Magnetic-activated Cell Sorting System Research Articles

Quantification of HSP70 Gene Expression and Determination of Capacitation Status of Magnetically Separated Cryopreserved Bovine Spermatozoa at Different Thawing Temperature and Time

The role of heat shock protein in reproduction is widely known as a molecular chaperone in aiding and repairing protein formation when stress occurred. The present objectives were to evaluate the effect of different thawing temperature and time on the expression of HSP70 gene expression and the capacitation status in cryopreserved bovine spermatozoa. Briefly, fresh ejaculates were obtained from three different adult bulls. The semen then underwent a sperm washing technique known as Magnetic Activated Cell Sorting System (MACS) and later on, cryopreserved. The sperm- containing straws were then thawed at five different thawing temperatures and time post-cryostorage; 20°C for 13 s, 37°C for 30 s, 40°C for 7 s, 60°C for 6 s and 80°C for 5 s. The RNA was extracted from each of the sperm’s pellets and converted to cDNA prior to the qPCR process. Capacitation status was then determined by means of CTC assay. The results showed that after the process of amplification, there is a significant different of HSP70 gene expression in MACS process samples when the thawing process was performed at 37°C for 30 s, with p<0.05. Furthermore, the CTC assay also showed that thawing at the same temperature gave less capacitated spermatozoa with p<0.05. As a conclusion, MACS yield spermatozoa with a better expression of HSP70 gene and less capacitated spermatozoa when thawing was done at 37°C for 30 s.

Changes in pulmonary endothelial cell properties during bleomycin-induced pulmonary fibrosis

BackgroundPulmonary fibrosis is a progressive and lethal disease characterized by damage to the lung parenchyma with excess extracellular matrix deposition. The involvement of endothelial cells in fibrosis development is unclear.MethodsWe isolated pulmonary endothelial cells, using a magnetic-activated cell sorting system, from mice with pulmonary fibrosis induced by intratracheal bleomycin. We characterized endothelial cells isolated at various times in the course of pulmonary fibrosis development.ResultsInflammatory cell infiltration was observed at 7 days after bleomycin administration, and fibrotic changes with increased collagen content were observed on day 21. Endothelial cells were isolated at these two timepoints. Levels of von Willebrand factor, plasminogen activator inhibitor-1 and matrix metalloproteinase-12 were elevated in lung endothelial cells isolated from bleomycin-treated mice at days 7 and 21. This indicated that intratracheal bleomycin administration induced endothelium injury. Expression of fibrogenic mediators, transforming growth factor (TGF)-β, connective tissue growth factor and platelet-derived growth factor-C was elevated in the cells from bleomycin-treated, compared with untreated, lungs. When endothelial cells were treated with TGF-β, α-smooth muscle actin (SMA) expression and collagen production were increased only in those cells from bleomycin-treated mouse lungs. Thapsigargin-induced prostaglandin I2 and nitric oxide production, decreased in endothelial cells from bleomycin-treated mouse lungs, compared with controls, was further suppressed by TGF-β.ConclusionBleomycin administration induced functional changes in lung endothelial cells, indicating potential involvement of endothelium in pulmonary fibrogenesis.

Identification of CD24 as a marker for tumorigenesis of melanoma.

ObjectiveCutaneous melanoma (CM) is a common skin cancer. Surgery is still the primary treatment for CM, as melanoma is resistant to chemotherapy. In the recent years, it has been found that cancer stem-like cells (CSCs) are responsible for this drug resistance. CD24 is a widely used marker to isolate CSCs. In this study, we aimed to analyze the properties of CD24+ and CD24− subpopulation of melanoma cells.Materials and methodsWe isolated CD24+ cells CSCs using magnetic-activated cell sorting system. We extracted total RNA and carried out reverse transcription polymerase chain reaction analysis. We counted the cell colonies using soft agar assay and assessed the cell invasion using cell migration assay. We implanted CD24+ or CD24− cells into the flank of non-obese diabetic severe combined immunodeficiency mice, and measured the tumor volumes every 5 days until the end of the experiment. We carried out immunohistochemical analysis to study the tissue sections.ResultsWe demonstrated that the CD24+ subpopulation has self-renewal properties in vitro and in vivo by using soft agar assay and xenograft tumor model. Furthermore, we confirmed that CD24 expression is accompanied by activation of Notch1 signaling pathway.ConclusionThis study provides new knowledge on the role of CD24 in the tumorigenic ability of melanoma.

