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

AbstractObjectiveNatural killer (NK) cells are the largest innate lymphocyte subset with potent antitumour and antiviral functions. However,clinical utilisation of human NK cells is hampered due to a lack of reliable methods to augment their antitumour potential. We demonstrated technology in which human NK cells were cocultured with osteoclasts in the presence of probiotic bacteria. This approach significantly augmented the antitumour cytotoxicity and polyfunctionality of human NK cells,resulting in the generation of supercharged NK (sNK) cells.Methods and analysisWe explored the proteomic,transcriptomic and functional characterisation of sNK cells using cell imaging,flow cytometric analysis,51-chromium release cytotoxicity assay,ELISA,ELIspot,IsoPLexis single-cell secretome analysis,proteomic analysis,RNA analysis,western blot and enzyme kinetics.ResultsWe found that sNK cells were less susceptible to split anergy and tumour-induced exhaustion. Proteomic analyses revealed that sNK cells significantly increased their cell motility and proliferation. Single-cell transcriptomes uncovered sNK cells undertaking a unique differentiation trajectory and turning on STAT1,JUN,BHLHE40,ELF1,MAX and MYC regulons essential for augmenting antitumour effector functions and proliferation,respectively. Both proteomic and single-cell transcriptomes revealed that an increase in Cathepsin C helped to augment the quantity and function of Granzyme B.ConclusionsThese results support that this unique method produces potent NK cells for clinical utilisation and delineate the molecular mechanisms associated with this process. 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

Hematopoietic stem cell (HSC) quiescence is a tightly regulated process crucial for hematopoietic regeneration,which requires a healthy and supportive microenvironmental niche within the bone marrow (BM). Here,we show that deletion of Ptpn21,a protein tyrosine phosphatase highly expressed in HSCs,induces stem cell egress from the niche due to impaired retention within the BM. Ptpn21-/- HSCs exhibit enhanced mobility,decreased quiescence,increased apoptosis,and defective reconstitution capacity. Ptpn21 deletion also decreased HSC stiffness and increased physical deformability,in part by dephosphorylating Spetin1 (Tyr246),a poorly described component of the cytoskeleton. Elevated phosphorylation of Spetin1 in Ptpn21-/- cells impaired cytoskeletal remodeling,contributed to cortical instability,and decreased cell rigidity. Collectively,these findings show that Ptpn21 maintains cellular mechanics,which is correlated with its important functions in HSC niche retention and preservation of hematopoietic regeneration capacity. View Publication

It has long been known that small changes to the structure of the R(2) side chain of nitrogen-containing bisphosphonates can dramatically affect their potency for inhibiting bone resorption in vitro and in vivo,although the reason for these differences in antiresorptive potency have not been explained at the level of a pharmacological target. Recently,several nitrogen-containing bisphosphonates were found to inhibit osteoclast-mediated bone resorption in vitro by inhibiting farnesyl diphosphate synthase,thereby preventing protein prenylation in osteoclasts. In this study,we examined the potency of a wider range of nitrogen-containing bisphosphonates,including the highly potent,heterocycle-containing zoledronic acid and minodronate (YM-529). We found a clear correlation between the ability to inhibit farnesyl diphosphate synthase in vitro,to inhibit protein prenylation in cell-free extracts and in purified osteoclasts in vitro,and to inhibit bone resorption in vivo. The activity of recombinant human farnesyl diphosphate synthase was inhibited at concentrations textgreater or = 1 nM zoledronic acid or minodronate,the order of potency (zoledronic acid approximately equal to minodronate textgreater risedronate textgreater ibandronate textgreater incadronate textgreater alendronate textgreater pamidronate) closely matching the order of antiresorptive potency. Furthermore,minor changes to the structure of the R(2) side chain of heterocycle-containing bisphosphonates,giving rise to less potent inhibitors of bone resorption in vivo,also caused a reduction in potency up to approximately 300-fold for inhibition of farnesyl diphosphate synthase in vitro. These data indicate that farnesyl diphosphate synthase is the major pharmacological target of these drugs in vivo,and that small changes to the structure of the R(2) side chain alter antiresorptive potency by affecting the ability to inhibit farnesyl diphosphate synthase. View Publication

