Optimizing dopaminergic differentiation of pluripotent stem cells for the manufacture of dopaminergic neurons for transplantation

Background aimsWe have previously described a xeno-free scalable system to generate transplantable dopaminergic neurons from human pluripotent stem cells. However, several important questions remain to be answered about our cell therapy efforts. These include determining the exact time at which cells should be transplanted and whether cells at this stage can be frozen, shipped, thawed and injected without compromising their ability to mature and survive the transplantation procedure. We also needed to determine whether further optimization of the culture process could shorten the development time and reduce variability and whether a current Good Manufacture Practice (CGMP) facility could manufacture cells with fidelity.MethodsWe developed an optimized protocol that included modulating the sonic hedgehog homolog gradient with bone morphogenetic proteins (BMP2) and addition of activin to the culture medium, which shortened the time to generate Lmx1A and FoxA2 immunoreactive cells by 4–6 days.ResultsWe showed that cells at this stage could be safely frozen and thawed while retaining an excellent ability to continue to mature in vitro and survive transplant in vivo. Importantly, we successfully adapted this process to a CGMP facility and manufactured two lots of transplant-ready dopaminergic neurons (>250 vials) under CGMP-compatible conditions. In vitro characterization, including viability/recovery on thawing, whole genome expression as well as expression of midbrain/dopaminergic markers, showed that the cells manufactured under GMP-compatible conditions were similar to cells produced at lab scale.ConclusionsOur results suggest that this optimized protocol can be used to generate dopaminergic neurons for Investigational New Drug enabling studies.     

基于生物阻抗谱的生物细胞活性免标记检测方法

目的 基于生物阻抗谱（bioelectrical impedance spectroscopy,BIS）技术,提出一种免标记的生物细胞活性实时检测方法。该方法依据不同浓度、生理、病理状态下细胞组织的电学特性差异来判断细胞是否具有活性,以协助医师在临床手术中快速精准定位患者烫伤组织并实现有效切除。方法 使用具有活性的斑马鱼胚胎干细胞来模拟人体烫伤组织,采用生物阻抗谱技术来鉴别细胞组织的生理状态。结果 在不同状态下,细胞的阻抗幅值变化有显著的差异,可以从中发现同等浓度下活性细胞的阻抗幅值比死亡细胞平均高出17.25%,活性细胞发生弛豫频率的时间也比死亡细胞早25%。结论 实验数据表明,生物阻抗谱法能有效区分胚胎干细胞的两类生理状态;从聚类区域中可以看出,BIS检测法具有明显的细胞活性及浓度区分能力,理论上能够快速地协助医师完成对患者烫伤组织检测。     

Predicting therapy response in live tumor cells isolated with the flexible micro spring array device

Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response. Here, we demonstrate how the flexible micro spring array (FMSA) device—a novel microfluidic device that enriches CTCs by two physical parameters: size and deformability—could be used in the rational development of treatment intervention and as a method to study the fundamental biology of CTCs. Cancer cells of different origins were spiked into healthy samples of donor blood to mimic blood samples of metastatic cancer patients. This spiked human blood was filtered using the FMSA device, and the recovered cells were successfully expanded in vitro and in a novel in vivo system. A series of experiments were performed to characterize these cells and to investigate the effect of chemotherapy on the resulting cultures. As few as 20 colon cancer cells in 7.5 mL blood could be isolated with the FMSA device, expanded both in vitro and in vivo and used at 25 cells per well to obtain significant and reliable chemosensitivity data. We also show that isolating a low number of viable patient CTCs and maintaining them in culture for a few weeks is possible. The isolation of viable cancer cells from human blood using the FMSA device provides a novel and realistic means for studying the biology of viable CTCs and for testing drug efficacy on these rare cells—a hypothesis that can be tested in future clinical trials.     

