Mesenchymal Stem/stromal Cells Research Articles

Abstract PO2-25-06: A hybrid breast cancer/mesenchymal stem cell population increases distant metastasis and chemoresistance of metaplastic breast carcinomas

Abstract Background: Metaplastic breast carcinoma is a heterogenous, highly aggressive and chemoresistant subtype of triple negative breast cancer (TNBC) with histological evidence of deregulated differentiation towards non-glandular components including spindle, squamous, and sarcomatoid. The role of the tumor microenvironment in the metastatic behavior and chemotherapy response of metaplastic carcinomas needs investigation to improve patient outcomes. Our lab has generated a model of spindle metaplastic breast carcinomas based on mammary epithelial cell-specific CCN6 knockout (MMTV-Cre;Ccn6fl/fl or CCN6KO). We have recently shown that a subset of TNBC cells engulf mesenchymal stem/stromal cells (MSC) generating a hybrid cell population with metastatic ability, which is detected in breast cancer tissue samples. In this study, we tested the hypothesis that hybrid cells may promote chemoresistant distant metastasis in spindle metaplastic breast carcinomas and that the tumor immune microenvironment may play a role in this function. Methods: Luc-GFP-CCN6KO cells were cultured with DsRED-MSCs for 7 days, yielding >90% of hybrid cells detected by flow cytometry. Hybrids or CCN6KO single cultures (1x10(5) cells) were injected intracardially in FVB/NJ syngeneic immune competent mice (n=20 mice per group). Four days after injection, each group was treated with Paclitaxel (PTX, 10 mg/kg twice a week, i.p.) or PBS control. The metastatic burden was evaluated by bioluminescence imaging twice a week. Mice were euthanized 23 days after heart injections, followed by necropsy and histopathological evaluation of the harvested organs. Results: All mice injected with single cultures of CCN6KO cells or hybrid cells formed distant metastasis. CCN6KO cells were highly invasive and spread to the lungs and lymph nodes in the chest,. We noted that 5 of 20 (25%) mice injected with hybrid cells showed metastasis in the liver (n=3 mice), pancreas (n=1), and abdominal lymph nodes (n=1). PTX treatment significantly reduced metastatic burden in mice injected with CCN6KO single cultures compared to vehicle (p=0.035) but had no effect on the metastatic burden of hybrid cells. Histologically, while the metastasis from CCN6KO cells and hybrids showed similar pathological features, consisting of spindle cancer cells with high grade nuclei and >10 mitoses/10 high power fields, hybrid-derived tumors had abundant infiltration by immune cells, mainly neutrophils, compared to single culture tumors. Conclusion: Our data show that hybrid cancer cell/MSC derived from CCN6KO spindle metaplastic carcinomas have higher ability to disseminate to distant organs, especially in the abdomen, compared to cancer cells. Our data show that hybrid cell-derived metastases have increased immune cell infiltration. We provide evidence that hybrid cells enhance Paclitaxel resistance in a mouse model of metaplastic breast carcinoma. Ongoing studies are aimed at elucidating the role of hybrids in the immune microenvironment of metaplastic breast carcinoma and clinical significance. Citation Format: Ahmad Eido, Maria Gonzalez, Celina Kleer. A hybrid breast cancer/mesenchymal stem cell population increases distant metastasis and chemoresistance of metaplastic breast carcinomas [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-06.

Cold Atmospheric Pressure Plasma-Activated Medium Modulates Cellular Functions of Human Mesenchymal Stem/Stromal Cells In Vitro.

Cold atmospheric pressure plasma (CAP) offers a variety of therapeutic possibilities and induces the formation of reactive chemical species associated with oxidative stress. Mesenchymal stem/stromal cells (MSCs) play a central role in tissue regeneration, partly because of their antioxidant properties and ability to migrate into regenerating areas. During the therapeutic application, MSCs are directly exposed to the reactive species of CAP. Therefore, the investigation of CAP-induced effects on MSCs is essential. In this study, we quantified the amount of ROS due to the CAP activation of the culture medium. In addition, cell number, metabolic activity, stress signals, and migration were analyzed after the treatment of MSCs with a CAP-activated medium. CAP-activated media induced a significant increase in ROS but did not cause cytotoxic effects on MSCs when the treatment was singular and short-term (one day). This single treatment led to increased cell migration, an essential process in wound healing. In parallel, there was an increase in various cell stress proteins, indicating an adaptation to oxidative stress. Repeated treatments with the CAP-activated medium impaired the viability of the MSCs. The results shown here provide information on the influence of treatment frequency and intensity, which could be necessary for the therapeutic application of CAP.

