic limb ischemia. ObjectivesA commercial blood filter used for autologous human PBMC transplantation procedures is characterized and used to collect PBMCs, that are then added to well-established 2D in vitro myogenic models assembled with a co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and skeletal myoblasts (hSkMs) whit the aim of investigating their potential contribution to stem cell myogenic commitment. MethodsA commercial blood filter was physically and chemically studied to understand its morphological characteristics and composition. PBMCs were concentrated using this system, further isolated by Ficoll-Paque density gradient centrifugation, and then added in an upper transwell chamber to a 2D co-culture of hBM-MSCs and hSkMs. Myogenic commitment was investigated by RT-PCR, immunofluorescence, and flow cytometry immunophenotyping. Cytokine levels were monitored by ELISA assay in culture media. ResultsThe blood filtration system was disassembled and appeared to be formed by twelve membranes of poly-butylene terephthalate fibers (diameters, 0.9–4.0 μm) with pore size distribution of 1–20 μm. Filter functional characterization was achieved by characterizing collected cells by flow cytometry. Subsequently, collected PBMCs fraction was added to an in-vitro model of hBM-MSC myogenic commitment. In the presence of PBMCs, stem cells significantly upregulated myogenic genes, such as Desmin and MYH2, as confirmed by qRT-PCR and expressed related proteins by immunofluorescence (IF) assay, while downregulated pro-inflammatory cytokines (IL12A at day 14) along the 21 days of culture. NoveltyOur work highlights chemical-physical properties of commercial blood filter and suggests that blood filtrated fraction of PBMC might modulate cytokine expression in response to muscle injury and promote myogenic events, supporting their clinical use in autologous transplantation.

Mesenchymal Stem Cell Secretome Alters Gene Expression and Upregulates Motility of Human Endometrial Stromal Cells.

Cyclic regeneration and repair of the endometrium are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) and umbilical cord (UC-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, mechanisms remain unclear. This study tested the hypothesis that the BM-MSC and UC-MSC secretomes upregulate human endometrial stromal cell (HESC) proliferation, migration, and invasion, and activate pathways to increase HESC motility. BM-MSCs were purchased from ATCC and cultured from the BM aspirate of three healthy female donors. UC-MSCs were cultured from umbilical cords of two healthy male term infants. Using indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system, we demonstrated that co-culture of HESCs with BM-MSCs or UC-MSCs from all donors significantly increased HESC migration and invasion, whereas effects on HESC proliferation varied among BM-MSC and UC-MSC donors. Analysis of gene expression by mRNA sequencing and RT-qPCR showed that expression of CCL2 and HGF was upregulated in HESCs that had been cocultured with BM-MSCs or UC-MSCs. Validation studies revealed that exposure to recombinant CCL2 for 48 hours significantly increased HESC migration and invasion. Increased HESC motility by the BM-MSC and UC-MSC secretome appears to be mediated in part by upregulated HESC CCL2 expression. Our data support the potential for leveraging MSC secretome as a novel cell-free therapy to treat disorders of endometrial regeneration.

Mesenchymal stem cells reversibly de-differentiate myofibroblasts to fibroblast-like cells by inhibiting the TGF-β-SMAD2/3 pathway