Transiently redirected T cells for adoptive transfer

Background aimsT cells can be redirected to reject cancer by retroviral transduction with a chimeric antigen receptor (CAR) or by administration of a bispecific T cell engager (BiTE). We demonstrate that transfection of T cells with messenger (m) RNA coding for CAR is an alternative strategy.MethodsWe describe the pre-clinical evaluation of a method based on transient modification of expanded T cells with a CD19 CAR directed against B-cell malignancies. CAR mRNA was generated under cell-free conditions in a scalable process using recombinant RNA polymerase. Efficient and non-toxic square-wave electroporation was used to load the mRNA into the cytoplasm of T cells with no risk of insertional mutagenesis.ResultsAfter transfection > 80% of T cells were viable, with 94% CAR expression. Transfected T cells were cytolytic to CD19⁺ targets and produced interferon (IFN)-γ in response. Killing of CD19⁺ target cells was demonstrated even at day 8 with undetectable CAR expression. Increasing the concentration of mRNA resulted in higher surface CAR expression, better killing and more IFN-γ release but at the expense of increased activation-induced cell death. Finally, we demonstrated that a second transgene could be introduced by co-electroporation of CXCR4 or CCR7 with CAR to also modify chemotactic responses.ConclusionsWe advocate the transient redirection approach as well suited to meet safety aspects for early phase studies, prior to trials using stably transduced cells once CAR has been proven safe. The simplicity of this methodology also facilitates rapid screening of candidate targets and novel receptors in pre-clinical studies.     

Glycosylation in viral hepatitis

BackgroundThe interaction between hepatitis viruses and host cells is regulated by glycans exposed on the surfaces of human and viruses cells. As the biosynthesis and degradation of human glycoproteins take place at the highest level in the liver, the changes in glycosylation of serum proteins may potentially be useful in the diagnosis of liver pathology. On the other hand, specific alterations in viruses envelope glycans could cause large changes in the entry process of hepatitis viruses into a host cells.Scope of reviewUnique alterations in glycosylation of specific proteins can be detected in HBV and HCV infected patients especially with confirmed fibrosis/cirrhosis. On the other hand, viral envelope proteins that bind to host cells are glycosylated. These glycosylated proteins play a key role in recognition, binding and penetration of the host cells. In this review we summarized the knowledge about significance of glycosylation for viral and host factors.Major conclusionsGlycosylation changes in single serum glycoproteins are noticed in the sera of patients with viral hepatitis. However, a more specific biomarker for the diagnosis of chronic hepatitis than that of a single glycosylated molecule is systemic investigation of complete set of glycan structures (N-glycome). Glycans play important roles in the viral biology cycle especially as a connecting element with host receptors.General significanceThe interaction between virus glycoproteins and cellular receptors, which are also glycoproteins, determines the possibility of virus penetration into host cells. Therefore these glycans can be the targets for the developing of novel treatment strategies of viral hepatitis.     

Extracellular cytochrome c as an intercellular signaling molecule regulating microglial functions

BackgroundCytochrome c is well known to be released from mitochondria into the cytosol where it can initiate apoptosis. Recent studies indicate that cytochrome c is also released into the extracellular space by both healthy and damaged cells, where its function is not well understood. We hypothesized that extracellular cytochrome c could function as an intercellular signaling molecule of the brain, which is recognized by brain microglia. These cells belong to the mononuclear phagocyte system and can be activated by endogenous substances associated with diverse pathologies including trauma, ischemic damage and neurodegenerative diseases.MethodsThree different cell types were used to model microglia. Respiratory burst activity, nitric oxide production and cytotoxic secretions were measured following exposure of microglial cells to cytochrome c.ResultsWe showed that extracellular cytochrome c primed the respiratory burst response of differentiated HL-60 cells, enhanced nitric oxide secretion by BV-2 cells, and augmented cytotoxicity of differentiated THP-1 cells. We demonstrated that the effects of cytochrome c on microglia-like cells were at least partially mediated by the toll-like receptor 4 (TLR4) and c-Jun N-terminal kinases (JNK) signaling pathway.ConclusionsExtracellular cytochrome c can interact with microglia TLR4 and modulate select functions of these brain immune cells.General significanceOur data identifies extracellular cytochrome c as a potential intercellular signaling molecule, which may be recognized by microglia causing or enhancing their immune activation. The data obtained support targeting TLR4 and JNK signaling as potential treatment strategies for brain diseases characterized by excessive cellular death and activation of microglia.     