Development of a novel high-throughput culture system for hypoxic 3D hydrogel cell culture.

Animal models lack physiologic relevance to the human system which results in low clinical translation of results derived from animal testing. Besides spheroids or organoids, hydrogel-based 3D in vitro models are used to mimic the in vivo situation increasing the relevance while reducing animal testing. However, to establish hydrogel-based 3D models in applications such as drug development or personalized medicine, high-throughput culture systems are required. Furthermore, the integration of oxygen-reduced (hypoxic) conditions has become increasingly important to establish more physiologic culture models. Therefore, we developed a platform technology for the high-throughput generation of miniaturized hydrogels for 3D cell culture. The Oli-Up system is based on the shape of a well-plate and allows for the parallel culture of 48 hydrogel samples, each with a volume of 15µl. As a proof-of-concept, we established a 3D culture of gelatin-methacryloyl (GelMA)-encapsulated mesenchymal stem/stromal cells (MSCs). We used a hypoxia reporter cell line to establish a defined oxygen-reduced environment to precisely trigger cellular responses characteristicof hypoxia in MSCs. In detail, the expression of hypoxia response element (HRE) increased dependent on the oxygen concentration and cell density. Furthermore, MSCs displayed an altered glucose metabolism and increased VEGF secretion upon oxygen-reduction. In conclusion, the Oli-Up system is a platform technology for the high-throughput culture of hydrogel-based 3D models in a defined oxygen environment. As it is amenable for automation, it holds the potential for high-throughput screening applications such as drug development and testing in more physiologic 3D in vitro tissue models.

Mesenchymal stromal/stem cells promote intestinal epithelium regeneration after chemotherapy-induced damage

BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for leukemia and a range of non-malignant disorders. The success of the therapy is hampered by occurrence of acute graft-versus-host disease (aGvHD); an inflammatory response damaging recipient organs, with gut, liver, and skin being the most susceptible. Intestinal GvHD injury is often a life-threatening complication in patients unresponsive to steroid treatment. Allogeneic mesenchymal stromal/stem cell (MSC) infusions are a promising potential treatment for steroid-resistant aGvHD. Data from our institution and others demonstrate rescue of approximately 40–50% of aGvHD patients with MSCs in Phase I, II studies and minor side effects. Although promising, better understanding of MSC mode of action and patient response to MSC-based therapy is essential to improve this lifesaving treatment.MethodsSingle cell human small intestine organoids were embedded in Matrigel, grown for 5 days and treated with busulfan for 48 h. Organoids damaged by treatment with busulfan or control organoids were co-cultured with 5000, 10,000, and 50,000 MSCs for 24 h, 48 h or 7 days and the analyses such as surface area determination, proliferation and apoptosis assessment, RNA sequencing and proteomics were performed.ResultsHere, we developed a 3D co-culture model of human small intestinal organoids and MSCs, which allows to study the regenerative effects of MSCs on intestinal epithelium in a more physiologically relevant setting than existing in vitro systems. Using this model we mimicked chemotherapy-mediated damage of the intestinal epithelium. The treatment with busulfan, the chemotherapeutic commonly used as conditioning regiment before the HSCT, affected pathways regulating epithelial to mesenchymal transition, proliferation, and apoptosis in small intestinal organoids, as shown by transcriptomic and proteomic analysis. The co-culture of busulfan-treated intestinal organoids with MSCs reversed the effects of busulfan on the transcriptome and proteome of intestinal epithelium, which we also confirmed by functional evaluation of proliferation and apoptosis.ConclusionsCollectively, we demonstrate that our in vitro co-culture system is a new valuable tool to facilitate the investigation of the molecular mechanisms behind the therapeutic effects of MSCs on damaged intestinal epithelium. This could benefit further optimization of the use of MSCs in HSCT patients.

