搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

To investigate intestinal health and its potential disruptors in vitro,representative models are required. Human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cells (IECs) more closely resemble the in vivo intestinal tissue than conventional in vitro models like human colonic adenocarcinoma Caco-2 cells. However,the potential of IECs to study immune-related responses upon external stimuli has not been investigated in detail yet. The aim of the current study was to evaluate immune-related effects of IECs by challenging them with a pro-inflammatory cytokine cocktail. Subsequently,the effects of Lactiplantibacillus plantarum WCFS1 were investigated in unchallenged and challenged IECs. All exposures were compared to Caco-2 cells and in vivo data where possible. Upon the inflammatory challenge,IECs and Caco-2 cells induced a pro-inflammatory response which was strongest in IECs. Heat-killed L. plantarum exerted the strongest effect on immune parameters in the IEC model,while L. plantarum in the stationary growth phase had most pronounced effects on immune-related gene expression in Caco-2 cells. Unfortunately,comparison to in vivo transcriptomics data showed limited similarities,which could be explained by essential differences in the study setups. Altogether,hiPSC-derived IECs show a high potential as a model to study immune-related responses in the intestinal epithelium in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-74802-w. View Publication

The sodium-iodine symporter (NIS) is expressed on the cell membrane of many thyroid cancer cells,and is responsible for the radioactive iodine accumulation. However,treatment of anaplastic thyroid cancer is ineffective due to the low expression of NIS on cell membranes of these tumor cells. Human embryonic stem cells (ESCs) provide a potential vehicle to study the mechanisms of NIS expression regulation during differentiation. Human ESCs were maintained on feeder-independent culture conditions. RT-qPCR and immunocytochemistry were used to study differentiation marker expression,(125)I uptake to study NIS function. We designed a two-step protocol for human ESC differentiation into thyroid-like cells,as was previously done for mouse embryonic stem cells. First,we obtained definitive endoderm from human ESCs. Second,we directed differentiation of definitive endoderm cells into thyroid-like cells using various factors,with thyroid stimulating hormone (TSH) as the main differentiating factor. Expression of pluripotency,endoderm and thyroid markers and (125)I uptake were monitored throughout the differentiation steps. These approaches did not result in efficient induction of thyroid-like cells. We conclude that differentiation of human ESCs into thyroid cells cannot be induced by TSH media supplementation alone and most likely involves complicated developmental patterns that are yet to be understood. View Publication

The development of acquired resistance to trastuzumab remains a prevalent challenge in the treatment of patients whose tumors express human epidermal growth factor 2 (HER2). We previously reported that HER2 overexpressing breast cancers are dependent on Y-box binding protein-1 (YB-1) for growth and survival. As YB-1 is also linked to drug resistance in other types of cancer,we address its possible role in trastuzumab insensitivity. Employing an in vivo model of acquired resistance,we demonstrate that resistant cell lines have elevated levels of P-YB-1(S102) and its activating kinase P-RSK and these levels are sustained following trastuzumab treatment. Further,to demonstrate the importance of YB-1 in mediating drug resistance,the expression of the active mutant YB-1(S102D) rendered the BT474 cell line insensitive to trastuzumab. Questioning the role of tumor-initiating cells (TIC) and their ability to escape cancer therapies,we investigate YB-1's role in inducing the cancer stem cell marker CD44. Notably,the resistant cells express more CD44 mRNA and protein compared with BT474 cells,which correlated with increased mammosphere formation. Expression of YB-1(S102D) in the BT474 cells increase CD44 protein levels,resulting in enhanced mammosphere formation. Further,exposing BT474 cells to trastuzumab selected for a resistant sub-population enriched for CD44. Conversely,small intefering RNA inhibition of CD44 restored trastuzumab sensitivity in the resistant cell lines. Our findings provide insight on a novel mechanism employed by tumor cells to acquire the ability to escape the effects of trastuzumab and suggest that targeting YB-1 may overcome resistance by eliminating the unresponsive TIC population,rendering the cancer sensitive to therapy. View Publication