Investigation into mechanisms mediating the inhibitory effect of 1,4-benzodiazepines on mast cells by gene expression profiling

AimsThis study aims to identify by a molecular genetic approach potential targets in mast cells at which 1,4-benzodiazepines may cause their inhibitory effect on mast cell activity.Main methodsGene expression analyses with microarray gene chip and/or quantitative PCR were performed using 1,4-benzodiazepine-treated human mast cell leukemia HMC-1.2 cells, promyelocytic leukemia HL-60 cells and human mast cells from healthy volunteers and patients with mast cell activation disease (MCAD). Pathway analysis was applied to search for enriched biological functions and canonical pathways within differentially regulated genes.Key findingsBoth neoplastic and normal human mast cells express several GABA_A receptor subunits at the mRNA level. In mast cells from MCAD patients expression of some GABA_A receptor subunits and expression of the translocator protein TSPO are increased compared with those from healthy controls. Expression of the protein tyrosine kinases Lyn, Fgr and Yes1 was increased in HMC-1.2 cells as compared with the ontogenetically related HL60 cells. Differences in gene regulation in HMC-1.2 cells after treatment with the 1,4-benzodiazepines clonazepam, flunitrazepam and 4-chlorodiazepam suggested that signaling and gene expression induced by clonazepam was similar to that of flunitrazepam but different from that of 4-chlorodiazepam. This conclusion is supported by the results of the pathway analysis.SignificanceA novel type of GABA_A receptors on mast cells appears to be involved in the inhibition of mast cell activity by 1,4-benzodiazepines. These receptors seem to be composed without γ subunits suggesting unique pharmacological properties. An action at Src-kinases, or at TSPO located in the plasma membrane may also be involved.     

A multimerized form of recombinant human CD40 ligand supports long-term activation and proliferation of B cells

Background aimsCD40-activated B cells have long been studied as potent antigen-presenting cells that can potentially be used for cancer immunotherapy. Nevertheless, their use in human clinical trials has been limited by the lack of a Good Manufacturing Practice–grade soluble human CD40 ligand that is able to induce activation and proliferation of primary B cells. We describe an in vitro method to effectively generate and expand B cells through the use of a multimerized form of human recombinant CD40 ligand (rCD40L).MethodsHuman B cells were isolated from healthy donors and cultivated with either rCD40L or on a monolayer of murine NIH3T3 cells stably expressing human CD40L (NIH3T3/tCD40L) as a widely used standard method. Morphology, expansion rate, immune phenotype and antigen presentation function were assessed.ResultsB cells efficiently proliferated in response to rCD40L over 14 days of culture in comparable amounts to NIH3T3/tCD40L. B-cell division in response to CD40L was also confirmed by carboxyfluorescein succinimidyl ester dilution. Moreover, rCD40L induced on B cells upregulation of co-stimulatory molecules essential for antigen presentation. Additionally, proliferation of T cells from allogeneic healthy volunteers confirmed the immunostimulatory capacities of CD40-activated B cells.ConclusionsWe demonstrated that B cells with potent antigen presentation capacity can be generated and expanded by use of a non-xenogeneic form of CD40L that could be implemented in future human clinical settings.     

