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

Sirtuins are NAD(+)-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date,several sirtuin inhibitors and activators have been identified,but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD(+)-dependent deacetylase activity with an IC(50) value of 22 microM. To provide insights into how sirtuin function is altered by inhibitors,we determined two crystal structures of SIRT5,one in complex with ADP-ribose,the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD(+),the product,and the substrate-binding site. Finally,our structures may enable the rational design of more potent inhibitors. View Publication

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference,we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM,RSeT,5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore,our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs,underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs. View Publication

The purpose of this study is to characterize the microRNA (miRNA) expression profiles of induced pluripotent stem (iPS) cells and retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE). MiRNAs have been demonstrated to play critical roles in both maintaining pluripotency and facilitating differentiation. Gene expression networks accountable for maintenance and induction of pluripotency are linked and share components with those networks implicated in oncogenesis. Therefore,we hypothesize that miRNA expression profiling will distinguish iPS cells from their iPS-RPE progeny. To identify and analyze differentially expressed miRNAs,RPE was derived from iPS using a spontaneous differentiation method. MiRNA microarray analysis identified 155 probes that were statistically differentially expressed between iPS and iPS-RPE cells. Up-regulated miRNAs including miR-181c and miR-129-5p may play a role in promoting differentiation,while down-regulated miRNAs such as miR-367,miR-18b,and miR-20b are implicated in cell proliferation. Subsequent miRNA-target and network analysis revealed that these miRNAs are involved in cellular development,cell cycle progression,cell death,and survival. A systematic interrogation of temporal and spatial expression of iPS-RPE miRNAs and their associated target mRNAs will provide new insights into the molecular mechanisms of carcinogenesis,eye differentiation and development. View Publication

STUDY QUESTION Can human embryonic stem cell-derived trophoblastic spheroids be used to study the early stages of implantation? SUMMARY ANSWER We generated a novel human embryonic stem cell-derived trophoblastic spheroid model mimicking human blastocysts in the early stages of implantation. WHAT IS KNOWN ALREADY Both human embryos and choriocarcinoma cell line derived spheroids can attach onto endometrial cells and are used as models to study the early stages of implantation. However,human embryos are limited and the use of cancer cell lines for spheroid generation remains sub-optimal for research. STUDY DESIGN,SIZE,DURATION Experimental induced differentiation of human embryonic stem cells into trophoblast and characterization of the trophoblast. PARTICIPANTS/MATERIALS,SETTING,METHODS Trophoblastic spheroids (BAP-EB) were generated by inducing differentiation of a human embryonic stem cell line,VAL3 cells with bone morphogenic factor-4,A83-01 (a TGF-$\$),and PD173074 (a FGF receptor-3 inhibitor) after embryoid body formation. The expressions of trophoblastic markers and hCG levels were studied by real-time PCR and immunohistochemistry. BAP-EB attachment and invasion assays were performed on different cell lines and primary endometrial cells. MAIN RESULTS AND THE ROLE OF CHANCE After 48 h of induced differentiation,the BAP-EB resembled early implanting human embryos in terms of size and morphology. The spheroids derived from embryonic stem cells (VAL3),but not from several other cell lines studied,possessed a blastocoel-like cavity. BAP-EB expressed several markers of trophectoderm of human blastocysts on Day 2 of induced differentiation. In the subsequent days of differentiation,the cells of the spheroids differentiated into trophoblast-like cells expressing trophoblastic markers,though at levels lower than that in the primary trophoblasts or in a choriocarcinoma cell line. On Day 3 of induced differentiation,BAP-EB selectively attached onto endometrial epithelial cells,but not other non-endometrial cell lines or an endometrial cell line that had lost its epithelial character. The attachment rates of BAP-EB was significantly higher on primary endometrial epithelial cells (EEC) taken from 7 days after hCG induction of ovulation (hCG+7 day) when compared with that from hCG+2 day. The spheroids also invaded through Ishikawa cells and the primary endometrial stromal cells in the co-culture. LIMITATIONS,REASONS FOR CAUTION The attachment rates of BAP-EB were compared between EEC obtained from Day 2 and Day 7 of the gonadotrophin stimulated cycle,but not the natural cycles. WIDER IMPLICATIONS OF THE FINDINGS BAP-EB have the potential to be used as a test for predicting endometrial receptivity in IVF cycles and provide a novel approach to study early human implantation,trophoblastic cell differentiation and trophoblastic invasion into human endometrial cells. View Publication

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent,quiescent limbal stem cells (LSCs) located at the limbus,where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood,which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study,we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover,TP63,KRT15,CXCL14,and ITGβ4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs),which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGβ4 could be used to enrich human corneal epithelial cell progenitors,which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1. View Publication

The T-cell receptor (TCR) initiates T-lymphocyte activation,but the mechanism of TCR activation remains uncertain. Here,we present cryogenic electron microscopy structures for the unliganded and human leukocyte antigen (HLA)-bound human TCR–CD3 complex in nanodiscs that provide a native-like lipid environment. Distinct from the open and extended conformation seen in detergent,the unliganded TCR–CD3 in nanodiscs adopts two related closed and compacted conformations that represent its physiologic resting state in vivo. By contrast,the HLA-bound complex adopts the open and extended conformation,and conformation-locking disulfide mutants show that ectodomain opening is necessary for maximal ligand-dependent T-cell activation. These structures also reveal conformation-dependent protein–lipid and glycan–glycan interactions within the TCR. Together,these results establish allosteric conformational change during TCR activation,reveal avenues for immunotherapeutic engineering,and highlight the importance of native-like lipid environments for membrane protein structure determination. The T-cell receptor (TCR) activation mechanism has remained uncertain. Here,the authors present molecular structures for the apo and ligand-bound human TCR–CD3 complex in lipid nanodiscs,revealing large conformational changes during activation. View Publication