BackgroundMyofibroblasts (MFB), one of the major effectors of pathologic fibrosis, mainly derived from the activation of fibroblast to myofibroblast transition (FMT). Although MFBs were historically considered terminally differentiated cells, their potential for de-differentiation was recently recognized and implied with therapeutic value in treating fibrotic diseases, for instance, idiopathic pulmonary fibrosis (IPF) and post allogeneic hematopoietic stem cell transplantation bronchiolitis obliterans (BO). During the past decade, several methods were reported to block or reverse MFB differentiation, among which mesenchymal stem cells (MSC) have demonstrated potential but undetermined therapeutic values. However, the MSC-mediated regulation of FMT and underlying mechanisms remained largely undefined.MethodBy identifying TGF-β1 hypertension as the pivotal landmark during the pro-fibrotic FMT, TGF-β1-induced MFB and MSC co-culture models were established and utilized to investigate regulations by MSC on FMT in vitro. Methods including RNA sequencing (RNA-seq), Western blot, qPCR and flow cytometry were used.ResultOur data revealed that TGF-β1 readily induced invasive signatures identified in fibrotic tissues and initiated MFB differentiation in normal FB. MSC reversibly de-differentiated MFB into a group of FB-like cells by selectively inhibiting the TGF-β-SMAD2/3 signaling. Importantly, these proliferation-boosted FB-like cells remained sensitive to TGF-β1 and could be re-induced into MFB.ConclusionOur findings highlighted the reversibility of MSC-mediated de-differentiation of MFB through TGF-β-SMAD2/3 signaling, which may explain MSC's inconsistent clinical efficacies in treating BO and other fibrotic diseases. These de-differentiated FB-like cells are still sensitive to TGF-β1 and may further deteriorate MFB phenotypes unless the pro-fibrotic microenvironment is corrected.

Co-Culture of Mesenchymal Stem Cells and Ligamentocytes on Triphasic Embroidered Poly(L-lactide-co-ε-caprolactone) and Polylactic Acid Scaffolds for Anterior Cruciate Ligament Enthesis Tissue Engineering

Successful anterior cruciate ligament (ACL) reconstructions strive for a firm bone-ligament integration. With the aim to establish an enthesis-like construct, embroidered functionalized scaffolds were colonized with spheroids of osteogenically differentiated human mesenchymal stem cells (hMSCs) and lapine (l) ACL fibroblasts in this study. These triphasic poly(L-lactide-co-ε-caprolactone) and polylactic acid (P(LA-CL)/PLA) scaffolds with a bone-, a fibrocartilage transition- and a ligament zone were colonized with spheroids directly after assembly (DC) or with 14-day pre-cultured lACL fibroblast and 14-day osteogenically differentiated hMSCs spheroids (=longer pre-cultivation, LC). The scaffolds with co-cultures were cultured for 14 days. Cell vitality, DNA and sulfated glycosaminoglycan (sGAG) contents were determined. The relative gene expressions of collagen types I and X, Mohawk, Tenascin C and runt-related protein (RUNX) 2 were analyzed. Compared to the lACL spheroids, those with hMSCs adhered more rapidly. Vimentin and collagen type I immunoreactivity were mainly detected in the hMSCs colonizing the bone zone. The DNA content was higher in the DC than in LC whereas the sGAG content was higher in LC. The gene expression of ECM components and transcription factors depended on cell type and pre-culturing condition. Zonal colonization of triphasic scaffolds using spheroids is possible, offering a novel approach for enthesis tissue engineering.

Adipose Tissue-Mesenchymal Stem Cells Caused to Change the Methylation Status of hTERT Gene Promoter CpG Islands of Molt-4 Leukemia Cells as Cell-based Therapy.

DNA methylation was considered as prognostic information in some hematological malignancies. Previous studies have reported the in vitro and in vivo biology role of mesenchymal stem cells (MSCs) on leukemic cells. The aim of this study was to investigate the effect of MSCs on the promoter methylation status of hTERT as a catalytic subunit of telomerase enzyme. In the experimental study, the Molt-4 leukemic cells were co-cultured with MSCs for 7 days. At the end of the co-culture period, the Molt-4 cells were collected, DNA and protein were extracted. Then methylation specific-PCR and western blotting were done for evaluating the hTERT gene promoter methylation status and cyclin D1 and hTERT protein expression, respectively. In the following, the flow cytometry was done for cell cycle distribution assay. It was found that MSCs resulted in a significant decrease in the cyclin D1 and hTERT protein expression levels. Also, MSCs caused changes in the methylation status of the CpG islands in the hTERT gene promoter region. The following results showed that MSCs caused a significant increase in the number of cells at G0/G1 phase and arrest the G0/G1 phase as well as decrease in the cell proliferation of Molt-4 cells. It is concluded that co-culture of MSCs with Molt-4 cells could be involved in changing the methylation status of hTERT gene promoter, cell cycle and hTERT protein expression; it could be potentially beneficial for further investigations regarding the cell transplantation and cell-based therapy.