In the present study,the trilineage differentiation capacity of human foreskin-derived precursor cells (hSKP) was evaluated upon exposure to various (non)commercial (i and ii) ectodermal,(iii) mesodermal and (iv) endodermal differentiation media. (i) Upon sequential exposure of the cells to keratinocyte growth (CnT-07® or CnT-057®) and differentiation (CnT-02® or Epilife®) media,keratinocyte-like cells (filaggrin(+)/involucrin(+)) were obtained. The preferred keratinocyte differentiation strategy was exposure to CnT-07®. (ii) When hSKP were subsequently exposed to NeuroCult® media,cells underwent a weak neuro-ectodermal differentiation expressing nestin,myelin binding protein (MBP),vimentin and alpha-foetoprotein (AFP). Sequential exposure to NPMM® and NPDM® generated cells with an inferior neuro-ectodermal phenotype (nestin(+)/vimentin(+)/MBP(-)/AFP(-)). (iii) Upon exposure of hSKP to insulin-transferrin-selenite (ITS) and dexamethasone,small lipid droplets were observed,suggesting their differentiation potential towards adipocyte-like cells. (iv) Finally,after sequential exposure to hepatogenic growth factors and cytokines,an immature hepatic cell population was generated. The presence of pre-albumin suggests that a sequential exposure strategy is here superior to a cocktail approach. In summary,a considerable impact of different (non)commercial media on the lineage-specific differentiation efficiency of hSKP is shown. In addition,we demonstrate here for the first time that,in a suitable keratinocyte stimulating micro-environment,hSKP can generate keratinocyte-like progeny in vitro. View Publication

Vascular abnormalities are a common component of eye diseases that often lead to vision loss. Vaso-obliteration is associated with inherited retinal degenerations,since photoreceptor atrophy lowers local metabolic demands and vascular support to those regions is no longer required. Given the degree of neurovascular crosstalk in the retina,it may be possible to use one cell type to rescue another cell type in the face of severe stress,such as hypoxia or genetically encoded cell-specific degenerations. Here,we show that intravitreally injected human endothelial colony-forming cells (ECFCs) that can be isolated and differentiated from cord blood in xeno-free media collect in the vitreous cavity and rescue vaso-obliteration and neurodegeneration in animal models of retinal disease. Furthermore,we determined that a subset of the ECFCs was more effective at anatomically and functionally preventing retinopathy; these cells expressed high levels of CD44,the hyaluronic acid receptor,and IGFBPs (insulin-like growth factor-binding proteins). Injection of cultured media from ECFCs or only recombinant human IGFBPs also rescued the ischemia phenotype. These results help us to understand the mechanism of ECFC-based therapies for ischemic insults and retinal neurodegenerative diseases. View Publication

Ikaros is expressed in early hematopoietic progenitors and is required for lymphoid differentiation. In the absence of Ikaros,there is a lack of markers defining fate restriction along lympho-myeloid pathways,but it is unclear whether formation of specific progenitors or expression of their markers is affected. Here we use a reporter based on Ikaros regulatory elements to separate early progenitors in wild-type and Ikaros-null mice. We found previously undetected Ikaros-null lympho-myeloid progenitors lacking the receptor tyrosine kinase Flt3 that were capable of myeloid but not lymphoid differentiation. In contrast,lack of Ikaros in the common myeloid progenitor resulted in increased formation of erythro-megakaryocytes at the expense of myeloid progenitors. Using this approach,we identify previously unknown pivotal functions for Ikaros in distinct fate 'decisions' in the early hematopoietic hierarchy. View Publication

Receptor type protein tyrosine phosphatase-sigma (PTPsigma) is primarily expressed by adult neurons and regulates neural regeneration. We recently discovered that PTPsigma is also expressed by hematopoietic stem cells (HSCs). Here,we describe small molecule inhibitors of PTPsigma that promote HSC regeneration in vivo. Systemic administration of the PTPsigma inhibitor,DJ001,or its analog,to irradiated mice promotes HSC regeneration,accelerates hematologic recovery,and improves survival. Similarly,DJ001 administration accelerates hematologic recovery in mice treated with 5-fluorouracil chemotherapy. DJ001 displays high specificity for PTPsigma and antagonizes PTPsigma via unique non-competitive,allosteric binding. Mechanistically,DJ001 suppresses radiation-induced HSC apoptosis via activation of the RhoGTPase,RAC1,and induction of BCL-XL. Furthermore,treatment of irradiated human HSCs with DJ001 promotes the regeneration of human HSCs capable of multilineage in vivo repopulation. These studies demonstrate the therapeutic potential of selective,small-molecule PTPsigma inhibitors for human hematopoietic regeneration. View Publication