Pharmacological inhibitors of glycogen synthase kinase-3 (GSK-3) and cyclin-dependent kinases have a promising potential for applications against several neurodegenerative diseases such as Alzheimer's disease. Indirubins,a family of bis-indoles isolated from various natural sources,are potent inhibitors of several kinases,including GSK-3. Using the cocrystal structures of various indirubins with GSK-3beta,CDK2 and CDK5/p25,we have modeled the binding of indirubins within the ATP-binding pocket of these kinases. This modeling approach provided some insight into the molecular basis of indirubins' action and selectivity and allowed us to forecast some improvements of this family of bis-indoles as kinase inhibitors. Predicted molecules,including 6-substituted and 5,6-disubstituted indirubins,were synthesized and evaluated as CDK and GSK-3 inhibitors. Control,kinase-inactive indirubins were obtained by introduction of a methyl substitution on N1. View Publication

Neuropilin-1 (Nrp1) is a multifunctional protein,identified principally as a receptor for the class 3 semaphorins and members of the vascular endothelial growth factor (VEGF) family,but it is capable of other interactions. It is a marker of regulatory T cells (Tr),which often carry Nrp1 and latency-associated peptide (LAP)-TGF-beta1 (the latent form). The signaling TGF-beta1 receptors bind only active TGF-beta1,and we hypothesized that Nrp1 binds the latent form. Indeed,we found that Nrp1 is a high-affinity receptor for latent and active TGF-beta1. Free LAP,LAP-TGF-beta1,and active TGF-beta1 all competed with VEGF165 for binding to Nrp1. LAP has a basic,arginine-rich C-terminal motif similar to VEGF and peptides that bind to the b1 domain of Nrp1. A C-terminal LAP peptide (QSSRHRR) bound to Nrp1 and inhibited the binding of VEGF and LAP-TGF-beta1. We also analyzed the effects of Nrp1/LAP-TGF-beta1 coexpression on T cell function. Compared with Nrp1(-) cells,sorted Nrp1+ T cells had a much greater capacity to capture LAP-TGF-beta1. Sorted Nrp1(-) T cells captured soluble Nrp1-Fc,and this increased their ability to capture LAP-TGF-beta1. Conventional CD4+CD25(-)Nrp1(-) T cells coated with Nrp1-Fc/LAP-TGF-beta1 acquired strong Tr activity. Moreover,LAP-TGF-beta was activated by Nrp1-Fc and also by a peptide of the b2 domain of Nrp1 (RKFK; similar to a thrombospondin-1 peptide). Breast cancer cells,which express Nrp1,also captured and activated LAP-TGF-beta1 in a Nrp1-dependent manner. Thus,Nrp1 is a receptor for TGF-beta1,activates its latent form,and is relevant to Tr activity and tumor biology. View Publication

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1,a cell surface glycoprotein expressed on MM cells. In preclinical models,elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein,we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1-CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary,human MM cells. Taken together,these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone. View Publication

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells,including functional ?-cells,have been based on a detailed understanding of the underlying developmental mechanisms. However,the final differentiation steps,leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells,remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells),which include a lack of ?-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP,INSeGFP/GCGmCHERRY) as well as ?-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive,insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions,we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and,using this approach,we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal ?-cells without affecting the number of ?-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets. View Publication

The clinical success of the immune checkpoint inhibitor (ICI) targeting programmed cell death protein 1 (PD-1) has revolutionized cancer treatment. However,the full potential of PD-1 blockade therapy remains unrealized,as response rates are still low across many cancer types. Interleukin-2 (IL-2)-based immunotherapies hold promise,as they can stimulate robust T cell expansion and enhance effector function - activities that could synergize potently with PD-1 blockade. Yet,IL-2 therapies also carry a significant drawback: they can trigger severe systemic toxicities and induce immune suppression by expanding regulatory T cells. To overcome the challenges of PD-1 blockade and IL-2 therapies while enhancing safety and efficacy,we have engineered a novel fusion protein,AWT020,combining a humanized anti-PD-1 nanobody and an engineered IL-2 mutein (IL-2c). The IL-2c component of AWT020 has been engineered to exhibit no binding to the IL-2 receptor alpha (IL-2Rα) subunit and attenuated affinity for the IL-2 receptor beta and gamma (IL-2Rβγ) complex,aiming to reduce systemic immune cell activation,thereby mitigating the severe toxicity often associated with IL-2 therapies. The anti-PD-1 antibody portion of AWT020 serves a dual purpose: it precisely delivers the IL-2c payload to tumor-infiltrating T cells while blocking the immune-inhibitory signals mediated by the PD-1 pathway. AWT020 showed significantly enhanced pSTAT5 signaling in PD-1 expressing cells and promoted the proliferation of activated T cells over natural killer (NK) cells. In preclinical studies using both anti-PD-1-sensitive and -resistant mouse tumor models,the mouse surrogate of AWT020 (mAWT020) demonstrated markedly enhanced anti-tumor efficacy compared to an anti-PD-1 antibody,IL-2,or the combination of an anti-PD-1 antibody and IL-2. In addition,the mAWT020 treatment was well-tolerated,with minimal signs of toxicity. Immune profiling revealed that mAWT020 preferentially expands CD8 + T cells within tumors,sparing peripheral T and NK cells. Notably,this selective tumoral T-cell stimulation enables potent tumor-specific T-cell responses,underscoring the molecule’s enhanced efficacy and safety. The AWT020 fusion protein offers a promising novel immunotherapeutic strategy by integrating PD-1 blockade and IL-2 signaling,conferring enhanced anti-tumor activity with reduced toxicity. View Publication