Abstract P2-21-12: Targeting ADAM8 dependent maintenance of the myCAF phenotype in the breast cancer tumor microenvironment

Abstract Background: Cancer associated fibroblasts (CAFs) regulate breast cancer growth, differentiation and metastasis. Targeting the signaling pathways between myofibroblast CAFs (myCAF) and cancer cells required for myCAF maintenance provides a mechanism to reverse engineer the tumor microenvironment to inhibit cancer growth and metastasis. Methods and Results: We co-cultured human triple negative breast cancer (MDA-MB-231) with human mesenchymal stem cells (MSC). After 72h, MSC mRNA expression of the myCAF markers, alpha-smooth muscle actin (α-SMA)/vimentin (Vim)/tenascin C (Ten-C) are all significantly increased. In parallel, MB-231 mRNA expression of stemness markers, SRY-Box Transcription factor 2 (Sox2)/Octamer-binding transcription factor 4 (Oct4)/Nanog, are also significantly increased. Migration assay and morphology imaging confirm the induction of the myCAF phenotype to promote MB-231 cancer stemness. shRNA lentivirus was used to knock down Sox2 gene expression in MB-231. We performed RNA-seq in MB-231 first comparing MB-231 vs. MB-231+MSC and then MB-231(-/-sox2)+MSC vs. MB-231+MSC. There are 104 overlapping genes in MB-231+MSC from the two analyses indicating upregulation with myCAF differentiation and cancer stemness. Using the Human Cancer Secretome Database, 9 genes are either membrane or secreted proteins. Using antibody blockade in co-culture, antibody depleted co-culture medium, and adding activated proteins to culture, A Disintegrin And Metalloprotease 8 (Adam8) gene was found to be required for myCAF maintenance. Using SELEX, we isolated 5 RNA aptamers targeting the human Adam8 soluble MP domain. Using in vitro quantification of Adam8 MP activity with human Adam8 fluorogenic peptide substrate and recombinant human Adam8 protein, Adam8 Apt-1 with IC50=33nM and Kd=29nM was selected. APt-1 does not inhibit human Adam 10 or 17 MP activity and remains extracellular. The core inhibitory domain of Apt-1 aptamer contains 23nt. Following induction of the myCAF phenotype and cancer stemness in MB-231+MSC coculture, addition of Apt-1 significantly decreased myCAF and cancer stemness markers. In murine NOD/scid xenograft model, 10^6 luciferase-MB-231 and 10^6 MSC were implanted in the R3 position at week 0. Apt1 was administered (10mg/kg via tail vein every 2 days) from week 3 to week 6. MB-231 volume (bioluminescence) was significantly decreased following Apt1. (Table) Conclusion: Adam8 is expressed in MB-231 breast cancer cells in the context of MSC/myCAF coculture and MB231 stemness and is required for MB-231 cancer growth. Aptamer ablation of Adam8 extracellular activity reverses MB-231 growth. MB-231 and MSC Murine Xenograft Model In murine NOD/scid xenograft model, 10^6 luciferase-MB-231 and 10^6 MSC were implanted in the R3 position at week 0. Apt1 was administered (10mg/kg via tail vein every 2 days) from week 3 to week 6. Citation Format: Zhiyong Mi, Paul C. Kuo. Targeting ADAM8 dependent maintenance of the myCAF phenotype in the breast cancer tumor microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-12.

Effect of Co-culture of mesenchymal stem cell and glomerulus endothelial cell to promote endothelialization under optimized perfusion flow rate in whole renal ECM scaffold.

