Non-conditioned Medium Research Articles

Deciphering the influence of mare oviductal organoid secretions on equine spermatozoa

The oviductal milieu assists fertilization by modulating sperm survival, supporting capacitation, and facilitating transport of early embryos. In many mammals, the direct relationship between the spermatozoa and the oviductal epithelial cells prolong sperm survival by inducing quiescence and suppressing metabolic activity. However, the detailed mechanisms remain unknown. In this study we sought to develop an equine organoid model to examine sperm-oviduct interactions. We aimed to determine whether oviductal secretions alone can induce quiescence, or alternatively, drive capacitation in preparation for fertilization. This was done by exposing spermatozoa to oviductal organoid-conditioned media and assessing sperm motility, viability, mitochondrial function, and capacitation. Oviductal organoids were cultured from single cells derived from the enzymatic digestion of freshly collected mare (n=3) infundibulum. Mature organoids (>21 days) with ciliated and secretory cells were cultured for 24 h with either fresh oviductal epithelial (OE) culture medium (containing Advanced DMEM-F12, Glutamax, HEPES, penicillin-streptomycin and WNT3A-RSPO3-NOGGIN) or Biggers-Whitten-Whittingham medium (BWW). Thereafter, culture media were collected and centrifuged (conditioned medium). Spermatozoa were collected from a stallion of known fertility, isolated using discontinuous gradient centrifugation and diluted to 20 × 106 cells/ml. Spermatozoa were then co-incubated with conditioned and non-conditioned (control) OE, and conditioned and non-conditioned BWW medium. Total (TM) and progressive (PM) motility were assessed at 1h and 24h; viability (via Live-Deadfluorescent stain/flow cytometry), mitochondrial membrane potential (MMP; via JC-1 probe/flow cytometry) and capacitation (via anti-tyrosine phosphorylation immunocytochemistry) were assessed at 24 h. Spermatozoa incubated with conditioned OE media showed significantly reduced motility at 24h (TM: conditioned vs control; 17.1±2.6% vs 43.2±5.5%; p=.025. and PM: conditioned vs control; 3.7±0.4% vs 27.1±2.8%; p=.008). Similar results were seen for BWW. Spermatozoa viability was not significantly affected (conditioned vs control; OE: 58.2±5.8% vs 84.6±5.9%; p>.05) nor was MMP (conditioned vs control; OE: 63.8±6.1% vs 66.9±7.5% of live cells with high MMP; p>.05). For BWW, similar results were recorded for viability (conditioned vs control; 84.5±6.2% vs 75.6±0.05%; p>.05), and MMP (conditioned vs control; 31.3±10.2% vs 56.2±13.5% of live cells with high MMP; p>.05). Observationally, MMP was suppressed in organoid-conditioned BWW exposed spermatozoa in 2 of the 3 replicates (from 41.3% to 6.3%, and from 89.1% to 42% cells with high MMP, concurrent with higher viability in exposed samples). Tyrosine phosphorylation, indicative of capacitation, showed no difference between organoid-conditioned medium exposed spermatozoa and controls. In summary, spermatozoa exposed to oviductal organoid-conditioned medium respond with suppressed motility but remain viable. These findings are consistent with the hypothesis that oviductal epithelial secretions can induce quiescence in the spermatozoa reservoir awaiting arrival of the oocyte.

The Effects of Co-Culture of Embryonic Stem Cells with Neural Stem Cells on Differentiation.

Researching the technology for in vitro differentiation of embryonic stem cells (ESCs) into neural lineages is very important in developmental biology, regenerative medicine, and cell therapy. Thus, studies on in vitro differentiation of ESCs into neural lineages by co-culture are expected to improve our understanding of this process. A co-culture system has long been used to study interactions between cell populations, improve culture efficiency, and establish synthetic interactions between populations. In this study, we investigated the effect of a co-culture of ESCs with neural stem cells (NSCs) in two-dimensional (2D) or three-dimensional (3D) culture conditions. Furthermore, we examined the effect of an NSC-derived conditioned medium (CM) on ESC differentiation. OG2-ESCs lost the specific morphology of colonies and Oct4-GFP when co-cultured with NSC. Additionally, real-time PCR analysis showed that ESCs co-cultured with NSCs expressed higher levels of ectoderm markers Pax6 and Sox1 under both co-culture conditions. However, the differentiation efficiency of CM was lower than that of the non-conditioned medium. Collectively, our results show that co-culture with NSCs promotes the differentiation of ESCs into the ectoderm.