The hexamethylene bisacetamide (HMBA) anticancer drug was dismissed due to limited efficacy in leukemic patients but it may re-enter into the clinics in HIV-1 eradication strategies because of its recently disclosed capacity to reactivate latent virus. Here,we investigated the impact of HMBA on the cytotoxicity of natural killer (NK) cells against acute T lymphoblastic leukemia (T-ALL) cells or HIV-1-infected T cells that exit from latency. We show that in T-ALL cells HMBA upmodulated MICB and ULBP2 ligands for the NKG2D activating receptor. In a primary CD4+ T cell-based latency model,HMBA did not reactivate HIV-1,yet enhanced ULBP2 expression on cells harboring virus reactivated by prostratin (PRO). However,HMBA reduced the expression of NKG2D and its DAP10 adaptor in NK cells,hence impairing NKG2D-mediated cytotoxicity and DAP10-dependent response to IL-15 stimulation. Alongside,HMBA dampened killing of T-ALL targets by IL-15-activated NK cells and impaired NK cell-mediated clearance of PRO-reactivated HIV-1+ cells. Overall,our results demonstrate a dominant detrimental effect of HMBA on the NKG2D pathway that crucially controls NK cell-mediated killing of tumors and virus-infected cells,providing one possible explanation for poor clinical outcome in HMBA-treated cancer patients and raising concerns for future therapeutic application of this drug. View Publication

Emerging evidence indicates that in myelodysplastic syndromes (MDS),the bone marrow (BM) microenvironment may also contribute to the ineffective,malignant haematopoiesis in addition to the intrinsic abnormalities of haematopoietic stem precursor cells (HSPCs). The BM microenvironment influences malignant haematopoiesis through indirect mechanisms,but the processes by which the BM microenvironment directly contributes to MDS initiation and progression have not yet been elucidated. Our previous data showed that BM-derived stromal cells (BMSCs) from MDS patients have an abnormal expression of focal adhesion kinase (FAK). In this study,we characterise the morpho-phenotypic features and the functional alterations of BMSCs from MDS patients and in FAK knock-downed HS-5 cells. The decreased expression of FAK or its phosphorylated form in BMSCs from low-risk (LR) MDS directly correlates with BMSCs' functional deficiency and is associated with a reduced level of haemoglobin. The downregulation of FAK in HS-5 cells alters their morphology,proliferation,and differentiation capabilities and impairs the expression of several adhesion molecules. In addition,we examine the CD34+ healthy donor (HD)-derived HSPCs' properties when co-cultured with FAK-deficient BMSCs. Both abnormal proliferation and the impaired erythroid differentiation capacity of HD-HSPCs were observed. Together,these results demonstrate that stromal adhesion mechanisms mediated by FAK are crucial for regulating HSPCs' homeostasis. View Publication

Breast Cancer Stem (BCS) cells play critical roles in self-renewal,Multi Drug Resistance (MDR),differentiation and generation of secondary tumors. Conventional chemotherapy may efficiently kill the bulk of differentiated drug sensitive breast cancer cells,but not the MDR self-renewable BCS cells,leading to enrichment of the MDR BCS cells. In order to target the MDR BCS cells,we have isolated: 1) BCS cells from either breast cancer cell lines or fresh breast cancer specimens; 2) ATP binding cassette (ABC) transporter group G number 2 (ABCG2)-specific aptamers; and 3) BCS cell-binding aptamers. Interestingly,ABCG2-specific aptamers labeled the membrane surface of the ABCG2-expressing baby hamster kidney (BHK) cells,but stained whole cells of the BCS cells derived from mammospheres,implying that BCS cells might have much higher rate of endocytosis than the ABCG2-expressing BHK cells. In addition,5D3,a monoclonal antibody that recognizes the extracellular loops of ABCG2 protein,also stained whole BCS cells. Furthermore,BCS cell-binding aptamers stained whole BCS cells,but not the differentiated breast cancer MCF-7 cells. All these results support above conclusion that BCS cells might have high rate of endocytosis. Further experiments performed with aptamers and human transferrin or lactosylceramide showed that BCS cells do have much higher endocytosis rate than the differentiated breast cancer cells. Interestingly,clathrin dependent endocytosis inhibitors,such as monodansylcadaverine or sucrose,or caveolin-dependent endocytosis inhibitors,such as methyl-$$-cyclodextrin or genistein,can inhibit the internalization of transferrin or lactosylceramide into the differentiated breast cancer cells,but cannot block the internalization of these compounds into the BCS cells,suggesting that BCS cells undergo clathrin-independent and caveolin-independent endocytosis. Taken together,our data suggest that BCS cells have high rate of endocytosis and open the possibilities for delivering therapeutic agents directly into the MDR BCS cells with aptamer-coated liposomes. View Publication