In recent era, many researches on implantable bio-artificial organs has been increased owing to large gap between donors and receivers. Comprehensive organ based researches on perfusion culture for cell injury using different flow rate have not been conducted at the cellular level. The present study investigated the co-culture of rat glomerulus endothelial cell (rGEC) and rat bone marrow mesenchymal stem cells (rBMSC) to develop micro vascularization in the kidney scaffolds culturing by bioreactor system. To obtain kidney scaffold, extracted rat kidneys were decellularized by 1% sodium dodecyl sulfate (SDS), 1% triton X-100, and distilled water. Expanded rGECs were injected through decellularized kidney scaffold artery and cultured using bioreactor system. Vascular endothelial cells adhered and proliferated on the renal ECM scaffold in the bioreactor system for 3, 7 and 14 days. Static, 1​ml/min and 2​ml/min flow rates (FR) were tested and among them, 1​ml/min flow rate was selected based on cell viability, glomerulus character, inflammation/endothelialization proteins expression level. However, the flow injury was still existed on primary cell cultured at vessel in kidney scaffold. Therefore, co-culture of rGEC​+​rBMSC found suitable to possibly solve this problem and resulted increased cell proliferation and micro-vascularization in the glomerulus, reducing inflammation and cell death which induced by flow injury. The optimized perfusion rate under rGEC​+​rBMSC co-culture conditions resulted in enhanced endocellularization to make ECM derived implantable renal scaffold and might be useful as a way of treatment of the acute renal failure.

Biomarkers of Clinical Resistance to Decitabine and Venetoclax Therapy Inferred By Transcriptome and Methylation Analyses

The combination of the selective BCL-2 inhibitor venetoclax (VEN) with the hypomethylating agent (HMA) decitabine (DEC) is highly effective in treating older ineligible for intensive chemotherapy AML patients. However, approximately 15-34% of newly diagnosed (ND) and 60% of relapsed/refractory (R/R) patients fail to achieve complete remission (CR) (DiNardo et al. New Engl J Med. 2020, DiNardo et al. Lancet Haematol. 2020). Factors that reduce efficacy remain to be elucidated. To dissect underlying mechanisms of HMA-VEN resistance, we performed RNA-seq utilizing primary samples from patients treated on a prospective clinical trial of 10-day DEC with VEN (NCT03404193). Previously, we reported that alterations of transcriptome signatures of energy-metabolism and cell migration/adhesion pathways were upregulated in primary resistant ND group, and immune-system-associated genes were downregulated in R/R group (Yamatani et al. ASH 2021). This study investigated transcriptomic and DNA methylation signatures associated with DEC10-VEN response or resistance in ND and R/R AML. Because DEC was reported to strongly induce interferon-stimulated genes (ISGs) through gene methylation modifications, we focused on 116 ISGs. An integrated analysis of DNA methylation status around the transcription start site (TSS) was performed using the Infinium methylated EPIC array (Illumina). Forty-four pre-treatment samples from 32 patients with AML from DEC-VEN clinical trial, ND patient group (CR/CRi 14; non-responder, NR 11) and R/R patient group (CR/CRi 6; NR 13), were analyzed. First, we performed the comparative analysis of NR vs CRs. In the ND group, NR cases showed significantly higher gene expression and lower TSS methylation of an urea cycle enzyme, ASS1 (Argininosuccinate Synthase 1) with RNA-seq Log2 fold-change (FC) 3.6 and methylation array Δβ (differential methylation β-value) -0.11, p < 0.05, and of an actin-based molecular motor, MYO1C (Myosin 1C), Log2FC 2.5, Δβ = -0.18, p< 0.05, than in CR cases. In the R/R group, gene expression levels of seven ISGs, including CCL3 (C-C motif chemokine 3), were significantly lower in NR than in CRs, with no difference in methylation status. Next, changes in gene expression levels and TSS methylation status after DEC-VEN treatment were investigated in non-responders in ND group (pre 19, post 12) and R/R group (pre 16, post 4). After DEC-VEN treatment, PPARG (Peroxisome Proliferator-Activated Receptor γ), a transcription factor that activates fatty acid metabolism and negatively regulates subsets of IFN-γ target genes, was significantly upregulated along with its TSS demethylation both in ND (Log2FC 4.9, Δβ = -0.14, p < 0.05), and R/R groups (Log2FC 3.7, Δβ = -0.06, p < 0.05). CDC42EP4, an effector protein of CDC42 involved in the organization of the actin cytoskeleton, was upregulated after the treatment in the R/R group (Log2FC 2.7, Δβ = -0.14, p < 0.05). Based on these findings, we validated transcriptional and methylation changes of ASS1, PPARG, MYO1C and CDC42EP4 by RT-qPCR and bisulfite pyrosequencing using HL60 and MV4-11 AML cell lines in which DEC (IC50, HL60, 249 nM; MV4-11, 2090 nM) and VEN (IC50, HL60, 18 nM; MV4-11, 64 nM) produced additive or synergistic combination effect. Cells were treated by DEC and/or VEN under BM-derived mesenchymal stem cells (MSCs) co-culture conditions to mimic the BM microenvironment. DEC upregulated gene expression levels with TSS demethylation of PPARG, MYO1C and CDC42EP4 both in HL60 and MV4-11, and of ASS1 only in HL-60. VEN treatment alone or in combination with DEC did not induce significant changes in the expression of these tested genes. Of note, PPARG expression was further upregulated under MSCs co-culture condition without TSS methylation status change in AML cells. DEC treatment induced no significant changes of PPARG in MSC. These findings suggest that not only TSS demethylation but also interaction with BM stromal cells may be involved in PPARG upregulation associated with energy production in AML cells. Taken together, DEC-VEN treatment stimulates two key factors, energy metabolism and interaction with the BM microenvironment, which may reduce therapeutic efficacy by increasing gene expression of PPARG,MYO1C,ASS1, and CDC42EP4 through TSS demethylation and/or interaction with BM stromal cells. Experimentally, the consequences of overexpression / knockdown of these genes will be further validated.