Predominance of CD14+ Cells in Burn Blister Fluids.

Burn blister fluid contains several angiogenic factors to promote wound neovascularization. In our previous study, we found that deep partial-thickness burn (DPTB) wounds showed higher expression levels of angiogenin to enhance vascularization compared with superficial partial-thickness burn wounds. Neovascularization is a complex process that involves an interaction between circulating angiogenic cells and mediators. We hypothesized that in addition to angiogenic factors burn blisters may contain specific cell types. The aim of the present study was to characterize the specific cells present in burn blisters. Twenty-four burn blister fluid samples were obtained with informed consent from patients with superficial partial-thickness burn (n = 16) or DPTB (n = 8) wounds. Blister cells were isolated from individual intact blisters and characterized with flow cytometry analysis using CD14, CD34, vascular endothelial growth factor receptor 2, and CD133 markers. CD14 and CD34 blister cells were also isolated using a magnetic-activated cell sorting system to examine their potential for endothelial differentiation. Angiogenin levels in the burn blister fluids were evaluated with enzyme-linked immunosorbent assay. CD14 cells were the most highly represented cell type in the burn fluids of both groups, although a significantly greater percentage of CD14 cells were observed in DPTB fluids. CD14 blister cells had a higher potency to differentiate into functional endothelial cells as compared with CD34 cells. The proportion of CD14 cells gradually increased after burn injury. In contrast to CD14 cells, angiogenin showed the highest expression levels at day 1 postburn. With regard to burn wound neovascularization, angiogenin expression was partially correlated with CD14 blister cells in the burn fluids. We provide the first report on the characterization of blister cells in burn fluids. Our data suggest that CD14 blister cells may play a role in burn wound neovascularization. Measurement of CD14 blister cells serves as a possible tool for assessing burn wound status.

Enhancing the anti-multiple myeloma efficiency in a cancer stem cell xenograft model by conjugating the ABCG2 antibody with microbubbles for a targeted delivery of ultrasound mediated epirubicin

BackgroundAlthough multiple myeloma (MM) treatment has improved in the last decade, it remains largely incurable. One of main reasons is that there are cancer stem cells (CSCs) in MM, which are responsible for MM’s drug resistance and relapse. In this study, we used the targeting microbubbles (MBs) conjugated with anti-ABCG2 monoclonal antibody (mAb) for ultrasound mediated epirubicin (EPI) delivery to evaluate the therapeutic effectiveness of the novel agent in MM CSC xenograft model. MethodsMM CSCs, marked by CD138−CD34− cell phenotypes were isolated from human MM RPMI8226 cell line using immune magnetic activated cell sorting system, and inoculated into nonobese diabetic/severe combined immunodeficient mice by subcutaneous or intravenous injection. After the mice developed MM, they were intravenous injection treated with EPI, EPI-MBs+mAb, and EPI-MBs+mAb with ultrasound exposure, respectively. ResultsAll treated mice showed inhibited tumor sizes or bone lesions, decreased renal damages and anemia, and increased MM bearing mice’ survival. In particular, the EPI-MBs+mAb plus ultrasound exhibited significantly enhanced therapeutic MM effectiveness by inducing apoptosis compared with other biologic agents. ConclusionThe data provide evidence that EPI-MBs+mAb with ultrasound exposure might be available for treatment MM patients in clinic.

PEGylated long-circulating liposomes deliver homoharringtonine to suppress multiple myeloma cancer stem cells.

