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Helper-dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off-target cleavage. Because of their large cloning capacity of 37 kb,helper-dependent adenoviral vectors with long homology arms are used for gene editing. However,this makes vector construction and recombinant analysis difficult. Conversely,insufficient homology may compromise targeting efficiency. Thus,we investigated the effect of homology length on helper-dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation. With 23.8 and 21.4 kb of homology,the frequencies of targeted recombinants were 50-64.6% after positive selection for vector integration,and 97.4-100% after negative selection against random integrations. With 14.8 kb,the frequencies were 26.9-57.1% after positive selection and 87.5-100% after negative selection. With 9.6 kb,the frequencies were 21.4 and 75% after positive and negative selection,respectively. With only 5.6 kb,the frequencies were 5.6-16.7% after positive selection and 50% after negative selection,but these were more than high enough for efficient identification and isolation of targeted clones. Furthermore,we demonstrate helper-dependent adenoviral vector-mediated footprintless correction of cystic fibrosis transmembrane conductance regulator mutations through piggyBac excision of the selectable marker. However,low frequencies (≤ 1 × 10(-3)) necessitated negative selection for piggyBac-excision product isolation. View Publication

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here,we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution,embryogenesis,and human disease,interspecies blastocyst complementation might allow human organ generation in animals whose organ size,anatomy,and physiology are closer to humans. To date,however,whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals,the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead,an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos. View Publication

Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium,we generated a diverse,comprehensive,and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities,β-globin gene (HBB) haplotypes,and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development,model disease,and perhaps eventually,treat patients. Here,we describe this unique resource for the study of sickle cell disease,including novel haplotype-specific polymorphisms that affect disease severity,as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library,and as proof of principle for future cell- and gene-based therapies,we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation. View Publication

OBJECTIVE In this work we set to develop and to validate a new in vivo frameless orthotopic Diffuse Intrinsic Pontine Glioma (DIPG) model based in the implantation of a guide-screw system. METHODS It consisted of a guide-screw also called bolt,a Hamilton syringe with a 26-gauge needle and an insulin-like 15-gauge needle. The guide screw is 2.6 mm in length and harbors a 0.5 mm central hole which accepts the needle of the Hamilton syringe avoiding a theoretical displacement during insertion. The guide-screw is fixed on the mouse skull according to the coordinates: 1mm right to and 0.8 mm posterior to lambda. To reach the pons the Hamilton syringe is adjusted to a 6.5 mm depth using a cuff that serves as a stopper. This system allows delivering not only cells but also any kind of intratumoral chemotherapy,antibodies or gene/viral therapies. RESULTS The guide-screw was successfully implanted in 10 immunodeficient mice and the animals were inoculated with DIPG human cell lines during the same anesthetic period. All the mice developed severe neurologic symptoms and had a median overall survival of 95 days ranging the time of death from 81 to 116 days. Histopathological analysis confirmed tumor into the pons in all animals confirming the validity of this model. CONCLUSION Here we presented a reproducible and frameless DIPG model that allows for rapid evaluation of tumorigenicity and efficacy of chemotherapeutic or gene therapy products delivered intratumorally to the pons. View Publication

Can photothermal gold nanoparticle mediated laser manipulation be applied to induce cardiac contraction? Based on our previous work,we present a novel concept of cell stimulation. A 532 nm picosecond laser was employed to heat gold nanoparticles on cardiomyocytes. This leads to calcium oscillations in the HL-1 cardiomyocyte cell line. As calcium is connected to the contractility,we aimed to alter the contraction rate of native and stem cell derived cardiomyocytes. A contraction rate increase was particularly observed in calcium containing buffer with neonatal rat cardiomyocytes. Consequently,the study provides conceptual ideas for a light based,nanoparticle mediated stimulation system. View Publication

Severe lupus often includes psychiatric and neurological sequelae,although the cellular contributors to CNS disease remain poorly defined. Using intravascular staining to discriminate tissue-localized from blood-borne cells,we find substantial accumulation of CD8+ T cells relative to other lymphocytes in brain tissue,which correlates with lupus disease and limited neuropathology. This is in contrast to all other affected organs,where infiltrating CD4+ cells are predominant. Brain-infiltrating CD8+ T cells represent an activated subset of those found in the periphery,having a resident-memory phenotype (CD69+CD122-PD1+CD44+CD62L-) and expressing adhesion molecules (VLA-4+LFA-1+) complementary to activated brain endothelium. Remarkably,infiltrating CD8+ T cells do not cause tissue damage in lupus-prone mice,as genetic ablation of these cells via $\beta$2 m deficiency does not reverse neuropathology,but exacerbates disease both in the brain and globally despite decreased serum IgG levels. Thus,lupus-associated inflammation disrupts the blood-brain barrier in a discriminating way biased in favor of non-pathogenic CD8+ T cells relative to other infiltrating leukocytes,perhaps preventing further tissue damage in such a sensitive organ. View Publication

During human brain development,multiple signaling pathways generate diverse cell types with varied regional identities. Here,we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor,neuronal,and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together,these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders. View Publication