Interaction between mesenchymal stem cells and myoblasts in the context of facioscapulohumeral muscular dystrophy contributes to the disease phenotype.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disease associated with ectopic expression of the DUX4 gene in skeletal muscle. Muscle degeneration in FSHD is accompanied by muscle tissue replacement with fat and connective tissue. Expression of DUX4 in myoblasts stimulates mesenchymal stem cells (MSC) migration via the CXCR4‐CXCL12 axis. MSCs participate in adipose and connective tissue formation and can contribute to fibrosis. Here we studied the interaction between myoblasts and MSCs and the consequences of this interaction in the FSHD context. We used cell motility assays and coculture of MSCs with myoblasts to study their mutual effects on cell migration, differentiation, proliferation, and extracellular matrix formation. The growth medium conditioned by FSHD myoblasts stimulated MSCs migration 1.6‐fold (p < 0.04) compared to nonconditioned medium. Blocking the CXCL12‐CXCR4 axis with the CXCR4 inhibitor (AMD3100) or neutralizing antibodies to CXCL12 abolished this effect. FSHD myoblasts stimulated MSC proliferation 1.5−2 times (p < 0.05) compared to control myoblasts, while the presence of MSCs impaired myoblast differentiation. Under inflammatory conditions, medium conditioned by FSHD myoblasts stimulated collagen secretion by MSCs 2.2‐fold as compared to the nonconditioned medium, p < 0.03. FSHD myoblasts attract MSCs via the CXCL12‐CXCR4 axis, stimulate MSC proliferation and collagen secretion by MSCs. Interaction between MSCs and FSHD myoblasts accounts for several important aspects of FSHD pathophysiology. The CXCL12‐CXCR4 axis may serve as a potential target to improve the state of the diseased muscles.

Abstract A27: Endothelial progenitor cells as drug-delivery Trojan horses for treatment and imaging of cancer

Abstract Introduction: Endothelial progenitor cells (EPCs) are a subtype of stem cells capable of differentiating into mature endothelial cells. Tumor-derived paracrine signals activate the bone marrow-residing EPCs to home to the tumor to promote angiogenesis. The aim of this study was to utilize the EPCs and their specific pathophysiologic homing to tumors as a biologically targeted drug-delivery system, as a novel “Trojan horse” cellular immunotherapy in cancer. Methods: To test the specificity of EPCs’ homing to cancer, chemotaxis of EPCs towards conditioned media derived from a series of cancer cell lines or towards nonconditioned media was performed in vitro using Boyden Chamber. In vivo biodistribution of EPCs was studied in diverse tumor models (localized and disseminated) of different cancers (myeloma, lymphoma, leukemia, breast, lung, pancreatic and glioma) after injecting EPCs intravenously, by analysis of the accumulation of EPCs in the tumors and other organs. To enhance the ability of EPCs to home to tumors, EPCs were “primed” in vitro by exposing them to conditioned media from hypoxic (1% O2) tumor cells, followed by testing the homing of “primed” EPCs to tumors, both in vitro and in vivo. Next, EPCs were loaded with LS-542 (NIR imaging dye), and with Titanocene (Tc) (a nontoxic compound used in phototherapy that can be activated by positrons to become toxic) and tested for the effect of the loading on viability and functionality of EPCs, using MTT and migration assays, respectively. Also, we tested the effect of Tc-loaded EPCs on killing of cancer cells when activated by 18FDG/PET. Results: We found that EPCs specifically homed to tumors in vitro and in vivo, in tumor size-dependent and time-dependent manner, and that EPCs selectively accumulated in tumor tissues, with significantly negligible amounts in other organs. Moreover, EPCs homed to primary tumors and followed the metastatic pattern of the tumor. Hypoxia “priming” of EPCs significantly improved their homing to cancer in vitro and in vivo. Loading EPCs with LS-542 or with Tc did not affect their viability or functionality. Additionally, Tc-loaded EPCs, when activated by 18FDG, induced a dose-dependent killing of cancer cells. Conclusions: EPCs have a highly specific and efficient biologic machinery to target tumors in as a pan-cancer phenomenon, especially when “primed” with hypoxic tumor media. Loading EPCs with LS-542 or Tc did not change their viability and functionality; thus, both are promising molecules for imaging and therapy of cancer. These results provide a proof of concept that EPCs can be used as a novel cellular immunotherapy for specific and efficacious delivery of theranostic agents in cancer. Also, unlike engineered/targeted immunotherapies such as cellular (CAR-T cells), bispecific T cell engagers, or monoclonal antibodies that target specific molecules on a specific tumor type, EPCs can be used as a pan-cancer “Trojan horse” drug-delivery platform due to the universality of (angiogenesis) in all cancer types. Citation Format: Barbara Muz, Pilar de la Puente, Shruti Shah, Cinzia Federico, Feda Azab, Justin King, Mark Fiala, Rui Tang, Partha Karmakar, Ravi Vij, Samuel Achilefu, Abdel Kareem Azab. Endothelial progenitor cells as drug-delivery Trojan horses for treatment and imaging of cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A27.

