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Oral facial cleft (OFC) comprises cleft lip with or without cleft palate (CL/P) or cleft palate only. Genome wide association studies (GWAS) of isolated OFC have identified common single nucleotide polymorphisms (SNPs) in many genomic loci where the presumed effector gene (for example,IRF6 in the 1q32 locus) is expressed in embryonic oral epithelium. To identify candidates for functional SNPs at eight such loci we conduct a massively parallel reporter assay in a fetal oral epithelial cell line,revealing SNPs with allele-specific effects on enhancer activity. We filter these SNPs against chromatin-mark evidence of enhancers and test a subset in traditional reporter assays,which support the candidacy of SNPs at loci containing FOXE1, IRF6, MAFB, TFAP2A,and TP63. For two SNPs near IRF6 and one near FOXE1,we engineer the genome of induced pluripotent stem cells,differentiate the cells into embryonic oral epithelium,and discover allele-specific effects on the levels of effector gene expression,and,in two cases,the binding affinity of transcription factors FOXE1 or ETS2. Conditional analyses of GWAS data suggest the two functional SNPs near IRF6 account for the majority of risk for CL/P at this locus. This study connects genetic variation associated with OFC to mechanisms of pathogenesis. Non-syndromic orofacial cleft is a relatively common congenital anomaly. Many non-coding genetic variants are associated with this disorder but only a subset is functional. Here the authors use reporter assays and stem cells to reveal members of this subset. View Publication

AbstractThe hippocampus is a brain area linked to cognition. The mechanisms that maintain cognitive activity in humans are poorly understood. Centenarians display extreme longevity which is generally accompanied by better quality of life,lower cognitive impairment,and reduced incidence of pathologies including neurodegenerative diseases. We performed transcriptomic studies in hippocampus samples from individuals of different ages (centenarians [?97 years],old,and young) and identified a differential gene expression pattern in centenarians compared to the other two groups. In particular,several isoforms of metallothioneins (MTs) were highly expressed in centenarians. Moreover,we identified that MTs were mainly expressed in astrocytes. Functional studies in human primary astrocytes revealed that MT1 and MT3 are necessary for their homeostasis maintenance. Overall,these results indicate that the expression of MTs specifically in astrocytes is a mechanism for protection during aging. Higher levels of MT1 and MT3 are detected in hippocampus of very old individuals (over 90) compared with old and young individuals. MTs colocalize with astrocytic markers. View Publication

Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches,we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis,the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture,accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development,we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach,we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases. The ability to differentiate human pluripotent stem cells (hPSCs) into insulin producing cells holds potential for diabetes treatments,but many of these approaches lack the complexity needed for in vitro disease modeling. Here they develop an hPSC-derived islet spheroid system,offering an experimental model to study pancreatic budding and islet morphogenesis with human cells. View Publication

SummaryThe human brain microenvironment undergoes dynamic changes during development,which have been incompletely characterized in in vitro models including neural organoids. Here,we used liquid chromatography-mass spectrometry to investigate proteome and secretome changes in human dorsal forebrain organoids derived from three hiPSC lines at days 20,35,and 50 of differentiation. Proteome and immunohistochemical analysis revealed reduced proliferation and increased differentiation of progenitor cells gradually over time. In contrast,secretome analysis showed distinct characteristics at each timepoint — notably,at day 35,the numbers of cell adhesion molecules,synaptic proteins,and proteases were increased. Taken together,we present a resource describing the dynamic features of a neural organoid proteome and secretome across different genetic backgrounds. We describe the unique niche composition of neural organoids during the period of neurogenesis and suggest that synaptic proteins may play a role in guiding neurogenesis. Graphical abstract Highlights•Proteomic analysis of DFOs on three time points shows neural differentiation•Protein secretion increases during peak neurogenesis at D35 and D50•Cell adhesion molecules,synapse proteins,and metalloproteases are mainly secreted at D35•Extracellular matrix proteins are predominantly secreted at D50 Natural sciences; Biological sciences; Neuroscience; Tissue Engineering View Publication

AG-490 is a member of the tyrphostin family of tyrosine kinase inhibitors. While AG-490 has been considered to be a Janus kinase (JAK)2-specific inhibitor,these conclusions were primarily drawn from acute lymphoblastic leukemia cells that lack readily detectable levels of JAK3. In the present study,evidence is provided that clearly demonstrates AG-490 potently suppresses IL-2-induced T cell proliferation,a non-JAK2-dependent signal,in a dose-dependent manner in T cell lines D10 and CTLL-2. AG-490 blocked JAK3 activation and phosphorylation of its downstream counterpart substrates,STATs. Inhibition of JAK3 by AG-490 also compromised the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways as measured by phosphorylation of Shc and extracellular signal-related kinase 1 and 2 (ERK1/2). AG-490 effectively inhibited tyrosine phosphorylation and DNA binding activities of several transcription factors including STAT1,-3,-5a,and -5b and activating protein-1 (AP-1) as judged by Western blot analysis and electrophoretic mobility shift assay. These data suggest that AG-490 is a potent inhibitor of the JAK3/STAT,JAK3/AP-1,and JAK3/MAPK pathways and their cellular consequences. Taken together,these findings support the notion that AG-490 possesses previously unrecognized clinical potential as an immunotherapeutic drug due to its inhibitory effects on T cell-derived signaling pathways. View Publication