Alterations of Human Bone Marrow Niche in Clonal Hematopoiesis and AML

Acute myeloid leukemia (AML) is a heterogeneous disease, predominantly caused by acquisition of mutations in the hematopoietic stem and progenitor cells. Some of these mutations are acquired in a preleukemic state termed clonal hematopoiesis of indeterminate potential (CHIP). Recent studies show that alterations in the bone marrow niche can support progression and therapy resistance of myeloid neoplasia. In our study, we aim to elucidate alterations in the bone marrow niche, in particular in mesenchymal stem cells (MSC), during the evolution from healthy to CHIP and further to AML. We collected primary human bone marrow (BM) and peripheral blood (pB) samples from hematologically healthy individuals who underwent hip replacement surgery at the LMU University Hospital as well from AML patients treated at our institution (LMU University Hospital Munich). Individuals were assigned to non-CHIP or CHIP cohorts based on targeted mutation analyses on pB samples. So far, we have included 105 healthy individuals and 92 have undergone targeted sequencing, revealing 27 individuals (29%) with CHIP. While there was no difference between the gender of the non-CHIP and CHIP groups, individuals with CHIP tended to be of older age (median, 64 y non-CHIP vs. 73.5 y CHIP). We detected DTA gene mutations as the most common CHIP alteration and 1.4 mutations per individual were found on average. Variant allele frequencies varied between 1% and 17%. The amount of mutations per individual increased with age. In the non-CHIP and CHIP individuals, we analyzed various blood parameters such as hemoglobin, leukocyte counts, mean corpuscular volume and others, as well as various comorbidities such as myocardial infarction, hypertesia, previously treated cancer, etc. We found a trend for more frequent myocardial infarction in the CHIP individuals. We did not find any significant differences between CHIP and non-CHIP for the blood parameters. MSC from donor BM samples were isolated via plastic adherence over 10-14 days of in vitro culture. MSC were characterized regarding their surface marker expression (FACS), differentiation capacity (osteogenic, adipogenic, chondrogenic) and metabolic activity (Seahorse XF). Analysis of MSCs from non-CHIP (n=5) and CHIP (n=5) individuals revealed no difference in surface marker expression over 10 passages. Also, differentiation assays of MSCs (non-CHIP=4, CHIP= 2) showed no differences in adipogenic and chondrogenic differentiation after 21 days. For osteogenesis, we noticed a trend towards an increased differentiation potential in the MSCs of CHIP individuals. This experiment is ongoing with the goal to increase the sample number up to 10 per condition, as well as including the AML controls. The data will be shown at the ASH meeting. To gain deeper insights into the BM niche we sorted 4 non-hematopoietic and 6 hematopoietic cell populations from non-CHIP (n=3), CHIP (n=4) and AML (n=4) individuals and performed bulk-RNA sequencing on each population. So far, changes in the composition of the bone marrow niche were only detected in AML samples. Currently, in a series of pilot experiments, we established primary MSC and HSPC cross co-cultures from non-CHIP, CHIP and AML samples. A preliminary experiment with one non-CHIP and one CHIP sample showed that both non-CHIP and CHIP HSC produced less hematopoietic cells and less colony forming units (CFUs) after co-culturing with CHIP MSCs, than with non-CHIP MSCs. The results of the main experiment with 3 individuals per condition and including the AML samples will be shown at the conference.