Impaired cardiac performance, protein synthesis, and mitochondrial function in tumor-bearing mice.

To understand the underlying mechanisms of cardiac dysfunction in cancer, we examined cardiac function, protein synthesis, mitochondrial function and gene expression in a model of heart failure in mice injected with Lewis lung carcinoma (LLC1) cells. Seven week-old C57BL/J6 male and female mice were injected with LLC1 cells or vehicle. Cardiac ejection fraction, ventricular wall and septal thickness were reduced in male, but not female, tumor-bearing mice compared to vehicle-injected control mice. Cardiac protein synthesis was reduced in tumor-bearing male mice compared to control mice (p = 0.025). Aspect ratio and form factor of cardiac mitochondria from the tumor-bearing mice were increased compared control mice (p = 0.042 and p = 0.0032, respectively) indicating a more fused mitochondrial network in the hearts of tumor-bearing mice. In cultured cardiomyocytes maximal oxygen consumption and mitochondrial reserve capacity were reduced in cells exposed to tumor cell-conditioned medium compared to non-conditioned medium (p = 0.0059, p = 0.0010). Whole transcriptome sequencing of cardiac ventricular muscle from tumor-bearing vs. control mice showed altered expression of 1648 RNA transcripts with a false discovery rate of less than 0.05. Of these, 54 RNA transcripts were reduced ≤ 0.5 fold, and 3 RNA transcripts were increased by ≥1.5-fold in tumor-bearing mouse heart compared to control. Notably, the expression of mRNAs for apelin (Apln), the apelin receptor (Aplnr), the N-myc proto-oncogene, early growth protein (Egr1), and the transcription factor Sox9 were reduced by >50%, whereas the mRNA for growth arrest and DNA-damage-inducible, beta (Gadd45b) is increased >2-fold, in ventricular tissue from tumor-bearing mice compared to control mice. Lung tumor cells induce heart failure in male mice in association with reduced protein synthesis, mitochondrial function, and the expression of the mRNAs for inotropic and growth factors. These data provide new mechanistic insights into cancer-associated heart failure that may help unlock treatment options for this condition.

Acetylcholinesterase is not a generic marker of extracellular vesicles

ABSTRACTAcetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood.

The Effects of Conditioned Medium Derived from Mesenchymal Stem Cells Cocultured with Hepatocytes on Damaged Hepatocytes and Acute Liver Failure in Rats.