Analysis of the National Cancer Institute Human Tumor Cell Line Anti-Cancer Drug Screen data using the COMPARE algorithm to detect similarities in the pattern of compound action to flavopiridol,a known inhibitor of cyclin-dependent kinases (CDKs),has suggested several possible novel CDK inhibitors. 9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one,NSC-664704 (kenpaullone),is reported here to be a potent inhibitor of CDK1/cyclin B (IC50,0.4 microM). This compound also inhibited CDK2/cyclin A (IC50,0.68 microM),CDK2/cyclin E (IC50,7.5 microM),and CDK5/p25 (IC50,0.85 microM) but had much less effect on other kinases; only c-src (IC50,15 microM),casein kinase 2 (IC50,20 microM),erk 1 (IC50,20 microM),and erk 2 (IC50,9 microM) were inhibited with IC50s less than 35 microM. Kenpaullone acts by competitive inhibition of ATP binding. Molecular modeling indicates that kenpaullone can bind in the ATP binding site of CDK2 with residue contacts similar to those observed in the crystal structures of other CDK2-bound inhibitors. Analogues of kenpaullone,in particular 10-bromopaullone (NSC-672234),also inhibited various protein kinases including CDKs. Cells exposed to kenpaullone and 10-bromopaullone display delayed cell cycle progression. Kenpaullone represents a novel chemotype for compounds that preferentially inhibit CDKs. View Publication

OBJECTIVE To investigate the inhibition of matrix metalloproteinase 1 (MMP-1),MMP-8,and MMP-13 by doxycycline,and to determine whether the variable hemopexin-like domain of each MMP was responsible for the differences in susceptibility to doxycycline inhibition among these collagenases. METHODS Recombinant human MMP-1 (collagenase 1),MMP-8 (collagenase 2),and MMP-13 (collagenase 3),truncated forms of MMP-8 and MMP-13 lacking the hemopexin-like domain,and a mutant form of truncated MMP-13 were used in these studies. The activity of the full-length MMP in the presence of doxycycline was tested against type II collagen,a natural substrate for the enzymes. A small peptolide substrate was used to determine which structural features of the MMPs were related to sensitivity to doxycycline inhibition. RESULTS The activity of MMP-13 and MMP-8 against type II collagen was inhibited by 50-60% by 30 microM doxycycline,while that of MMP-1 was inhibited only 18% by 50 microM doxycycline. In contrast,in experiments with the peptolide substrate,neither full-length nor truncated MMP-13 was inhibited until the concentration of the drug exceeded 90 microM. MMP-8 and truncated MMP-8 were sensitive to inhibition by 30 microM doxycycline,while MMP-1 was slightly inhibited (14%) by 90 microM doxycycline. For MMP-8,inhibition was reversible upon dilution and was independent of the order in which the reagents were added. Kinetic analysis of the inhibition constant (K(i)) of MMP-8 (K(i) = 36 microM) and truncated MMP-8 (K(i) = 77 microM) indicated that inhibition was noncompetitive. CONCLUSION Significant inhibition of MMP-13 and MMP-8 activity against collagen occurred in vitro at concentrations that were near the concentrations achieved in serum after oral dosing. Studies with truncated enzymes and 2 substrates suggest that doxycycline disrupts the conformation of the hemopexin-like domain of MMP-13 and the catalytic domain of MMP-8. View Publication

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects are in part determined by p53-mediated apoptosis,temporary suppression of p53 has been suggested as a therapeutic strategy to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility,a small molecule was isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound,pifithrin-alpha,protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors. Thus,inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated with p53 induction. View Publication

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted,self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani,K. Hobson,and M. S. Rao,1997,Dev. Biol. 186,202-223; M. S. Rao and M. Mayer-Proschel,1997,Dev. Biol. 188,48-63; M. Mayer-Proschel,A. J. Kalyani,T. Mujtaba,and M. S. Rao,1997,Neuron 19,773-785). We now show that cells identical to rat NEPs,NRPs,and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs,respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive,undergo self-renewal in defined medium,and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus,lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells. View Publication

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells,up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking,however,decreased to 0.1% or lower within 2 weeks. In contrast,EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks,the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis,and EGFP expression was observed in CD4(+),CD8(+),CD20(+),and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452) View Publication

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1),EP(2),EP(3),EP(4),DP,FP,IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed,for the first time,an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should,therefore,result in a greater understanding of prostanoid receptor biology. View Publication

The Dishevelled (Dvl) gene family encodes cytoplasmic proteins that are necessary for Wnt signal transduction. Utilizing the yeast two-hybrid system,we identified protein phosphatase 2Calpha (PP2C) as a Dvl-PDZ domain-interacting protein. PP2C exists in a complex with Dvl,beta-catenin,and Axin,a negative regulator of Wnt signaling. In a Wnt-responsive LEF-1 reporter gene assay,expression of PP2C activates transcription and also elicits a synergistic response with beta-catenin and Wnt-1. In addition,PP2C expression relieves Axin-mediated repression of LEF-1-dependent transcription. PP2C utilizes Axin as a substrate both in vitro and in vivo and decreases its half-life. These results indicate that PP2C is a positive regulator of Wnt signal transduction and mediates its effects through the dephosphorylation of Axin. View Publication