Low-grade,chronic inflammation has been associated with many diseases of aging,but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress,and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β,nucleotide metabolism dysfunction,elevated oxidative stress,high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β,who lack these characteristics. Adenine and N(4)-acetylcytidine,nucleotide-derived metabolites that are detectable in the blood of the former group,prime and activate the NLRC4 inflammasome,induce the production of IL-1β,activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age,the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus,targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions. View Publication

Mutations within the FUS gene (Fused in Sarcoma) are known to cause Amyotrophic Lateral Sclerosis (ALS),a neurodegenerative disease affecting upper and lower motoneurons. The FUS gene codes for a multifunctional RNA/DNA-binding protein that is primarily localized in the nucleus and is involved in cellular processes such as splicing,translation,mRNA transport and DNA damage response. In this study,we analyzed pathophysiological alterations associated with ALS related FUS mutations (mFUS) in human induced pluripotent stem cells (hiPSCs) and hiPSC derived motoneurons. To that end,we compared cells carrying a mild or severe mFUS in physiological- and/or stress conditions as well as after induced DNA damage. Following hyperosmolar stress or irradiation,mFUS hiPS cells recruited significantly more cytoplasmatic FUS into stress granules accompanied by impaired DNA-damage repair. In motoneurons wild-type FUS was localized in the nucleus but also deposited as small punctae within neurites. In motoneurons expressing mFUS the protein was additionally detected in the cytoplasm and a significantly increased number of large,densely packed FUS positive stress granules were seen along neurites. The amount of FUS mislocalization correlated positively with both the onset of the human disease (the earlier the onset the higher the FUS mislocalization) and the maturation status of the motoneurons. Moreover,even in non-stressed post-mitotic mFUS motoneurons clear signs of DNA-damage could be detected. In summary,we found that the susceptibility to cell stress was higher in mFUS hiPSCs and hiPSC derived motoneurons than in controls and the degree of FUS mislocalization correlated well with the clinical severity of the underlying ALS related mFUS. The accumulation of DNA damage and the cellular response to DNA damage stressors was more pronounced in post-mitotic mFUS motoneurons than in dividing hiPSCs suggesting that mFUS motoneurons accumulate foci of DNA damage,which in turn might be directly linked to neurodegeneration. View Publication

p53-related protein kinase (TP53RK,also known as PRPK) is an upstream kinase which phosphorylates (Ser15) and mediates p53 activity. Here we show that TP53RK confers poor prognosis in MM patients; and conversely,that TP53RK knockdown inhibits p53 phosphorylation and triggers multiple myeloma (MM) cell apoptosis,associated with downregulation of c-Myc and E2F-1-mediated upregulation of pro-apoptotic Bim. We further demonstrate that TP53RK downregulation also triggers growth inhibition in p53-deficient (KMS-11) and p53-mutant (U266) MM cell lines,and identify novel downstream targets of TP53RK including ribonucleotide reductase-1,telomerase reverse transcriptase,and cyclin dependent kinase inhibitor 2C (CDKN2C). Our previous studies showed that immunomodulatory drugs (IMiDs) downregulate p21 and trigger apoptosis in wt-p53 MM.1S cells,Importantly we here demonstrate by pull-down,nuclear magnetic resonance spectroscopy,differential scanning fluorimetry,and isothermal titration calorimetry,that IMiDs bind and inhibit TP53RK,with biologic sequelae similar to TP53RK knockdown. Our studies therefore demonstrate that either genetic or pharmacological inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and -independent pathways,validating TP53RK as a novel therapeutic target in patients with poor prognosis MM. View Publication

One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry,its exact role during the first steps of HIV-1 infection is not well characterized. Here,we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall,our data suggest that dynamin-2,as a tetramer,might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion. View Publication

Numerous studies have demonstrated that administration of antigen (Ag)-pulsed dendritic cells (DCs) is an effective strategy for enhancing immunity to tumors and infectious disease organisms. However,the generation and/or isolation of DCs can require substantial time and expense. Therefore,using inactivated F. tularensis (iFt) Ag as a model immunogen,we first sought to determine if DCs could be replaced with peripheral blood mononuclear cells (PBMCs) during the ex-vivo pulse phase and still provide protection against Ft infection. Follow up studies were then conducted using the S. pneumoniae (Sp) vaccine Prevnar {\textregistered}13 as the Ag in the pulse phase followed by immunization and Sp challenge. In both cases,we demonstrate that PBMCs can be used in place of DCs when pulsing with iFt and/or Prevnar {\textregistered}13 ex vivo and re-administering the Ag-pulsed PBMCs as a vaccine. In addition,utilization of the i.n. route for Ag-pulsed PBMC administration is superior to use of the i.v. route in the case of Sp immunization,as well as when compared to direct injection of Prevnar {\textregistered}13 vaccine i.m. or i.n. Furthermore,this PBMC-based vaccine strategy provides a more marked and enduring protective immune response and is also capable of serving as a multi-organism vaccine platform. The potential for this ex-vivo vaccine strategy to provide a simpler,less time consuming,and less expensive approach to DC-based vaccines and vaccination in general is also discussed. View Publication