Mesenchymal stem cells (MSCs) and hepatocytes are two attractive sources of cell-based therapies for acute liver failure (ALF). The cotransplantation of hepatocytes with MSCs can improve the therapeutic performance for the treatment of ALF. However, the therapeutic potential of conditioned medium (CM) derived from MSCs cocultured with hepatocytes (MSC-H-CM) remains unclear. The purpose of this study was to investigate the effects of MSC-H-CM on damaged hepatocytes in vitro and on D-galactosamine-induced ALF in vivo. D-Galactosamine-treated L02 cells cultured in MSC-H-CM exhibited higher of cell viability and total protein synthesis than L02 cells cultured in MSC-CM, CM derived from hepatocytes (H-CM), MSC-CM + H-CM, or with nonconditioned medium (NCM). Lactate dehydrogenase and aspartate aminotransferase levels were lower in the supernatant of damaged L02 cells cultured in MSC-H-CM than in that of L02 cells cultured in other types of CM. The lowest percentage of apoptotic cells was observed after the MSC-H-CM treatment. When CM was injected into the tail vein of rats with ALF, MSC-H-CM was the most successful at preventing the release of liver injury biomarkers and in promoting the recovery of liver structure. The greatest survival rate 7 days after the first treatment was observed in the MSC-H-CM-treated rats. Our results reveal that the delivery of MSC-H-CM could be a novel strategy for integrating the therapeutic potentials of hepatocytes and MSCs for the treatment of ALF.

Abstract P1-01-18: Disrupting interactions between human breast cancer cells, adipocytes, and macrophages

Abstract An increasing number of studies have found that other cells in the tumor microenvironment can influence tumor cells. Adipocytes, which were once thought to function only in energy storage, are now considered an active endocrine organ that secretes pro-inflammatory cytokines and proteases. In human breast cancer patients, invading tumor cells are surrounded by mature adipocytes. The breast cancer cells cause the adipocytes to undergo phenotypic changes and overexpress pro-inflammatory cytokines and matrix metalloproteinases (MMPs), such as MMP-3 and MMP-11. The presence of the changed adipocytes then increases the invasion capacity of breast cancer cells. Obesity is an important breast cancer risk factor. It causes changes to adipocytes and the surrounding microenvironment by invasion of macrophage cells. The effects of obesity-induced macrophage invasion have not been well characterized in the sector of breast cancer-adipocyte crosstalk. A novel 3D co-culture system was used to investigate how macrophage-induced inflammation can worsen this crosstalk, and what changes are involved in cytokine expression, adipocyte de-differentiation, breast cancer aggressiveness, and extracellular matrix (ECM) composition. When macrophage conditioned media was added to adipocytes and breast cancer cells in co-culture, the breast cancer cells showed increased cell proliferation and migration abilities. Experimental co-culture (adipocytes, breast cancer cells, macrophage conditioned media) showed almost twice the number of cells than the control co-culture (adipocytes, breast cancer cells, non-conditioned media). Interestingly, breast cancer cells alone in macrophage conditioned media did not show an increase in proliferation. This shows that macrophages may not affect breast cancer proliferation directly, as all three cell types are needed for increased proliferation of breast cancer cells. Introducing macrophages into this crosstalk also resulted in increased migration abilities. After 24 hours, breast cancer cells grown in experimental co-culture migrated further to close a wound than breast cancer cells grown in control co-culture. These results show that macrophages can affect adipocyte-breast cancer cell crosstalk. This may help explain why obese breast cancer patients have worse prognosis than non-obese breast cancer patients. Further investigation into these effects may identify targets to improve prognosis and abate breast cancer aggressiveness in obese breast cancer patients. Citation Format: Vallega KA, Sang Q-XA. Disrupting interactions between human breast cancer cells, adipocytes, and macrophages [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-18.

High-quality human preimplantation embryos actively influence endometrial stromal cell migration

PurposeThe purpose of this paper is to study whether human preimplantation embryos regulate endometrial stromal cell (hESC) migration.MethodsPrimary hESCs were isolated from fertile patients undergoing hysterectomy for benign conditions (uterine scar niche n = 3, dysmenorrhea n = 2; no hormonal treatment). Migration and proliferation assays were performed by culturing decidualized or non-decidualized hESCs in the presence of embryo conditioned medium (ECM) from high-quality embryos (fragmentation ≤ 20%) or from low-quality embryos (fragmentation > 20%) or in non-conditioned medium from the same dishes (control). ECM samples from 425 individually cultured human embryos were used in this study.ResultsECM from high-quality embryos, i.e., with a low percentage of fragmentation, actively stimulated decidualized hESC migration (p < 0.001). This effect was consistent throughout embryonic development from cleavage stage embryos with 2–7 cells (high quality vs. control; p = 0.036), 8–18 cells (high quality vs. control; p < 0.001) to morulae (high quality vs. control; p = 0.003). Additionally, linear regression analysis showed that hESC migration was influenced by embryo quality (fragmentation, β − 0.299; p = 0.025) and not developmental stage (cell number, β 0.177; p = 0.176) or maternal age (β − 0.036; p = 0.78). Opposite to decidualized hESCs, the migration response of non-decidualized hESCs was inhibited by ECM from high-quality embryos (p = 0.019). ECM from low-quality embryos, i.e., with a high percentage of fragmentation, did not cause an altered migration response in decidualized hESCs (p = 0.860) or non-decidualized hESCs (p = 0.986). Furthermore, ECM of both high- and low-quality human embryos did not influence the number of proliferating cells (p = 0.375) and the cell cycle time (p = 0.297) of non-decidualized or decidualized hESCs.ConclusionThis study reveals a mechanism by which high-quality human preimplantation embryos actively interact with the endometrium to increase their chances of successful implantation.