We describe a new procedure for large-scale CB processing in the collection bag,thus minimizing the risk of CB contamination. A solution of 6% hydroxyethyl starch (HES) was added directly to the CB containing bag. After RBC sedimentation at 4 degrees C,the WBC-rich supernatant was collected in a satellite bag and centrifuged. After supernatant removal,the cell pellet was resuspended and the percent recovery of total WBC,CD34+ progenitor cells,CFU-GM and cobblestone area-forming cells (CAFC) evaluated. Results obtained with three different types of CB collection bags (300,600 and 1000 ml) were analyzed and compared with those of an open system in 50 ml tubes. CB processing procedures in 300 and 1000 ml bags were associated with better WBC,CFU,CD34+ cell and CAFC recovery (83-93%). This novel CB processing procedure appears to be easy,effective and particularly suitable for large-scale banking under GMP conditions. View Publication

The introduction of antibody–drug conjugates represents a significant advancement in targeted therapy of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Our study aims to investigate the role of the DNA damage response pathway and the impact of PARP1 inhibition,utilizing talazoparib,on the response of AML and ALL cells to Gemtuzumab ozogamicin (GO) and Inotuzumab ozogamicin (INO),respectively. AML and ALL cells were treated with GO,INO and γ-calicheamicin in order to induce severe DNA damage and activate the G2/M cell-cycle checkpoint in a dose- and time-dependent manner. The efficacy of PARP1 inhibitors and,in particular,talazoparib in enhancing INO or GO against ALL or AML cells was assessed through measurements of cell viability,cell death,cell cycle progression,DNA damage repair,accumulation of mitotic DNA damage and inhibition of clonogenic capacity. We observed that both ALL and AML cell lines activate the G2/M cell-cycle checkpoint in response to γ-calicheamicin-induced DNA damage,highlighting a shared cellular response mechanism. Talazoparib significantly enhanced the efficacy of INO against ALL cell lines,resulting in reduced cell viability,increased cell death,G2/M cell-cycle checkpoint override,accumulation of mitotic DNA damage and inhibition of clonogenic capacity. Strong synergism was observed in primary ALL cells treated with the combination. In contrast,AML cells exhibited a heterogeneous response to talazoparib in combination with GO. Our findings suggest a potential link between the differential responses of ALL and AML cells to the drug combinations and the ability of talazoparibto override G2/M cell-cycle arrest induced by antibody–drug conjugates. PARP1 emerges as a key player in the response of ALL cells to INO and represents a promising target for therapeutic intervention in this leukemia setting. Our study sheds light on the intricate interplay between the DNA damage response pathway,PARP1 inhibition,and response of γ-calicheamicin-induced DNA damages in AML and ALL. These findings underscore the importance of targeted therapeutic strategies and pave the way for future research aimed at optimizing leukemia treatment approaches. The online version contains supplementary material available at 10.1186/s12967-024-05838-9. View Publication

PURPOSE Imatinib,the breakpoint cluster region protein (BCR)/Abelson murine leukemia viral oncogene homolog (ABL) inhibitor,is widely used to treat chronic myeloid leukemia (CML). However,imatinib resistance develops in many patients. Therefore,new drugs with improved therapeutic effects are urgently needed. Berberine (BBR) is a potent BCR-ABL inhibitor for imatinib-sensitive and -resistant CML. EXPERIMENTAL DESIGN Protein structure analysis and virtual screening were used to identify BBR targets in CML. Molecular docking analysis,surface plasmon resonance imaging,nuclear magnetic resonance assays,and thermoshift assays were performed to confirm the BBR target. The change in BCR-ABL protein expression after BBR treatment was assessed by Western blotting. The effects of BBR were assessed in vitro in cell lines,in vivo in mice,and in human CML bone marrow cells as a potential strategy to overcome imatinib resistance. RESULTS We discovered that BBR bound to the protein tyrosine kinase domain of BCR-ABL. BBR inhibited the activity of BCR-ABL and BCR-ABL with the T315I mutation,and it also degraded these proteins via the autophagic lysosome pathway by recruiting E3 ubiquitin-protein ligase LRSAM1. BBR inhibited the cell viability and colony formation of CML cells and prolonged survival in CML mouse models with imatinib sensitivity and resistance. CONCLUSIONS The results show that BBR directly binds to and degrades BCR-ABL and BCR-ABL T315I via the autophagic lysosome pathway by recruiting LRSAM1. The use of BBR is a new strategy to improve the treatment of patients with CML with imatinib sensitivity or resistance. View Publication