The goal of this investigation was to evaluate the inhibiting effect of high proportion polyethyleneglycol of long-circulating homoharringtonine liposomes on RPMI8226 multiple myeloma cancer stem cells. The CD138-CD34- multiple myeloma cancer stem cells isolated from RPMI8226 cell line using magnetic activated cell sorting system were, respectively, incubated with the optimized formulation of polyethyleneglycol of long-circulating homoharringtonine liposomes and the homoharringtonine invitro, and the multiple myeloma cancer stem cell proliferation, colony formation, and cell cycle were analyzed. The inhibition of the multiple myeloma CD138-CD34- cancer stem cell growth was investigated in non-obese-diabetic/severe-combined-immunodeficiency mice that were implanted with multiple myeloma RPMI 8226 cancer stem cells and treated with the LCL-HHT-H-PEG. The results showed that the polyethyleneglycol of long-circulating homoharringtonine liposomes significantly inhibited MM cancer stem cell proliferation, colony formation, and induced cancer stem cell apoptosis invitro as well as MM cancer stem cell growth in non-obese-diabetic/severe-combined-immunodeficiency mice compared with the homoharringtonine. In addition, the mouse bone mineral density and the red blood cell count were significantly increased in polyethyleneglycol of long-circulating homoharringtonine liposomes group. In conclusion, the data demonstrated that the developed polyethyleneglycol of long-circulating homoharringtonine liposomes formulation may serve as an efficient therapeutic drug for suppressing CD138-CD34- multiple myeloma cancer stem cell growth by inducing cancer stem cell apoptosis in non-obese-diabetic/severe-combined-immunodeficiency mouse model. Impact statement Multiple myeloma (MM) remains largely incurable until now. One of the main reasons is that there are cancer stem cells (CSCs) in MM, which are responsible for MM's drug resistance and relapse. In this study, we wanted to extend our previous investigation22 that whether we developed the LCL-HHT-H-PEG formulation have an inhibitory effect on MM CD138-CD34-CSCs in MM CSC engrafted NOD/SCID mouse model. Our data from the present study have demonstrated the therapeutic effect of LCL-HHT-H-PEG on MM-bearing mouse model. The study represents the first attempt to demonstrate that the LCL-HHT-H-PEG formulation is available for treatment MM patients in clinic. Therefore, this finding is important and deserves publication in Experimental Biology and Medicine.

Salinomycin reduces stemness and induces apoptosis on human ovarian cancer stem cell.

ObjectiveCancer stem cells (CSCs) represent a subpopulation of undifferentiated tumorigenic cells thought to be responsible for tumor initiation, maintenance, drug resistance, and metastasis. The role of CSCs in drug resistance and relapse of cancers could significantly affect outcomes of ovarian cancer patient. Therefore, therapies that target CSCs could be a promising approach for ovarian cancer treatment. The antibiotic salinomycin has recently been shown to deplete CSCs. In this study, we evaluated the effect of salinomycin on ovarian cancer stem cells (OCSCs), both alone and in combination with paclitaxel (PTX).MethodsThe CD44+CD117+CSCs were obtained from the ascitic fluid of patients with epithelial ovarian cancer by using an immune magnetic-activated cell sorting system. OCSCs were treated with PTX and salinomycin either singly or in combination. Cell viability and apoptosis assays were performed and spheroid-forming ability was measured. The expression of sex determining region Y-box 2 (SOX2) and octamer-binding transcription factor 3/4 (OCT3/4) mRNA was determined using reverse transcription polymerase chain reaction, and protein expression was observed using western blot analysis.ResultsTreatment with salinomycin alone reduced the stemness marker expression and spheroid-forming ability of OCSCs. Treatment with PTX alone did not decrease the viability of OCSCs. Treatment with a combination of salinomycin decreased the viability of OCSCs and promoted cell apoptosis. The enhancement of combination treatment was achieved through the apoptosis as determined by annexin V/propidium iodide (PI) staining, caspase-3 activity, and DNA fragmentation assay.ConclusionBased on our findings, combining salinomycin with other anti-cancer therapeutic agents holds promise as an ovarian cancer treatment approach that can target OCSCs.

Differentiation of bone marrow-derived stage-specific embryonic antigen 1 positive pluripotent stem cells into male germ cells.

Studies published in recent years have changed the outlook on sterility and germ cell development by producing gametes from stem cells. In present study, a novel approach on differentiation of bone marrow-derived stage-specific embryonic antigen 1 positive (SSEA-1+ ) pluripotent stem cells into male germ cells has been addressed. SSEA-1+ stem cells were separated from murine bone marrow using magnetic-activated cell sorting (MACS) system and propagated on a feeder layer cells. To evaluate the pluripotency characteristic of the purified cells, they were differentiated toward cells of three germ layers. Later the SSEA-1+ stem cells were induced to differentiate along male germ cell lineage with retinoic acid. Flowcytometric analysis of SSEA-1+ stem cells revealed purity of about 62% which increased to 91% after cultivation over feeder cells. Expression of specific transcripts of Oct4, SSEA-1, Nanog, Dppa3, fragilis, Rex-1, SOX-2, and alkaline-phosphatase and immunofluorescence evaluation of Oct4 and SSEA-1 expression showed the differentiation of purified stem cells toward the cells of three germ layers. Differentiation potential of purified cells was positively evidenced by expression markers specific for primordial germ cells, spermatogonial stem cells and spermatogonia including Mvh, fragilis, Dppa3, Stra8, DAZL, Piwil2, β1, and α6-integrins as well as meiotic-specific marker SYCP3. Our results showed that SSEA-1+ pluripotent stem cells are able to differentiate into male germ cells. The results of the present study are encouraging enough to merit further investigation, provide a new hope for those suffering from infertility and introduce a novel platform for research on germ cell development.