The shift of macrophages toward M1 phenotype promotes aortic valvular calcification

ObjectiveThe purpose of the present study was to comprehensively compare the phenotype profile of infiltrated macrophages in human noncalcified and calcific aortic valves, and to determine whether the shift of macrophage polarization modulates valvular calcification in vitro. MethodsCell surface markers of macrophages and inflammatory cytokines expression in 90 cases of human noncalcified and calcific aortic valve leaflets were analyzed. The normal aortic valve interstitial cells were isolated and cultured in vitro. After incubation with nonconditioned medium and conditioned medium from unstimulated or lipopolysaccharide-stimulated U937 monocytes, valve interstitial cells were evaluated by osteogenic differentiation markers. ResultsInfiltration of macrophages was enhanced in the calcific aortic valves, and M1 phenotype was the predominant macrophage subsets. In addition, both proinflammatory and anti-inflammatory cytokines were significantly upregulated in the calcific aortic valves. Furthermore, lipopolysaccharide-stimulated monocytes presented with increased expression of inducible nitric oxide synthase and high proportional CD11c-positive (M1) macrophages. Conditioned medium from unstimulated monocytes promoted the osteogenic differentiation of valve interstitial cells in vitro, as evidenced by increased markers such as bone morphogenetic protein 2, osteopontin, and alkaline phosphatase. Conditioned medium from M1 macrophages further enhanced valve interstitial cells calcification. Enzyme-linked immunosorbent assay showed that M1 phenotype macrophages secreted tumor necrosis factors α and interleukin 6, and neutralizing antibodies to these 2 proinflammatory cytokines attenuated induction of osteogenic differentiation and calcification by the conditioned media. ConclusionsBoth total numbers and polarization of macrophage influence the process of calcification in human aortic valve. The shift toward M1 phenotype might promote valve interstitial cell calcification.

Chitosan/β-1,3-glucan/hydroxyapatite bone scaffold enhances osteogenic differentiation through TNF-α-mediated mechanism

The role of TNF-α in bone healing process is still unclear and controversial. Although it is commonly believed that TNF-α inhibits osteogenic differentiation, there are few reports that identified a crucial role of TNF-α in enhancing bone regeneration process. The aim of this study was to prove that novel chitosan/β-1,3-glucan/HA scaffold (chit/glu/HA) may promote osteogenic differentiation via TNF-α-mediated mechanism and an autocrine stimulation of osteoblasts. It was demonstrated that normal human fetal osteoblasts (hFOB 1.19) maintained in conditioned medium containing increased level of TNF-α and harvested from hFOB 1.19 cells cultured on the chit/glu/HA scaffold (CM-chit/glu/HA) were in more advanced phase of osteogenic differentiation compared to the osteoblasts cultured in non-conditioned osteogenic medium and conditioned medium harvested from hFOB 1.19 cells cultured on the polystyrene plate. Cells cultured in CM-chit/glu/HA produced significantly more Col I protein, revealed 2-fold higher bALP activity, deposited 3-fold more calcium phosphate, and formed mineralized nodules. Thus, it was demonstrated that novel chit/glu/HA scaffold is promising material for bone regeneration applications to stimulate accelerated new bone formation as it enhances osteogenic differentiation via increasing TNF-α production by osteoblasts.