Mesenchymal Stem Cell Exosomes Enhance Posterolateral Spinal Fusion in a Rat Model.

Spinal fusion, a common surgery performed for degenerative lumbar conditions, often uses recombinant human bone morphogenetic protein 2 (rhBMP-2) that is associated with adverse effects. Mesenchymal stromal/stem cells (MSCs) and their extracellular vesicles (EVs), particularly exosomes, have demonstrated efficacy in bone and cartilage repair. However, the efficacy of MSC exosomes in spinal fusion remains to be ascertained. This study investigates the fusion efficacy of MSC exosomes delivered via an absorbable collagen sponge packed in a poly Ɛ-caprolactone tricalcium phosphate (PCL-TCP) scaffold in a rat posterolateral spinal fusion model. Herein, it is shown that a single implantation of exosome-supplemented collagen sponge packed in PCL-TCP scaffold enhanced spinal fusion and improved mechanical stability by inducing bone formation and bridging between the transverse processes, as evidenced by significant improvements in fusion score and rate, bone structural parameters, histology, stiffness, and range of motion. This study demonstrates for the first time that MSC exosomes promote bone formation to enhance spinal fusion and mechanical stability in a rat model, supporting its translational potential for application in spinal fusion.

Frailty: Focus on Regenerative Medicine

Frailty, as a specific condition of increased vulnerability and deterioration of general health associated with aging in the elderly, is an emerging problem worldwide with serious implications for clinical practice and public health. Frailty increases the risk of falls, hospitalization, disability, and death. Muscle aging is associated with progressive loss of skeletal muscle mass and function. Loss of muscle mass with age can be related to muscle fiber atrophy and loss of muscle fibers. With increasing human age, remodeling of bone tissue becomes more prone to slow and steady loss of bone mass, which is primarily associated with loss of bone formation by osteoblasts. Recent preclinical and clinical studies have confirmed the safety of mesenchymal stem/stromal cells (MSCs) in the treatment of frailty. MSCs are attracted to sites of damage where they act to reduce inflammation and promote cell repair. Thus, there is an assumption that frailty can be treated with cell therapy

Protection of neutrophils by bone marrow mesenchymal stromal cells is enhanced by tumor-associated inflammatory cytokines.

Mesenchymal stromal/stem cells (MSCs), which are distributed in many tissues including bone marrow, have been reported to play a critical role in tumor development. While bone marrow, the primary site for hematopoiesis, is important for establishing the immune system, whether MSCs in the bone marrow can promote tumor growth via influencing hematopoiesis remains unclear. We observed that the numbers of MSCs and neutrophils were increased in bone marrow in tumor-bearing mice. Moreover, co-culture assay showed that MSCs strongly protected neutrophils from apoptosis and induced their maturation. G-CSF and GM-CSF have been well-documented to be associated with neutrophil formation. We found a remarkably increased level of G-CSF, but not GM-CSF, in the supernatant of MSCs and the serum of tumor-bearing mice. The G-CSF expression can be enhanced with inflammatory cytokines (IFNγ and TNFα) stimulation. Furthermore, we found that IFNγ and TNFα-treated MSCs enhanced their capability of promoting neutrophil survival and maturation. Our results indicate that MSCs display robustly protective effects on neutrophils to contribute to tumor growth in bone niches.

Defining mesenchymal stem/stromal cell-induced myeloid-derived suppressor cells using single-cell transcriptomics