Hematopoietic stem cell (CD34+) uptake of superparamagnetic iron oxide is enhanced by but not dependent on a transfection agent

Background aimsTracking the fate of cells after infusion would be a valuable asset for many stem cell therapies, but very few (cell) labels are approved for human therapeutic use. Superparamagnetic iron oxide particles (SPIO) can be internalized into stem cells in vitro to allow real-time tracking with gradient echo magnetic resonance imaging, but SPIO are approved for (diagnostic) imaging and not for (therapeutic) cell labeling in vivo. In this study, we investigated the possibility of labeling stem cells with an SPIO approved for patient use, albeit in a novel manner by enhancing uptake with the use of a transfection agent, also approved for patient use. Although there are many reports of hematopoietic stem cells being labeled with SPIO, there is some controversy regarding the efficiency of this and whether undifferentiated CD34+ progenitor (stem) cells are able to take up iron in the absence of a transfection agent to enhance the process.MethodsHuman CD34+ cells were treated in vitro as follows: incubation with (i) medium only (control), (ii) ferumoxide (Endorem) and (iii) ferumoxide (Endorem) plus exposure to a transfection agent (protamine sulfate). Cells were incubated for 2, 4 and 24 hours and assessed for viability, differentiation capacity and visualized in vitro with 3-T magnetic resonance imaging. The cells were also analyzed by means of flow cytometry and morphology examined by electron microscopy.ResultsCD34+ hematopoietic progenitor cells can internalize ferumoxide (Endorem) independently of a transfection agent. However, uptake of ferumoxide is enhanced after exposure to protamine sulfate. Iron labeling of CD34+ cells in this manner does not affect cell viability and does not appear to affect the potential of the cells to grow in culture. Iron-labeled CD34+ cells can be visualized in vitro on 3-T magnetic resonance image scanning.ConclusionsEndorem and protamine sulfate can be combined to promote iron oxide nanoparticle uptake by CD34+ cells, and this methodology can potentially be used to track the fate of cells in a clinical trial setting because both compounds are (separately) approved for clinical use.     

Treatment of patients with advanced cancer with the natural killer cell line NK-92

Background aimsNatural killer (NK) cells, either naive or genetically engineered, are increasingly considered for cellular therapy of patients with malignancies. When using NK cells from peripheral blood, the number of expanded NK cells can be highly variable and the need for NK cell enrichment can make the process expensive. The NK-92 cell line (CD56+/CD3?) that was isolated from a patient with lymphoma has predictable high cytotoxic activity and can be expanded under good manufacturing practice conditions in recombinant interleukin-2.MethodsFifteen patients (age, 9–71 years) with advanced, treatment-resistant malignancies, either solid tumors/sarcomas (n = 13) or leukemia/lymphoma (n = 2), received two infusions of NK-92 cells, given 48 h apart. Three cohorts of patients were treated with escalating doses of NK-92 cells (n = 7 at 1 × 10⁹, n = 6 at 3 × 10⁹ and n = 2 at 1 × 10¹⁰ cells/m²).ResultsNo infusion-related or long-term side effects were observed. The dose of 10¹⁰ cells/m² was considered the maximum expandable cell dose with the use of an established culture bag system. Three fourths of patients with lung cancer had some anti-tumor response. Only one patient of seven had development of human leukocyte antigen antibodies. The persistence of NK-92 cells (male origin) in the circulation was confirmed by Y chromosome–specific polymerase chain reaction in two female patients.ConclusionsInfusions of NK-92 cells up to 10¹⁰ cells/m² were well tolerated. Despite the allogeneic nature of NK-92, development of human leukocyte antigen antibodies in these patients with cancer appears to be rare. The cells can persist in the recipient's circulation for at least 48 h. Some encouraging responses were seen in patients with advanced lung cancer.     

Uptake and delivery of antigens by mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSCs) are multipotent stem cells with immunosuppressive properties. Nevertheless, it has been previously reported that MSCs might also trigger the immune response. We studied whether MSCs may act as carriers, capturing antigens that can be endocytosed by antigen-presenting cells later on.MethodsWe measured the cellular uptake of mannose receptor-mediated fluid phase macropinocytosis, assessed as cellular uptake of fluorescein isothiocyanate-dextran, and PKH-67-labeled cell lysates as a surrogate marker for antigen capture among dendritic cells (DCs, positive control), T lymphocytes (negative control) and MSCs.ResultsAll experiments confirmed that MCSs displayed pinocytic and endocytic capacities, which were lower than those observed for DCs but significantly higher than those observed for T cells. We also demonstrated that MSCs release previously endocytosed antigens, which subsequently can be captured by DCs.ConclusionsMSCs have the ability to capture and release antigens.     