Infrapatellar fat pad aggravates degeneration of acute traumatized cartilage: a possible role for interleukin-6

The infrapatellar fat pad (IPFP), which is located underneath the patella, close to cartilage surfaces, functions in distributing mechanical load and has been shown to produce cytokines. This study aims to assess the involvement of the IPFP in the progression of post-traumatic osteoarthritis (OA) through investigating the crosstalk between the IPFP and injured cartilage invitro. A single blunt impact (36MPa) on healthy bovine articular cartilage explants was used to generate traumatized cartilage. Conditioned media from IPFP and traumatized cartilage (FP-CM and TC-CM) were prepared separately. After culturing in FP-CM, the posttraumatic cartilage explants were analyzed for expression of cartilage degeneration associated genes and secretion of the interleukin (IL)-6, into the culture medium. The effect of traumatized cartilage on IPFP was studied by treating IPFP-derived adipocytes and IPFP adipose-derived stromal cells (ADSC) with TC-CM followed by analysis of cytokine expression. FP-CM aggravated glycosaminoglycan (GAG) release in traumatized cartilage, but did not significantly affect healthy cartilage. FP-CM raised gene expression of cyclooxygenase-2, inducible nitric oxide synthase, and IL-6 in traumatized cartilage explants, and lowered expression of tissue inhibitor of metalloproteinases-1, 2, 3, compared to non-conditioned medium. Of particular significance is that medium IL-6 levels increased substantially in both FP-CM and FP-CM treated traumatized cartilage cultures. Extrinsic IL-6 treatment of traumatized cartilage simulated part of the effects of FP-CM. TC-CM elevated levels of IL-6 expression in IPFP derived adipocytes and ADSCs. IPFP aggravates post-traumatized cartilage degeneration, and IL-6 is a candidate tissue degeneration mediator.

Activation of platelet protease-activated receptor-1 induces epithelial-mesenchymal transition and chemotaxis of colon cancer cell line SW620

Oncol Rep 33: [Related article:] 2681-2688, 2015; DOI: 10.3892/or.2015.3897 After the publication of the article, it has been brought to the authors' attention by an interested reader that we had made an error regarding the colon cancer cell line in the manuscript. The error relates to Materials and methods, as well as Results, the colon cancer cell line in the Transwell migration assay and Flow cytometric detection of CXCR4 expression is HCT-116 rather than SW620. Accordingly, the correct legends in Figs. 3 and 6 in the paper are HCT-116 cells. This error does not affect the overall conclusions reported in the present study. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this error may have caused.

Short term exposure of BMP4 created novel trophoblast progenitor cells from human pluripotent stem cells

Short term exposure of BMP4 created novel trophoblast progenitor cells from human pluripotent stem cells

Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure

Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24-36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.

Activation of platelet protease-activated receptor-1 induces epithelial-mesenchymal transition and chemotaxis of colon cancer cell line SW620.

The aim of the present study was to examine the role of protease-activated receptor-1 (PAR1)-stimulated platelet activation in the epithelial-mesenchymal transition (EMT) and migration of colon cancer cells, and to identify the underlying mechanisms. TFLLR-NH2, a PAR1 agonist, was used to activate platelets and the platelet supernatants were used to treat the SW620 colon cancer cell line. Expression of E-cadherin and vimentin on SW620 cells was detected by immunofluorescence and western blotting, and the level of the transforming growth factor β1 (TGF-β1) was measured using ELISA following the activation of platelets by TFLLR-NH2. miR-200b expression was detected using quantitative PCR in SW620 cells. In order to investigate the chemotactic ability of the SW620 cells, the expression of CXC chemokine receptor type 4 (CXCR4) was measured by flow cytometry. Transwell migration assays were performed following exposure of the cells to the supernatant of PAR1-activated platelets. SW620 cells cultured in the supernatant of TFLLR-NH2-activated platelets upregulated E-cadherin expression and downregulated the vimentin expression. In the in vitro platelet culture system, a TFLLR-NH2 dose-dependent increase of secreted TGF-β1 was detected in the supernatant. The activation of PAR1 on the platelets led to the inhibition of miR-200b expression in the SW620 cells that were cultured in platelet-conditioned media. The number of SW620 cells that penetrated through the Transwell membrane increased with the dose of TFLLR-NH2 used to treat the platelets. The percentage of CXCR4-positive SW620 cells was significantly higher when they were exposed to the supernatant of platelets cultured for 24 h with PAR1 agonist than when cultured in non-conditioned media (40.89±6.74 vs. 3.47±1.40%, P<0.01). Platelet activation with a PAR1 agonist triggered TGF-β secretion, which induced EMT of SW620 human colon cancer cells via the downregulation of miR-200b expression, and activated platelets had a chemotactic effect on colon cancer cells mediated by the upregulation of CXCR4 on the cell surface.