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here,human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ωtextperiodcenteredcm(2). By assessing the permeabilities of several known drugs,a benchmarking system to evaluate brain permeability of drugs was established. Furthermore,relationships between TEER and permeability to both small and large molecules were established,demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype,and the benchmarks established here are useful for assessing functionality of other in vitro BBB models. View Publication

In recent years,immunotherapy has shown considerable promise in the management of several malignancies. However,the majority of preclinical studies have been conducted in rodents,the results of which often translate poorly to patients given the substantial differences between murine and human immunology. As the porcine immune system is far more analogous to that of humans,pigs may serve as a supplementary preclinical model for future testing of such therapies. We have generated the genetically modified Oncopig with inducible tumor formation resulting from concomitant KRAS(G12D) and TP53(R167H) mutations under control of an adenoviral vector Cre-recombinase (AdCre). The objective of this study was to characterize the tumor microenvironment in this novel animal model with respect to T-cell responses in particular and to elucidate the potential use of Oncopigs for future preclinical testing of cancer immunotherapies. In this study,we observed pronounced intratumoral T-cell infiltration with a strong CD8$\beta$(+) predominance alongside a representation of highly differentiated $\gamma$$\delta$ T cells. The infiltrating CD8$\beta$(+) T cells displayed increased expression of the cytotoxic marker perforin when compared with the peripheral T-cell pool. Similarly,there was robust granzyme B staining localizing to the tumors; affirming the presence of cytotoxic immune cells within the tumor. In parallel with this antitumor immune response,the tumors displayed enrichment in FOXP3-expressing T cells and increased gene expression of indoleamine 2,3-dioxygenase 1 (IDO1),cytotoxic T-lymphocyte-associated protein 4 (CTLA4),and programmed death-ligand 1 (PDL1). Finally,we investigated the Oncopig immune system in mediating antitumor immunity. We observed pronounced killing of autologous tumor cells,which demonstrates the propensity of the Oncopig immune system to recognize and mount a cytotoxic response against tumor cells. Together,these findings suggest innate and adaptive recognition of the induced tumors with a concomitant in vivo suppression of T-cell effector functions. Combined,the data support that the Oncopig may serve as a valuable model for future preclinical testing of immunotherapies aimed at reactivating tumor-directed cytotoxicity in vivo. View Publication

Mesenchymal stem cells have been implicated as playing an important role in stem cell engraftment. Recently,a new pluripotent population of umbilical cord blood (UCB) cells,unrestricted somatic stem cells (USSCs),with intrinsic and directable potential to develop into mesodermal,endodermal,and ectodermal fates,has been identified. In this study,we evaluated the capacity of ex vivo expanded USSCs to influence the homing of UCB-derived CD34(+) cells into the marrow and spleen of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. USSCs induced a significant enhancement of CD34(+) cell homing to both bone marrow and spleen (2.2 +/- 0.3- and 2.4 +/- 0.6-fold,respectively; p textless .05),with a magnitude similar to that induced by USSCs that had been thawed prior to transplantation. The effect of USSCs was dose-dependent and detectable at USSC:CD34(+) ratios of 1:1 and above. Enhanced marrow homing by USSCs was unaltered by extensive culture passaging of the cells,as similar enhancement was observed for both early-passage (passage 5 [p5]) and late-passage (p10) USSCs. The homing effect of USSCs was also reflected in an increased proportion of NOD/SCID mice exhibiting significant human cell engraftment 6 weeks after transplantation,with a similar distribution of myeloid and lymphoid components. USSCs enhanced the homing of cellular products of ex vivo expanded UCB lineage-negative (lin(-)) cells,generated in 14-day cultures by Selective Amplification. The relative proportion of homing CD34(+) cells within the culture-expanded cell population was unaltered by USSC cotransplantation. Production of stromal-derived factor-1 (SDF-1) by USSCs was detected by both gene expression and protein released into culture media of these cells. Knockdown of SDF-1 production by USSCs using lentiviral-SiRNA led to a significant (p textless .05) reduction in USSC-mediated enhancement of CD34(+) homing. Our findings thus suggest a clinical potential for using USSCs in facilitating homing and engraftment for cord blood transplant recipients. View Publication