Mesenchymal stem cells and differentiated insulin producing cells are new horizons for pancreatic regeneration in type I diabetes mellitus

BackgroundDiabetes mellitus has become the third human killer following cancer and cardiovascular disease. Millions of patients, often children, suffer from type 1 diabetes (T1D). Stem cells created hopes to regenerate damaged body tissues and restore their function.AimThis work aimed at clarifying and comparing the therapeutic potential of differentiated and non-differentiated mesenchymal stem cells (MSCs) as a new line of therapy for T1D.Methods40 Female albino rats divided into group I (control): 10 rats and group II (diabetic), III and IV, 10 rats in each, were injected with streptozotocin (50 mg/kg body weight). Group III (MSCs) were transplanted with bone marrow derived MSCs from male rats and group IV (IPCs) with differentiated insulin producing cells. Blood and pancreatic tissue samples were taken from all rats for biochemical and histological studies.ResultsMSCs reduced hyperglycemia in diabetic rats on day 15 while IPCs normalizes blood glucose level on day 7. Histological and morphometric analysis of pancreas of experimental diabetic rats showed improvement in MSCs-treated group but in IPCs-treated group, β-cells insulin immunoreactions were obviously returned to normal, with normal distribution of β-cells in the center and other cells at the periphery. Meanwhile, most of the pathological lesions were still detected in diabetic rats.ConclusionMSCs transplantation can reduce blood glucose level in recipient diabetic rats. IPCs initiate endogenous pancreatic regeneration by neogenesis of islets. IPCs are better than MSCs in regeneration of β-cells. So, IPCs therapy can be considered clinically to offer a hope for patients suffering from T1D.     

Bicistronic CAR-T cells targeting CD123 and CLL1 for AML to reduce the risk of antigen escape

PurposeAcute myeloid leukemia (AML) is a highly heterogeneous neoplastic disease with a poor prognosis that relapses even after its treatment with chimeric antigen receptor (CAR)-T cells targeting a single antigen. CD123 and CLL1 are expressed in most AML blasts and leukemia stem cells, and their low expression in normal hematopoietic stem cells makes them ideal targets for CAR-T. In this study, we tested the hypothesis that a new bicistronic CAR targeting CD123 and CLL1 can enhance antigenic coverage and prevent antigen escape and subsequent recurrence of AML.MethodsCD123 and CLL1 expressions were evaluated on AML cell lines and blasts. Then, in addition to concentrating on CD123 and CLL1, we introduced the marker/suicide gene RQR8 with a bicistronic CAR. Xenograft models of disseminated AML and in vitro coculture models were used to assess the anti-leukemia efficacy of CAR-T cells. The hematopoietic toxicity of CAR-T cells was evaluated in vitro by colony cell formation assays. It was demonstrated in vitro that the combination of rituximab and NK cells caused RQR8-mediated clearance of 123CL CAR-T cells.ResultsWe have successfully established bicistronic 123CL CAR-T cells that can target CD123 and CLL1. 123CL CAR-T cells effectively cleared AML cell lines and blasts. They also demonstrated appreciable anti-AML activity in animal transplant models. Moreover, 123CL CAR-T cells can be eliminated in an emergency by a natural safety switch and don't target hematopoietic stem cells.ConclusionsThe bicistronic CAR-T cells targeting CD123 and CLL1 may be a useful and secure method for treating AML.     