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for many diverse blood and immune diseases. However,HSCT regimens currently commonly utilize genotoxic chemotherapy and/or total body irradiation (TBI) conditioning which causes significant morbidity and mortality through inducing broad tissue damage triggering infections,graft vs. host disease,infertility,and secondary cancers. We previously demonstrated that targeted monoclonal antibody (mAb)-based HSC depletion with anti(α)-CD117 mAbs could be an effective alternative conditioning approach for HSCT without toxicity in severe combined immunodeficiency (SCID) mouse models,which has prompted parallel clinical αCD117 mAbs to be developed and tested as conditioning agents in clinical trials starting with treatment of patients with SCID. Subsequent efforts have built upon this work to develop various combination approaches,though none are optimal and how any of these mAbs fully function is unknown. To improve efficacy of mAb-based conditioning as a stand-alone conditioning approach for all HSCT settings,it is critical to understand the mechanistic action of αCD117 mAbs on HSCs. Here,we compare the antagonistic properties of αCD117 mAb clones including ACK2,2B8,and 3C11 as well as ACK2 fragments in vitro and in vivo in both SCID and wildtype (WT) mouse models. Further,to augment efficacy,combination regimens were also explored. We confirm that only ACK2 inhibits SCF binding fully and prevents HSC proliferation in vitro. Further,we verify that this corresponds to HSC depletion in vivo and donor engraftment post HSCT in SCID mice. We also show that SCF-blocking αCD117 mAb fragment derivatives retain similar HSC depletion capacity with enhanced engraftment post HSCT in SCID settings,but only full αCD117 mAb ACK2 in combination with αCD47 mAb enables enhanced donor HSC engraftment in WT settings,highlighting that the Fc region is not required for single-agent efficacy in SCID settings but is required in immunocompetent settings. This combination was the only non-genotoxic conditioning approach that enabled robust donor engraftment post HSCT in WT mice. These findings shed new insights into the mechanism of αCD117 mAb-mediated HSC depletion. Further,they highlight multiple approaches for efficacy in SCID settings and optimal combinations for WT settings. This work is likely to aid in the development of clinical non-genotoxic HSCT conditioning approaches that could benefit millions of people world-wide. The online version contains supplementary material available at 10.1186/s13287-024-03981-0. View Publication

Regulated adhesion of T cells by the integrins LFA-1 (lymphocyte function-associated antigen-1) and VLA-4 (very late antigen-4) is essential for T-cell trafficking. The small GTPase Rap1 is a critical activator of both integrins in murine lymphocytes and T-cell lines. Here we examined the contribution of the Rap1 regulatory pathway in integrin activation in primary CD3(+) human T cells. We demonstrate that inactivation of Rap1 GTPase in human T cells by expression of SPA1 or Rap1GAP blocked stromal cell-derived factor-1alpha (SDF-1alpha)-stimulated LFA-1-ICAM-1 (intercellular adhesion molecule-1) interactions and LFA-1 affinity modulation but unexpectedly did not significantly affect binding of VLA-4 to its ligand VCAM-1 (vascular cell adhesion molecule 1). Importantly,silencing of the Rap1 guanine exchange factor CalDAG-GEFI inhibited SDF-1alpha- and phorbol 12-myristate 13-acetate (PMA)-induced adhesion to ICAM-1 while having no effect on adhesion to VCAM-1. Pharmacologic inhibition of Phospholipase C (PLC) blocked Rap1 activation and inhibited cell adhesion and polarization on ICAM-1 and VCAM-1. Protein kinase C (PKC) inhibition led to enhanced levels of active Rap1 concomitantly with increased T-cell binding to ICAM-1,whereas adhesion to VCAM-1 was reduced. Thus,PLC/CalDAG-GEFI regulation of Rap1 is selectively required for chemokine- and PMA-induced LFA-1 activation in human T cells,whereas alternate PLC- and PKC-dependent mechanisms are involved in the regulation of VLA-4. View Publication