Characterization of the Hematopoietic Supporting Activity of Osteoblasts Derived from Bone Marrow Mesenchymal Stromal Cells

Osteoblasts (OST) found within the endosteal niche are important regulators of Hematopoietic Stem and Progenitor Cells (HSPC) under steady state and during hematopoietic reconstitution. OST are derived from mesenchymal stromal cells (MSC) following osteogenic differentiation. MSC and OST secrete a wide array of soluble factors that sustain hematopoiesis. Recently, we showed that media conditioned with OST derived from MSC (referred as M-OST) after 6 days of osteogenic differentiation were superior to MSC conditioned media (CM) for the expansion of cord blood (CB) progenitors, and CB cells expanded with M-OST CM supported a more robust engraftment of platelets in NSG mice after transplantation. These findings raised the possibility that M-OST could be superior to MSC for the ex vivoexpansion HSPC.In this study, we set out to test the hypothesis that the growth modulatory activity of M-OST would vary as a function of their maturation status. The objectives were to first monitor the impact of M-OST differentiation and maturation status on the expression of soluble factors that promote HSPC expansion and in second, to investigate the capacity of M-OST CMs prepared from M-OST at distinct stages of differentiation to support the expansion and differentiation of HSPCs in culture. M-OST at distinct stages of differentiation were derived by culturing bone marrow MSC in osteogenic medium for various length of time (3 to 21 days). All CB CD34+ enriched (92±7% purity) cell cultures were done with serum free media conditioned or not with MSC or M-OST and supplemented with cytokines SCF, TPO and FL.We first confirmed the progressive differentiation and maturation of M-OST as a function of osteogenic culture length, which was evident by the induction of the osteogenic transcription factors Osterix, Msx2 and Runx2 mRNAs, the gradual increase in osteopontin and alkaline phosphatase positive cells and quantitative increases in calcium deposit. Next, we investigated the expression in MSC and M-OSTs of genes known to collaborate for the expansion of HSPCs by Q-PCR. Transcript copy numbers for IGFBP-2 increased swiftly during osteogenic differentiation, peaking at day-3 (˃100-fold vs MSC, n=2) and returning below MSC level by day-21. In contrast, ANGPTL members (ANGPTL-1, -2, -3 and -5) remained superior in M-OSTs throughout osteogenic differentiation with expression levels peaking around day 6 (n=2).Next, we tested the capacity of media conditioned with primitive (day-3, -6), semi-mature (day-10, -14) and mature M-OST (day-21) to support the growth of CB cells. All M-OST CMs increased (p˂0.03) the growth of total nucleated cells (TNC) after 6 days of culture compared to non-conditioned medium used as control (mean 2.0-fold, n=4). Moreover, there was a positive correlation between cell growth and M-OST maturation status though differences between the different M-OST CMs tested were not significant. The capacity of M-OST CMs to increase (mean 2-fold, n=4) the expansion of CD34+ cells was also shared by all M-OST CMs (p˂0.05), as supported by significant increases with immature day-3 (mean ± SD of 18 ± 6, p˂0.02) and mature day-21 M-OST CMs (14 ± 5, p˂0.05) vs. control (8 ± 3, n=4). Conversely, expansions of TNC and CD34+ cells in MSC CM cultures were in-between that of control and M-OST CMs cultures. Interestingly, M-OST CMs also modulated the expansion of the HSPC compartment. Indeed, while the expansion of multipotent progenitors defined as CD34+CD45RA+ was promoted in control culture (ratio of 4.5 for CD34+CD45RA+/CD34+CD45RA- cells), M-OST CMs supported greater expansion of the more primitive CD34+CD45RA- HSPC subpopulation reducing the ratio to 3.3±0.4 for M-OST cultures (cumulative mean of 10 cultures, n=2). Moreover, the expansions of CD34+CD38- cells and of the long term HSC-enriched subpopulation (CD34+CD38-CD45RA-Thy1+) in M-OST CM cultures were respectively 2.7- and 2.8-fold greater than those measured in control cultures (n=2-4). Finally, the impact of M-OST CMs on the expansion of myeloid progenitors was investigated using a colony forming assay; expansion of myeloid progenitors were superior in all M-OST CM cultures (1.6±0.2 fold, n=2).In conclusion, our results demonstrate that M-OST rapidly acquire the expression of growth factors known to promote HSPC expansion. Moreover, the capacity of M-OST CMs to support the expansion of HSPCs appears to be a property shared by M-OST at various stages of maturation. DisclosuresNo relevant conflicts of interest to declare.

Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells.

The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 μm). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV.

Human trophoblasts confer resistance to viruses implicated in perinatal infection

Primary human trophoblasts were previously shown to be resistant to viral infection, and able to confer this resistance to nontrophoblast cells. Can trophoblasts protect nontrophoblastic cells from infection by viruses or other intracellular pathogens that are implicated in perinatal infection? Isolated primary term human trophoblasts were cultured for 48-72 hours. Diverse nonplacental human cell lines (U2OS, human foreskin fibroblast, TZM-bl, MeWo, and Caco-2) were preexposed to either trophoblast conditioned medium, nonconditioned medium, or miR-517-3p for 24 hours. Cells were infected with several viral and nonviral pathogens known to be associated with perinatal infections. Cellular infection was defined and quantified by plaque assays, luciferase assays, microscopy, and/or colonization assays. Differences in infection were assessed by Student t test or analysis of variance with Bonferroni correction. Infection by rubella and other togaviruses, human immunodeficiency virus-1, and varicella zoster was attenuated in cells preexposed to trophoblast-conditioned medium (P < .05), and a partial effect by the chromosome 19 microRNA miR-517-3p on specific pathogens. The conditioned medium had no effect on infection by Toxoplasma gondii or Listeria monocytogenes. Our findings indicate that medium conditioned by primary human trophoblasts attenuates viral infection in nontrophoblastic cells. Our data point to a trophoblast-specific antiviral effect that may be exploited therapeutically.

A deletion in chromosome 6q is associated with human abdominal aortic aneurysm

Current efforts to identify the genetic contribution to abdominal aortic aneurysm (AAA) have mainly focused on the assessment of germ-line variants such as single-nucleotide polymorphisms. The aim of the present study was to assess the presence of acquired chromosomal aberrations in human AAA. Microarray data of ten biopsies obtained from the site of main AAA dilatation (AAA body) and three control biopsies obtained from the macroscopically non-dilated neck of the AAA (AAA neck) were initially compared with identified chromosomal aneuploidies using the Chromosomal Aberration Region Miner (ChARM) software. A commonly deleted segment of chromosome bands 6 (q22.1-23.2) was predicted within AAA biopsies. This finding was confirmed by quantitative real-time PCR (qPCR)-based DNA copy number assessments of an independent set of six AAA body and neck biopsies which identified a fold copy number change (∆KCt) of -1±0.35, suggesting the loss of one copy of the long interspersed nucleotide element type 1 (LINE-1) mapped to chromosome 6 (q22.1-23.2). The median relative genomic content of LINE-1 DNA was also reduced in AAA body compared with AAA neck biopsies (1.540 compared with 3.159; P=0.031). A gene important for vascular homoeostasis mapped to 6q23.1, connective tissue growth factor (CTGF), was assessed and found to be significantly down-regulated within AAA bodies compared with AAA necks (0.261 compared with 0.627; P=0.031), as determined by reverse transcription qPCR using total RNA as a template. Histology demonstrated marked staining for macrophages within AAA body biopsies. We found in vitro that the median relative genomic content of LINE-1 DNA in aortic vascular smooth muscle cells (AoSMCs) exposed to pro-inflammatory medium was ~1.5 times greater than that measured in control AoSMCs exposed to non-conditioned medium (3.044 compared with 2.040; P=0.015). Our findings suggest that acquired chromosomal aberrations associated with retrotransposon propagation may predispose to sporadic AAA.