Phosphorylation-dependent subcellular redistribution of small myosin light chain kinase

BackgroundMyosin light chain kinase (MLCK) is a Ca²⁺-calmodulin-dependent enzyme dedicated to phosphorylate and activate myosin II to provide force for various motile processes. In smooth muscle cells and many other cells, small MLCK (S-MLCK) is a major isoform. S-MLCK is an actomyosin-binding protein firmly attached to contractile machinery in smooth muscle cells. Still, it can leave this location and contribute to other cellular processes. However, molecular mechanisms for switching the S-MLCK subcellular localization have not been described.MethodsSite-directed mutagenesis and in vitro protein phosphorylation were used to study functional roles of discrete in-vivo phosphorylated residues within the S-MLCK actin-binding domain. In vitro co-sedimentation analysis was applied to study the interaction of recombinant S-MLCK actin-binding fragment with filamentous actin. Subcellular distribution of phosphomimicking S-MLCK mutants was studied by fluorescent microscopy and differential cell extraction.ResultsPhosphorylation of S-MLCK actin-binding domain at Ser25 and/or Thr56 by proline-directed protein kinases or phosphomimicking these posttranslational modifications alters S-MLCK binding to actin filaments both in vitro and in cells, and induces S-MLCK subcellular translocation with no effect on the enzyme catalytic properties.ConclusionsPhosphorylation of the amino terminal actin-binding domain of S-MLCK renders differential subcellular targeting of the enzyme and may, thereby, contribute to a variety of context-dependent responses of S-MLCK to cellular and tissue stimuli.General significanceS-MLCK physiological function can potentially be modulated via phosphorylation of its actin recognition domain, a regulation distinct from the catalytic and calmodulin regulatory domains.     

Morphological and mechanical stability of bladder cancer cells in response to substrate rigidity

BackgroundMorphology of cells can be considered as an interplay between the accessibility of substrate anchoring sites, cytoskeleton properties and cellular deformability. To withstand tension induced by cell's environment, cells tend to spread out and, simultaneously, to remodel actin filament organization.MethodsIn this context, the use of polyacrylamide hydrogel substrates with a surface coated with laminin allows to trace remodeling of actin cytoskeleton during the interaction of cells with laminin-rich basement membrane. Reorganization of actin cortex can be quantified by a surface spreading area and deformability of single cells.ResultsIn our study, we demonstrated that morphological and mechanical alterations of bladder cancer cells in response to altered microenvironment stiffness are of biphasic nature. Threshold-dependent relations are induced by mechanical properties of cell microenvironment. Initially, fast alterations in cellular capability to spread and to deform are followed by slow-rate changes. A switch provided by cellular deformability threshold, in the case of non-malignant cells, triggers the formation of thick actin bundles accompanied by matured focal adhesions. For cancer cells, cell spreading and deformability thresholds switch between slow and fast rate of changes with weak reorganization of actin filaments and focal adhesions formation.ConclusionsThe presence of transition region enables the cells to achieve a morphological and mechanical stability, which together with altered expression of vinculin and integrins, can contribute to invasiveness of bladder cancers.General significanceOur findings show that morphological and mechanical stability is directly related to actin filament organization used by cancer cells to adapt to altered laminin-rich microenvironment.     

Survival and engraftment of dopaminergic neurons manufactured by a Good Manufacturing Practice-compatible process

Background aimsWe have previously reported a Good Manufacturing Practice (GMP)-compatible process for generating authentic dopaminergic neurons in defined media from human pluripotent stem cells and determined the time point at which dopaminergic precursors/neurons (day 14 after neuronal stem cell [NSC] stage) can be frozen, shipped and thawed without compromising their viability and ability to mature in vitro. One important issue we wished to address is whether dopaminergic precursors/neurons manufactured by our GMP-compatible process can be cryopreserved and engrafted in animal Parkinson disease (PD) models.MethodsIn this study, we evaluated the efficacy of freshly prepared and cryopreserved dopaminergic neurons in the 6-hydroxydopamine-lesioned rat PD model.ResultsWe showed functional recovery up to 6 months post-transplantation in rats transplanted with our cells, whether freshly prepared or cryopreserved. In contrast, no motor improvement was observed in two control groups receiving either medium or cells at a slightly earlier stage (day 10 after NSC stage). Histologic analysis at the end point of the study (6 months post-transplantation) showed robust long-term survival of donor-derived tyrosine hydroxylase (TH)⁺ dopaminergic neurons in rats transplanted with day 14 dopaminergic neurons. Moreover, TH⁺ fibers emanated from the graft core into the surrounding host striatum. Consistent with the behavioral analysis, no or few TH⁺ neurons were detected in animals receiving day 10 cells, although human cells were present in the graft. Importantly, no tumors were detected in any grafted rats, but long-term tumorigenic studies will need to determine the safety of our products.ConclusionsDopaminergic neurons manufactured by a GMP-compatible process from human ESC survived and engrafted efficiently in the 6-OHDA PD rat model.     