CXCL1 from tumor-associated lymphatic endothelial cells drives gastric cancer cell into lymphatic system via activating integrin β1/FAK/AKT signaling

Crosstalk between lymphatic endothelial cells (LECs) and tumor cells in the tumor microenvironment plays a crucial role in tumor metastasis. Our previous study indicated chemokine (C-X-C motif) ligand 1 (CXCL1) from LECs stimulates the metastasis of gastric cancer. However, the mechanism is still unclear. Here, we successfully isolated tumor-associated LECs (T-LECs) and normal LECs (N-LECs) from clinical samples by magnetic-activated cell sorting system (MACS) and proved that CXCL1 expression was elevated in T-LECs compared with N-LECs in situ and vitro. Besides, we demonstrated that CXCL1 secreted by T-LECs promoted the migration, invasion, and adhesion of gastric cancer cells by upregulating integrin β1, MMP2, and MMP9. Furthermore, CXCL1 induced MMP2/9 expression by activating integrin β1-FAK-AKT signaling. In the animal model, CXCL1 overexpressed in LECs increased the lymph node metastasis of gastric cancer. In conclusion, CXCL1 expression in T-LECs was upregulated, and CXCL1 secreted by T-LECs promoted the lymph node metastasis of gastric cancer through integrin β1/FAK/AKT signaling, leading to MMP2 and MMP9 expression. Therefore, CXCL1 produced in T-LECs represents a potentially promising target for treating gastric cancer.

The flavonoid apigenin reduces prostate cancer CD44+ stem cell survival and migration through PI3K/Akt/NF-κB signaling

AimsCancer stem cells (CSCs) are involved in drug resistance, metastasis and recurrence of cancers. The efficacy of apigenin on cell survival, apoptosis, migration and stemness properties were analyzed in CSCs. Main methodsProstate CSCs (CD44+) were isolated from human prostate cancer (PCa) PC3 cells using a magnetic-activated cell sorting system. PC3 and CSCs were treated with various concentrations of apigenin, docetaxel and their combinations for 48h. Key findingsApigenin dose dependently inhibited CSCs and PC3 cell survival, and this was accompanied with a significant increase of p21 and p27. Apigenin induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mRNA expressions of caspases-8, -3 and TNF-α, but failed to regulate the intrinsic pathway as determined by the Bax, cytochrome c (Cyt-c) and APAF-1 in CSCs. In contrary to CSCs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of Bax, Cyt-c and caspase-3 while caspase-8, TNF-α and Bcl-2 levels remained unchanged in PC3 cells. The flavonoid strongly suppressed the migration rate of CSCs compared to untreated cells. Significant downregulation of matrix metallopeptidases-2, -9, Snail and Slug exhibits the ability of apigenin treatment to suppress invasion. The expressions of NF-κB p105/p50, PI3K, Akt and the phosphorylation of pAkt were decreased after apigenin treatment. Moreover, apigenin treatment significantly reduced pluripotency marker Oct3/4 protein expression which might be associated with the down-regulation of PI3K/Akt/NF-κB signaling. SignificanceOur data indicated that, apigenin could be a useful compound to prevent proliferation and migration of cancer cells as well as CSCs.

The effect of salinomycin on ovarian cancer stem-like cells.