Mesenchymal stem/stromal cells (MSCs) modulate the immune response through interactions with innate immune cells. We previously demonstrated that MSCs alleviate ocular autoimmune inflammation by directing BM cell differentiation from pro-inflammatory CD11bhiLy6ChiLy6Glo cells into immunosuppressive CD11bmidLy6CmidLy6Glo cells. Herein, we analyzed MSC-induced CD11bmidLy6Cmid cells using single-cell RNA sequencing and compared them with CD11bhiLy6Chi cells. Our investigation revealed 7 distinct immune cell types including myeloid-derived suppressor cells (MDSCs) in the CD11bmidLy6Cmid cells, while CD11bhiLy6Chi cells included mostly monocytes/macrophages with a small cluster of neutrophils. These MSC-induced MDSCs highly expressed Retnlg, Cxcl3, Cxcl2, Mmp8, Cd14 and Csf1r as well as Arg1. Comparative analyses of CSF-1RhiCD11bmidLy6Cmid and CSF-1RloCD11bmidLy6Cmid cells demonstrated that the former had a homogeneous monocyte morphology and produced elevated levels of IL-10. Functionally, these CSF-1RhiCD11bmidLy6Cmid cells, compared with the CSF-1RloCD11bmidLy6Cmid cells, inhibited CD4+ T cell proliferation and promoted CD4+CD25+Foxp3+ Treg expansion in culture and in a mouse model of experimental autoimmune uveoretinitis. RELM-γ encoded by Retnlg, one of the highly-upregulated genes in MSC-induced MDSCs, had no direct effects on T cell proliferation, Treg expansion or splenocyte activation. Together, our study revealed a distinct transcriptional profile of MSC-induced MDSCs and identified CSF-1R as a key cell-surface marker for detection and therapeutic enrichment of MDSCs.

Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.

This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 μg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and IL-17A gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group (P = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 (P = 0.022) and IL-17A (P = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the IL-1β, IL-36α, and TNF-α genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.

Human Wharton's jelly-derived mesenchymal stromal stem cells preconditioned with valproic acid promote cell migration and reduce renal inflammation in ischemia/reperfusion injury by activating the AKT/P13K and SDF1/CXCR4 pathways

ObjectiveTo determine whether WJ-MSCs pretreated with VPA would enhance their migration to improve functional recovery of renal IRI in rats. Methods150 Sprague-Dawley rats were distributed into 5 groups; Sham, IRI, WJ-MSC, VPA, and WJ-MSCs + VPA. 10 rats were sacrificed after 3, 5, and 7 days. Role of WJ-MSCs pretreated with VPA was evaluated by assessment of renal function, antioxidant enzymes together with renal histopathological and immunohistopathological analyses and finally by molecular studies. ResultsWJ-MSCs and VPA significantly improved renal function and increased antioxidants compared to IRI group. Regarding gene expression, WJ-MSCs and VPA decreased BAX and TGF-β1, up-regulated Akt, PI3K, BCL2, SDF1α, and CXCR4 related to IRI. Additionally, WJ-MSCs pretreated with VPA improved the measured parameters more than either treatment alone. ConclusionWJ-MSCs isolated from the umbilical cord and pretreated with VPA defended the kidney against IRI by more easily homing to the site of injury.

Mesenchymal stem/stromal cells armored by FGF21 ameliorate alcohol-induced liver injury through modulating polarization of macrophages.

Alcohol-associated liver disease (ALD) is a major health care challenge worldwide with limited therapeutic options. Although mesenchymal stem/stromal cells (MSCs) represent a newly emerging therapeutic approach to treat ALD, thus far, there have been extensive efforts to try and enhance their efficacy, including genetically engineering MSCs. FGF21, an endocrine stress-responsive hormone, has been shown to regulate energy balance, glucose, and lipid metabolism and to enhance the homing of MSCs toward injured sites. Therefore, the purpose of this study was to investigate whether MSCs that overexpress FGF21 (FGF21-MSCs) improve the therapeutic effect of MSCs in treating ALD. Human umbilical cord-derived MSCs served as the gene delivery vehicle for the FGF21 gene. Human umbilical cord-derived MSCs were transduced with the FGF21 gene using lentiviral vectors to mediate FGF21 overexpression. We utilized both chronic Lieber-DeCarli and Gao-binge models of ethanol-induced liver injury to observe the therapeutic effect of FGF21-MSCs. Liver injury was phenotypically evaluated by performing biochemical methods, histology, and inflammatory cytokine levels. Compared with MSCs alone, administration of MSCs overexpressing FGF21(FGF21-MSCs) treatment significantly enhanced the therapeutic effect of ALD in mice, as indicated by the alleviation of liver injury with reduced steatosis, inflammatory infiltration, oxidative stress, and hepatic apoptosis, and the promotion of liver regeneration. Mechanistically, FGF21 could facilitate the immunomodulatory function of MSCs on macrophages by setting metabolic commitment for oxidative phosphorylation, which enables macrophages to exhibit anti-inflammatory inclination. Our data elucidate that MSC modification by FGF21 could enhance their therapeutic effect in ALD and may help in the exploration of effective MSCs-based cell therapies for the treatment of ALD.