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.     

人脐带间充质干细胞条件培养基联合白藜芦醇对人滋养层细胞的作用

目的:探讨人脐带间充质干细胞条件培养基联合白藜芦醇对人绒毛膜外滋养层细胞凋亡的影响。方法:通过CCK8细胞活力检测试剂盒测定白藜芦醇及其与人脐带间充质干细胞条件培养基共同处理人绒毛膜外滋养层细胞HTR8后对细胞增殖及活性的影响;细胞迁移试验检测白藜芦醇和人脐带间充质干细胞条件培养基对细胞迁移能力的影响;显微镜观察细胞形态,并用流式细胞仪检测细胞凋亡率的变化;Western blot检测白藜芦醇和人脐带间充质干细胞条件培养基对细胞凋亡相关蛋白Bax、Bcl-2以及迁移相关蛋白MMP-9表达的影响。结果:白藜芦醇能够抑制HTR8细胞增殖,抑制细胞迁移及MMP-9蛋白的表达,改变Bax和Bcl-2蛋白表达诱导细胞凋亡的作用。而人脐带间充质干细胞条件培养基能够逆转白藜芦醇对细胞的抑制作用。结论:人脐带间充质干细胞条件培养基能够通过调控Bax、Bcl-2、MMP-9的蛋白表达逆转白藜芦醇对人绒毛膜外滋养层细胞的抑制作用。人脐带间充质干细胞条件培养基可作为潜在的治疗人绒毛膜外滋养层细胞功能障碍的临床手段,孕妇需要小心使用白藜芦醇。     

Glycosylation-dependent binding of galectin-8 to activated leukocyte cell adhesion molecule (ALCAM/CD166) promotes its surface segregation on breast cancer cells

BackgroundWe previously demonstrated that the activated leukocyte cell adhesion molecule (ALCAM/CD166) can interact with galectin-8 (Gal-8) in endothelial cells. ALCAM is a member of the immunoglobulin superfamily that promotes homophilic and heterophilic cell–cell interactions. Gal-8 is a “tandem-repeat”-type galectin, known as a matricellular protein involved in cell adhesion. Here, we analyzed the physical interaction between both molecules in breast cancer cells and the functional relevance of this phenomenon.MethodsWe performed binding assays by surface plasmon resonance to study the interaction between Gal-8 and the recombinant glycosylated ALCAM ectodomain or endogenous ALCAM from MDA-MB-231 breast cancer cells. We also analyzed the binding of ALCAM-silenced or control breast cancer cells to immobilized Gal-8 by SPR. In internalization assays, we evaluated the influence of Gal-8 on ALCAM surface localization.ResultsWe showed that recombinant glycosylated ALCAM and endogenous ALCAM from breast carcinoma cells physically interacted with Gal-8 in a glycosylation-dependent fashion displaying a differential behavior compared to non-glycosylated ALCAM. Moreover, ALCAM-silenced breast cancer cells exhibited reduced binding to Gal-8 relative to control cells. Importantly, exogenously added Gal-8 provoked ALCAM segregation, probably trapping this adhesion molecule at the surface of breast cancer cells.ConclusionsOur data indicate that Gal-8 interacts with ALCAM at the surface of breast cancer cells through glycosylation-dependent mechanisms.General significanceA novel heterophilic interaction between ALCAM and Gal-8 is demonstrated here, suggesting its physiologic relevance in the biology of breast cancer cells.