ObjectiveThe identification of cancer stem-like cells is a recent development in ovarian cancer. Compared to other cancer cells, cancer stem-like cells present more chemo-resistance and more aggressive characteristics. They play an important role in the recurrence and drug resistance of cancer. Therefore, the target therapy of cancer stem-like cell may become a promising and effective approach for ovarian cancer treatment. It may also help to provide novel diagnostic and therapeutic strategies.MethodsThe OVCAR3 cell line was cultured under serum-free conditions to produce floating spheres. The CD44+CD117+ cell line was isolated from the human ovarian cancer cell line OVCAR3 by using immune magnetic-activated cell sorting system. The expression of stemness genes such as OCT3/4, NANOG and SOX2 mRNA were determined by reverse transcription polymerase chain reaction. OVCAR3 parental and OVCAR3 CD44+CD117+ cells were grown in different doses of paclitaxel and salinomycin to evaluate the effect of salinomycin. And growth inhibition of OVCAR3 CD44+CD117+ cells by paclitaxel combined with salinomycin was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.ResultsTumor spheroids generated from the OVCAR3 cell line are shown to have highly enriched CD44 and CD117 expression. Treatment with a combination of paclitaxel and salinomycin demonstrated growth inhibition of OVCAR3 CD44+CD117+ cells.ConclusionThe present study is a detailed investigation on the expression of CD44 and CD117 in cancer stem cells and evaluates their specific tumorigenic characteristics in ovarian cancer. This study also demonstrates significant growth inhibition of cancer stem-like cells by paclitaxel combined with salinomycin. Identification of these cancer stem-like cell markers and growth inhibition effect of salinomycin may be the next step to the development of novel target therapy in ovarian cancer.

Effect of targeted ovarian cancer immunotherapy using ovarian cancer stem cell vaccine.

BackgroundAccumulating evidence has shown that different immunotherapies for ovarian cancer might overcome barriers to resistance to standard chemotherapy. The vaccine immunotherapy may be a useful one addition to conditional chemotherapy regimens. The present study investigated the use of vaccine of ovarian cancer stem cells (CSCs) to inhibit ovarian cancer growth.MethodsCD117+CD44+CSCs were isolated from human epithelial ovarian cancer (EOC) SKOV3 cell line by using a magnetic-activated cell sorting system. Pre-inactivated CD117+CD44+CSC vaccine was vacccinated into athymic nude mice three times, and then the mice were challenged subcutaneously with SKOV3 cells. The anti-tumor efficacy of CSC vaccine was envaluated by in vivo tumorigenicity, immune efficient analysis by flow cytometer, and enzyme-linked immunosorbent assays, respectively.ResultsThe CD117+ CD44+CSC vaccine increased anti-ovarian cancer efficacy in that it depressed ovarian cancer growth in the athymic nude mice. Vaccination resulted in enhanced serum IFN-γ, decreased TGF-β levels, and increased cytotoxic activity of natural killer cells in the CD117+ CD44+CSC vaccine immunized mice. Moreover, the CSC-based vaccine significantly reduced the CD117+CD44+CSC as well as the aldehyde dehydrogenase 1 positive cell populations in the ovarian cancer tissues in the xenograft mice.ConclusionThe present study provided the first evidence that human SKOV3 CD117+ CD44+CSC-based vaccine may induce the anti-ovarian cancer immunity against tumor growth by reducing the CD117+CD44+CSC population.

Isolation of lung multipotent stem cells using a novel microfluidic magnetic activated cell sorting system.

In recent years, more and more research has shown that the lung is an organ of regenerative potential, with several types of stem/progenitor cells undergoing proliferation and differentiation after lung injury and participating the injury repair process. Mouse lung multipotent stem cells (MLSCs) have extensive self-renewal ability in culture and could differentiate into endothelial and lung epithelial (alveolar epithelial type 1, 2, and Clara) cells in vitro. But the research of MLSCs was limited due to its rarity. In this study, we introduced a novel microfluidic magnetic activated cell sorting system in the isolation of MLSCs. The sorted MLSCs had better viability and purity. They were identified by colony formation efficiency and differentiation ability and they have self-renewal and differentiation capacities, highlighting their stem cell properties.