PEDF peptide plus hyaluronic acid stimulates cartilage regeneration in osteoarthritis via STAT3-mediated chondrogenesis.

Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA). Mesenchymal stem/stromal cells (MSCs) were isolated from rat bone marrow (BM) and used to evaluate the impact of 29-mer on chondrogenic differentiation of BM-MSCs in culture. Knee OA was induced in rats by a single intra-articular injection of monosodium iodoacetate (MIA) in the right knees (set to day 0). The 29-mer dissolved in 5% hyaluronic acid (HA) was intra-articularly injected into right knees at day 8 and 12 after MIA injection. Subsequently, the therapeutic effect of the 29-mer/HA on OA was evaluated by the Osteoarthritis Research Society International (OARSI) histopathological scoring system and changes in hind paw weight distribution, respectively. The regeneration of chondrocytes in damaged AC was detected by dual-immunostaining of 5-bromo-2'-deoxyuridine (BrdU) and chondrogenic markers. The 29-mer promoted expansion and chondrogenic differentiation of BM-MSCs cultured in different defined media. MIA injection caused chondrocyte death throughout the AC, with cartilage degeneration thereafter. The 29-mer/HA treatment induced extensive chondrocyte regeneration in the damaged AC and suppressed MIA-induced synovitis, accompanied by the recovery of cartilage matrix. Pharmacological inhibitors of PEDF receptor (PEDFR) and signal transducer and activator of transcription 3 (STAT3) signalling substantially blocked the chondrogenic promoting activity of 29-mer on the cultured BM-MSCs and injured AC. The 29-mer/HA formulation effectively induces chondrocyte regeneration and formation of cartilage matrix in the damaged AC.

Optimizing cryopreservation conditions for use of fucosylated human mesenchymal stromal cells in anti-inflammatory/immunomodulatory therapeutics.

Mesenchymal stem/stromal cells (MSCs) are being increasingly used in cell-based therapies due to their broad anti-inflammatory and immunomodulatory properties. Intravascularly-administered MSCs do not efficiently migrate to sites of inflammation/immunopathology, but this shortfall has been overcome by cell surface enzymatic fucosylation to engender expression of the potent E-selectin ligand HCELL. In applications of cell-based therapies, cryopreservation enables stability in both storage and transport of the produced cells from the manufacturing facility to the point of care. However, it has been reported that cryopreservation and thawing dampens their immunomodulatory/anti-inflammatory activity even after a reactivation/reconditioning step. To address this issue, we employed a variety of methods to cryopreserve and thaw fucosylated human MSCs derived from either bone marrow or adipose tissue sources. We then evaluated their immunosuppressive properties, cell viability, morphology, proliferation kinetics, immunophenotype, senescence, and osteogenic and adipogenic differentiation. Our studies provide new insights into the immunobiology of cryopreserved and thawed MSCs and offer a readily applicable approach to optimize the use of fucosylated human allogeneic MSCs as immunomodulatory/anti-inflammatory therapeutics.

Standard Radio-Iodine Labeling Protocols Impaired the Functional Integrity of Mesenchymal Stem/Stromal Cell Exosomes.