Mesenchymal stem cells: A novel treatment option for primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a progressive liver disease for which there is no effective therapy. Hepatocytes and cholangiocytes from a PSC patient were cocultured with mesenchymal stem cells (MSCs) to assess in vitro change. A single patient with progressive PSC was treated with 150 million MSCs via direct injection into the common bile duct. Coculture of MSCs with cholangiocytes and hepatocytes showed in vitro improvement. Local delivery of MSCs into a single patient with progressive PSC was safe. Radiographic and endoscopic evaluation showed stable distribution of multifocal structuring in the early postoperative period. MSCs may be effective for the treatment of PSC.

Antifibrotic TSG-6 Expression Is Synergistically Increased in Both Cells during Coculture of Mesenchymal Stem Cells and Macrophages via the JAK/STAT Signaling Pathway.

The pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1β upregulate TNF-α-stimulated gene 6 (TSG-6); however, current knowledge about the optimal conditions for TSG-6 expression in mesenchymal stem cells (MSCs) is limited. Here, we investigated whether TSG-6 expression varies depending on the polarization state of macrophages co-cultured with adipose tissue-derived stem cells (ASCs) and analyzed the optimal conditions for TSG-6 expression in ASCs. TSG-6 expression increased in ASCs co-cultured with M0, M1, and M2 macrophages indirectly; among them, M1 macrophages resulted in the highest increase in TSG-6 expression in ASCs. TSG-6 expression in ASCs dramatically increased by combination (but not single) treatment of TNF-α, IL-1β, interferon-gamma (IFN-γ), and lipopolysaccharide (LPS). In addition, phosphorylation of signal transducer and activator of transcription (STAT) 1/3 was observed in response to IFN-γ and LPS treatment but not TNF-α and/or IL-1β. STAT1/3 activation synergistically increased TNF-α/IL-1β-dependent TSG-6 expression, and JAK inhibitors suppressed TSG-6 expression both in ASCs and macrophages. In LX-2 hepatic stellate cells, TSG-6 inhibited TGF-β-induced Smad3 phosphorylation, resulting in decreased α-smooth muscle actin (SMA) expression. Moreover, fibrotic activities of LX-2 cells induced by TGF-β were dramatically decreased after indirect co-culture with ASCs and M1 macrophages. These results suggest that a comprehensive inflammatory microenvironment may play an important role in determining the therapeutic properties of ASCs by increasing TSG-6 expression through STAT1/3 activation.