Antitumor immunostimulatory activity of polysaccharides from Salvia chinensis Benth

Ethnopharmacological relevanceSalvia chinensis Benth (S. chinensis) is a traditional herb applied in the treatment of hepatocellular carcinoma (HCC). Polysaccharides abundantly exist in this plant. However, it remains poorly understood if polysaccharides from S. chinensis (PSSC) contribute to its anti-HCC activity. Materials and methodsThe in vivo anti-HCC activity of PSSC was evaluated in Kunming mice bearing H22 ascitic hepatoma cells. An array of physiological indexes was measured to evaluate toxicological effects on host animals. Subgroups of immune cells were purified by a magnetic-activated cell sorting system and analyzed by flow cytometry. Reverse transcription real-time PCR and immunoblotting were recruited to determine the effects of PSSC on the cellular signaling of different subgroup of immune cells. ResultsPSSC suppressed in vivo proliferation of H22 cells with undetectable toxic effects on tumor-bearing mice. PSSC alleviated tumor transplantation-induced CD4+ T cell apoptosis and dysregulation of serum cytokine profiles, which elevated cytotoxic activities of natural killer and CD8+ T cells. PSSC reduced serum levels of prostaglandin E2 (PGE2). Injection of exogenous PGE2 completely abrogated the antitumor immunostimulatory activity of PSSC. Cyclic adenosine monophosphate (cAMP) is the second messager of PGE2. In CD4+ T cells, PSSC substantially declined intracellular cAMP. This event elevated protein levels of JAK3, enhancing STAT5 phosphorylation and STAT5-depedent expression of anti-apoptotic genes. Cyclooxygenase-2 is the key enzyme mediating biosynthesis of PGE2. PSSC suppressed the transcription and translation of cyclooxygenase-2 in tumor associated macrophages. ConclusionOur data clearly showed antitumor immunostimulatory activity of PSSC against transplanted H22 HCC cells. Suppressing tumor transplantation-induced PGE2 production was implicated in the anti-tumor immunostimulatory activity of PSSC. These works provides novel insights into the traditional application of S. chinensis against HCC and supported considering PSSC as an adjuvant reagent in clinical HCC treatment.

Cell proliferation-inducing protein 52/mitofilin is a surface antigen on undifferentiated human dental pulp stem cells.

Dental pulp is a soft tissue located inside the hard part of a tooth and it contains a stem cell population that can regenerate damaged dentin and/or pulp itself. Human dental pulp stem cells (hDPSCs) are multipotent adult stem cells that have the potential to be differentiated into a variety of cell types. Although cells cultured primarily from pulp tissue show heterogeneous phenotypes and variable efficiency in their dentinogenic differentiation, proper selection markers, which are specific to hDPSCs, are essential for the osteo/dentinogenic study of human dental pulp cells. We had previously screened a set of undifferentiation-specific cell surface antibodies of hDPSCs through decoy immunization. In this study, we show that one of these surface monoclonal antibodies, 3C4, is bound to intact pulp cells in a highly undifferentiation-specific manner. The surface antigen protein bound specifically to 3C4 antibody was identified through direct immunoprecipitation and liquid chromatography-tandem mass spectrometry as the cell proliferation-inducing protein 52/Mitofilin, which is a protein of the inner mitochondrial membrane and is a possible antagonist to maintaining mitochondrial activation during differentiation. The expression of mitofilin/3C4 antigen dramatically decreased during differentiation, and the depletion of mitofilin/3C4 antigen induced the expression of osteogenic/dentinogenic markers earlier than during normal differentiation. The 3C4-positive cells isolated by a magnetic-activated cell sorting system were differentiated with a higher efficiency than 3C4-negative cells. These results indicate that finding mitochondria-related stem cell markers is valuable to be able to identify and isolate primitive stem cells.

Downregulated lincRNA HOTAIR expression in ovarian cancer stem cells decreases its tumorgeniesis and metastasis by inhibiting epithelial-mesenchymal transition.

BackgroundEmerging evidence indicates that dysregulated long intervening non-coding RNA (lincRNA) HOTAIR correlates highly with tumor invasion and metastasis but a link between the high expression of HOTAIR and the metastatic cascade of cancer stem cells (CSCs) needs to be further studied. The purpose of this study was to investigate the effect of down-regulated HOTAIR expression on tumorgeniesis and metastasis of epithelial ovarian cancer (EOC) CSCs. CD117+CD44+CSCs were isolated from human EOC SKOV3 cell line by using a magnetic-activated cell sorting system, and were then transfected with the expression vector-based small hairpin RNA targeting HOTAIR; the stably transfected cells were selected for the study. Colony-forming, wound-healing, cellular metastasis and tumorigenicity assays were performed.ResultsThe results demonstrated that the HOTAIR expression in clinical EOC tissues and SKOV3 CD117+CD44+CSCs was higher than in SKOV3 tumor tissues and non-CD117+CD44+CSCs. The CD117+CD44+-shHOTAIR showed an inhibited HOTAIR expression, reduced cell migration and invasion than CD117+CD44+- scramble, suggesting the inhibition of an epithelial-mesenchymal transition. Moreover, the downregulated HOTAIR expression in CD117+CD44+ CSCs significantly decreased the tumor growth and lung metastasis in xenograft mice.ConclusionOur findings demonstrated the shHOTAIR-mediated down-regulation of the HOTAIR expression in CD117+CD44+ CSCs can be a promising new opportunity for future clinical trials.