Mesenchymal stem/stromal cells (MSCs) are an extensively studied cell type in clinical trials due to their easy availability, substantial ex vivo proliferative capacity, and therapeutic efficacy in numerous pre-clinical animal models of disease. The prevailing understanding suggests that their therapeutic impact is mediated by the secretion of exosomes. Notably, MSC exosomes present several advantages over MSCs as therapeutic agents, due to their non-living nature and smaller size. However, despite their promising therapeutic potential, the clinical translation of MSC exosomes is hindered by an incomplete understanding of their biodistribution after administration. A primary obstacle to this lies in the lack of robust labels that are highly sensitive, capable of directly and easily tagging exosomes with minimal non-specific labeling artifacts, and sensitive traceability with minimal background noise. One potential candidate to address this issue is radioactive iodine. Protocols for iodinating exosomes and tracking radioactive iodine in live imaging are well-established, and their application in determining the biodistribution of exosomes has been reported. Nevertheless, the effects of iodination on the structural or functional activities of exosomes have never been thoroughly examined. In this study, we investigate these effects and report that these iodination methods abrogate CD73 enzymatic activity on MSC exosomes. Consequently, the biodistribution of iodinated exosomes may reflect the biodistribution of denatured exosomes rather than functionally intact ones.

Bispecific aptamer-decorated and light-triggered nanoparticles targeting tumor and stromal cells in breast cancer derived organoids: implications for precision phototherapies

BackgroundBased on the established role of cancer-stroma cross-talk in tumor growth, progression and chemoresistance, targeting interactions between tumor cells and their stroma provides new therapeutic approaches. Dual-targeted nanotherapeutics selectively acting on both tumor and stromal cells may overcome the limits of tumor cell-targeting single-ligand nanomedicine due to the complexity of the tumor microenvironment.MethodsGold-core/silica-shell nanoparticles embedding a water-soluble iridium(III) complex as photosensitizer and luminescent probe (Iren-AuSiO2_COOH) were efficiently decorated with amino-terminated EGFR (CL4) and PDGFRβ (Gint4.T) aptamers (Iren-AuSiO2_Aptamer). The targeting specificity, and the synergistic photodynamic and photothermal effects of either single- and dual-aptamer-decorated nanoparticles have been assessed by confocal microscopy and cell viability assays, respectively, on different human cell types including mesenchymal subtype triple-negative breast cancer (MES-TNBC) MDA-MB-231 and BT-549 cell lines (both EGFR and PDGFRβ positive), luminal/HER2-positive breast cancer BT-474 and epidermoid carcinoma A431 cells (only EGFR positive) and adipose-derived mesenchymal stromal/stem cells (MSCs) (only PDGFRβ positive). Cells lacking expression of both receptors were used as negative controls. To take into account the tumor-stroma interplay, fluorescence imaging and cytotoxicity were evaluated in preclinical three-dimensional (3D) stroma-rich breast cancer models.ResultsWe show efficient capability of Iren-AuSiO2_Aptamer nanoplatforms to selectively enter into target cells, and kill them, through EGFR and/or PDGFRβ recognition. Importantly, by targeting EGFR+ tumor/PDGFRβ+ stromal cells in the entire tumor bulk, the dual-aptamer-engineered nanoparticles resulted more effective than unconjugated or single-aptamer-conjugated nanoparticles in either 3D spheroids cocultures of tumor cells and MSCs, and in breast cancer organoids derived from pathologically and molecularly well-characterized tumors.ConclusionsOur study proposes smart, novel and safe multifunctional nanoplatforms simultaneously addressing cancer-stroma within the tumor microenvironment, which are: (i) actively delivered to the targeted cells through highly specific aptamers; (ii) localized by means of their luminescence, and (iii) activated via minimally invasive light, launching efficient tumor death, thus providing innovative precision therapeutics. Given the unique features, the proposed dual targeted nanoformulations may open a new door to precision cancer treatment.

The antiapoptotic effects of conditioned medium from bone marrow-derived mesenchymal stromal stem cells on cyclophosphamide-induced testicular damage in rat: An experimental study.