In vitro functions of natural killer cells in peripheral blood of patients with severe aplastic anemia

To observe the in vitro killing functions of natural killer (NK) cells for K562 cells in peripheral blood of severe aplastic anemia (SAA) patients and explore the role of NK cells in the immunological pathogenesis of SAA. NK cells (CD3(-)CD56(+)CD16(+)) as effector cells were sorted from peripheral blood mononuclear cells (PBMNC) of 12 SAA patients and 10 normal controls by magnetic activated cell sorting system (MACS)from June 2013 to August 2014. K562 cells as target cells were co-cultured with the effector cells (1: 1, 1: 5, 1: 10). Using Annexin V and PI double staining markers, the apoptotic rate of K562 cells were measured by flow cytometry after culturing. The apoptotic rate of K562 cells in SAA patients was (10.30% ± 6.08%) according to the ratio of effector-target (1: 1). And it was significantly higher than that in normal controls (6.30% ± 3.46%) (P < 0.05). The apoptotic rate of K562 cells in SAA patients were 10.30% ± 6.08%, 16.47% ± 8.29% and 25.45% ± 9.88% respectively according to the ratio of effector-target(1: 1, 5: 1, 10: 1). The higher ratio of effector-target, the higher apoptotic rate of K562 cells. There were statistic differences among three groups (P < 0.05). There are enhanced in vitro functions of NK cells in peripheral blood of SAA patients NK cells may play an important role in abnormal immune tolerance mechanisms of SAA patients.

Ex Vivo Expansion and Characterization of Human Umbilical Cord Blood CD34+ Stem Cells

Background: Human umbilical cord blood (hUCB) has been considered as an alternative source of hematopoietic stem cells (HSCs) for allogeneic cell transplantation owing to its easy availability and less stringent requirement for HLA matching. However, the absolute number of HSCs in hUCB is much smaller than that in bone marrow or mobilized peripheral blood. Therefore, there is a need to develop a reliable and efficient approach for the expansion of HSCs of hUBC in vitro.Method: CD34+ cells were purified from hUCB using magnetic-activated cell sorting system and cultured in IMDM basal medium supplemented with cytokine cocktails. CD34+ cell population was identified by flow cytometry after in vitro expansion. Nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice were randomly allocated into four groups (n=4 for each group). Twenty-four hours after exposure to sub-lethal irradiation of 60Co, CD34+ cells obtained directly from hUCB or from in vitro expansion were transplanted into NOD/SCID mice. Four groups of mice were injected with saline, 0.4 million unexpanded CD34+ cells, 0.4 million in vitro expanded CD34+ cells, and 2.9 million in vitro expanded CD34+ cells, respectively. Multi-lineage differentiation was assessed using antibodies against a panel of cell surface markers (e.g., CD34, CD45, CD11, CD19, CD15, CD71, CD66, CD41a, and CD42b) at 1, 3, and 8 weeks post transplantation. Circulation peripheral blood was also collected for various analyses.Result: Human CD34+ cells were increased about 7.3 folds after 4 days’ culture in vitro. For in vivo mouse study, no cells expressing human cell surface marker were detected in mice injected with saline (negative control). One week after injection with CD34+ cells, human surface markers including CD34, CD45, and CD15 were easily detectable in all three groups. Three weeks after cell injection, CD34+ cells were decreased below 1% in group 2 and 3 but not in group 4. On the other hand, CD45+ and CD15+ populations were increased in groups 2, 3, and 4. There was a strong correlation between the numbers of injected CD34+ cells and differentiated cell populations. This trend was also observed with cells expressing megakaryocytic marker CD41 and erythroid progenitor cell marker CD71 in mouse groups 2 to 4. Eight weeks post transplantation, human CD34+ cells were hardly detected in any groups. However, peripheral blood and bone marrow cells expressing human cell surface markers of various hematopoietic lineages were detectable in groups 2-4. These results indicated that CD34+ cells from hUCB either before or after expansion in vitro are capable of differentiation into hematopoietic cells of various lineages in vivo. Our study also indicates that in vitro expanded CD34+ cells are successfully engrafted to bone marrow of NOD/SCID mice.Conclusion: An efficient experimental method has been developed for the expansion of hUBC CD34+ cells in vitro and these cells can be potentially explored for various applications in the clinic. DisclosuresJiang:Biopharmagen.corp: Employment. Ren:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.

Abstract 217: Salinomycin have antiproliferative and apoptotic effects on ovarian cancer stem-like cell

Abstract OBJECTIVE: Cancer stem cells (CSCs) represent a subpopulation of undifferentiated tumorigenic cells responsible for tumor initiation, maintenance, drug resistance and metastasis of tumors. CSCs involved in drug resistance and relapse of cancers can significantly affect ovarian cancer therapy. The antibiotics salinomycin has recently been shown to be a potent compound to deplete chemoresistant cells in adenocarcinoma. The objective of the present study was to evaluate the effect of salinomycin on ovarian CSC whereby salinomycin mono-and combination treatment with paclitaxol regimend were analyzed. METHODS: The CD44+CD117+CSCs were isolated from the primary ovarian cancer cells derived from ascites fluids of patients with epithelial ovarian cancer by using immune magnetic-activated cell sorting system. To evaluate the effect of salinomycin on ovarian CSC, cells were treated in single and combined treatment. The expression was Nanog, Oct3/4, Sox2 and ABCG2 mRNA was determined by RT-PCR and protein expression was detected by Western blot analysis. The cell viability assay, gelatin zymography and apoptosis assay were applied to evaluate the effects of salinomycin compared with parental tumor cells and CSCs. RESULTS: The combination of salinomycin with paclitaxel (PTX) was enhanced change cell viability or activating apoptosis in CD44+CD117+ cells, whereas the salinomycin monotreatment did not cause significant changes. Salinomycin treatment reduced stemness gene expression and suppressed invasion of CSCs. CONCLUSIONS: Based on our findings, we concluded that anti-cancer effect of salinomycin with PTX reduced stemness and induced apoptosis in ovarian cancer and cancer stem cells. Citation Format: So-Jin Shin, Jin-Young Kim, Hyun-Gyo Lee, Eun-Ji Nam, Chi-Heum Cho. Salinomycin have antiproliferative and apoptotic effects on ovarian cancer stem-like cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 217. doi:10.1158/1538-7445.AM2014-217

Retinoic acid induces mouse bone marrow‐derived CD15+, Oct4+ and CXCR4+ stem cells into male germ‐like cells in a two‐dimensional cell culture system

We have examined the effect of retinoic acid (RA) on differentiation of bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells into male germ cells. Bone marrow stem cells (BMSCs) were isolated from the femur of 3-4-week-old male C57BL/6 mice. Magnetic-activated cell sorting (MACS) system was used to sort CD15(+) , Oct4(+) and CXCR4(+) cells. RT-PCR was used to follow the expression of pluripotency markers. Sorted CD15(+) , Oct4(+) and CXCR4(+) cells were cultured in an undifferentiated condition on a feeder layer of mitomycin C-inactivated C2C12. The embryoid-like bodies were differentiated into male germ cells by retinoic acid. To identify the expression of male germ specific markers, differentiated cells were analysed by means of reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence staining. RT-PCR and immunofluorescence show that bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells express pluripotency markers, Oct4, Nanog, Rex-1, SOX-2 and AP. The purified CD15(+) , Oct4(+) and CXCR4(+) formed structures like embryoid bodies when plated over a feeder layer; these bodies were alkaline phosphatase positive. When cells were induced by RA, bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) were positive for Mvh, Dazl, Piwil2, Dppa3 and Stra8, that known molecular markers of male germ cells. Thus RA can induce differentiation of mouse bone marrow-derived CD15(+) , Oct4(+) and CXCR4(+) cells into male germ cells in vitro. Negative results for the gene expression analysis of female germ cells markers, GDF9 and ZP3, confirmed this conclusion.