Cyclophosphamide (CP) has some negative effects on the reproductive system. Stem cells and their metabolites are being utilized to enhance fertility after chemotherapy. This study aimed to investigate the impact of conditioned medium (CM) derived from bone marrow mesenchymal stromal stem cells (BM-MSCs) on the toxic effects of CP on testicles. BM-MSCs were isolated, a CM was collected and 25-fold concentrated. 24 male Wistar rats (8 wk, 200-250 gr) were randomly divided into following groups: control, CP, CP+ Dulbecco's Modified Eagle Medium (DMEM), CP+CM. CP was given at a single dose of 100 mg/kg. 2 wk after the CP administration, CM was injected into the testicular efferent duct. Sperm parameters, testicular histopathology, and the level of testosterone were analyzed 2 months after treatment. The expression of B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) genes were evaluated by real-time polymerase chain reaction. CP had a negative effect on testis histology (p 0.001) and sperm quality (p 0.001). It changed the expression of genes associated with apoptosis (p 0.001). Treatment with CM reduced the expression of Bax (p 0.001), while significantly increasing the expression of Bcl2 (p = 0.01). It improved sperm count (p = 0.03), viability (p 0.001), motility (p 0.001), spermatogonial count (p 0.001), and epithelial thickness of testicular tubules (p = 0.02). These findings suggest that CM produced from BM-MSCs may be valuable for therapeutic approaches in reproductive medicine and may lessen the side effects of CP.

Chemical etching of Ti-6Al-4V biomaterials fabricated by selective laser melting enhances mesenchymal stromal cell mineralization.

Porous titanium scaffolds fabricated by powder bed fusion additive manufacturing techniques have been widely adopted for orthopedic and bone tissue engineering applications. Despite the many advantages of this approach, topological defects inherited from the fabrication process are well understood to negatively affect mechanical properties and pose a high risk if dislodged after implantation. Consequently, there is a need for further post-process surface cleaning. Traditional techniques such as grinding or polishing are not suited to lattice structures, due to lack of a line of sight to internal features. Chemical etching is a promising alternative; however, it remains unclear if changes to surface properties associated with such protocols will influence how cells respond to the material surface. In this study, we explored the response of bone marrow derived mesenchymal stem/stromal cells (MSCs) to Ti-6Al-4V whose surface was exposed to different durations of chemical etching. Cell morphology was influenced by local topological features inherited from the SLM fabrication process. On the as-built surface, topological nonhomogeneities such as partially adhered powder drove a stretched anisotropic cellular morphology, with large areas of the cell suspended across the nonhomogeneous powder interface. As the etching process was continued, surface defects were gradually removed, and cell morphology appeared more isotropic and was suggestive of MSC differentiation along an osteoblastic-lineage. This was accompanied by more extensive mineralization, indicative of progression along an osteogenic pathway. These findings point to the benefit of post-process chemical etching of additively manufactured Ti-6Al-4V biomaterials targeting orthopedic applications.

A division-of-labor mode contributes to the cardioprotective potential of mesenchymal stem/stromal cells in heart failure post myocardial infarction.

Treatment of heart failure post myocardial infarction (post-MI HF) with mesenchymal stem/stromal cells (MSCs) holds great promise. Nevertheless, 2-dimensional (2D) GMP-grade MSCs from different labs and donor sources have different therapeutic efficacy and still in a low yield. Therefore, it is crucial to increase the production and find novel ways to assess the therapeutic efficacy of MSCs. hUC-MSCs were cultured in 3-dimensional (3D) expansion system for obtaining enough cells for clinical use, named as 3D MSCs. A post-MI HF mouse model was employed to conduct in vivo and in vitro experiments. Single-cell and bulk RNA-seq analyses were performed on 3D MSCs. A total of 125 combination algorithms were leveraged to screen for core ligand genes. Shinyapp and shinycell workflows were used for deploying web-server. 3D GMP-grade MSCs can significantly and stably reduce the extent of post-MI HF. To understand the stable potential cardioprotective mechanism, scRNA-seq revealed the heterogeneity and division-of-labor mode of 3D MSCs at the cellular level. Specifically, scissor phenotypic analysis identified a reported wound-healing CD142+ MSCs subpopulation that is also associated with cardiac protection ability and CD142- MSCs that is in proliferative state, contributing to the cardioprotective function and self-renewal, respectively. Differential expression analysis was conducted on CD142+ MSCs and CD142- MSCs and the differentially expressed ligand-related model was achieved by employing 125 combination algorithms. The present study developed a machine learning predictive model based on 13 ligands. Further analysis using CellChat demonstrated that CD142+ MSCs have a stronger secretion capacity compared to CD142- MSCs and Flow cytometry sorting of the CD142+ MSCs and qRT-PCR validation confirmed the significant upregulation of these 13 ligand factors in CD142+ MSCs. Clinical GMP-grade 3D MSCs could serve as a stable cardioprotective cell product. Using scissor analysis on scRNA-seq data, we have clarified the potential functional and proliferative subpopulation, which cooperatively contributed to self-renewal and functional maintenance for 3D MSCs, named as "division of labor" mode of MSCs. Moreover, a ligand model was robustly developed for predicting the secretory efficacy of MSCs. A user-friendly web-server and a predictive model were constructed and available (https://wangxc.shinyapps.io/3D_MSCs/).

Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal.

During the progression of knee osteoarthritis (OA), the synovium and infrapatellar fat pad (IFP) can serve as source for Substance P (SP) and calcitonin gene-related peptide (CGRP), two important pain-transmitting, immune, and inflammation modulating neuropeptides. Our previous studies showed that infrapatellar fat pad-derived mesenchymal stem/stromal cells (MSC) acquire a potent immunomodulatory phenotype and actively degrade Substance P via CD10 both in vitro and in vivo. On this basis, our hypothesis is that CD10-bound IFP-MSC sEVs can be engineered to target CGRP while retaining their anti-inflammatory phenotype. Herein, human IFP-MSC cultures were transduced with an adeno-associated virus (AAV) vector carrying a GFP-labelled gene for a CGRP antagonist peptide (aCGRP). The GFP positive aCGRP IFP-MSC were isolated and their sEVs' miRNA and protein cargos were assessed using multiplex methods. Our results showed that purified aCGRP IFP-MSC cultures yielded sEVs with cargo of 147 distinct MSC-related miRNAs. Reactome analysis of miRNAs detected in these sEVs revealed strong involvement in the regulation of target genes involved in pathways that control pain, inflammation and cartilage homeostasis. Protein array of the sEVs cargo demonstrated high presence of key immunomodulatory and reparative proteins. Stimulated macrophages exposed to aCGRP IFP-MSC sEVs demonstrated a switch towards an alternate M2 status. Also, stimulated cortical neurons exposed to aCGRP IFP-MSC sEVs modulate their molecular pain signaling profile. Collectively, our data suggest that yielded sEVs can putatively target CGRP in vivo, while containing potent anti-inflammatory and analgesic cargo, suggesting the promise for novel sEVs-based therapeutic approaches to diseases such as OA.

PLOD2, a key factor for MRL MSC metabolism and chondroprotective properties

BackgroundInitially discovered for its ability to regenerate ear holes, the Murphy Roth Large (MRL) mouse has been the subject of multiple research studies aimed at evaluating its ability to regenerate other body tissues and at deciphering the mechanisms underlying it. These enhanced abilities to regenerate, retained during adulthood, protect the MRL mouse from degenerative diseases such as osteoarthritis (OA). Here, we hypothesized that mesenchymal stromal/stem cells (MSC) derived from the regenerative MRL mouse could be involved in their regenerative potential through the release of pro-regenerative mediators.MethodTo address this hypothesis, we compared the secretome of MRL and BL6 MSC and identified several candidate molecules expressed at significantly higher levels by MRL MSC than by BL6 MSC. We selected one candidate, Plod2, and performed functional in vitro assays to evaluate its role on MRL MSC properties including metabolic profile, migration, and chondroprotective effects. To assess its contribution to MRL protection against OA, we used an experimental model for osteoarthritis induced by collagenase (CiOA).ResultsAmong the candidate molecules highly expressed by MRL MSC, we focused our attention on procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2). Plod2 silencing induced a decrease in the glycolytic function of MRL MSC, resulting in the alteration of their migratory and chondroprotective abilities in vitro. In vivo, we showed that Plod2 silencing in MRL MSC significantly impaired their capacity to protect mouse from developing OA.ConclusionOur results demonstrate that the chondroprotective and therapeutic properties of MRL MSC in the CiOA experimental model are in part mediated by PLOD2.