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1.
Cell ; 185(14): 2523-2541.e30, 2022 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-35738284

RESUMEN

Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.


Asunto(s)
Virus Hendra , Virus Nipah , Células Madre Pluripotentes , Arterias , Células Endoteliales , Virus Hendra/genética , Humanos , Tropismo
2.
Gene Ther ; 27(10-11): 525-534, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-32704085

RESUMEN

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promising potential for opening new avenues in regenerative medicine. However, since the tumorigenic potential of undifferentiated pluripotent stem cells (PSCs) is a major safety concern for clinical transplantation, inducible Caspase-9 (iC9) is under consideration for use as a fail-safe system. Here, we used targeted gene editing to introduce the iC9 system into human iPSCs, and then interrogated the efficiency of inducible apoptosis with normal iPSCs as well as diseased iPSCs derived from patients with acute myeloid leukemia (AML-iPSCs). The iC9 system induced quick and efficient apoptosis to iPSCs in vitro. More importantly, complete eradication of malignant cells without AML recurrence was shown in disease mouse models by using AML-iPSCs. In parallel, it shed light on several limitations of the iC9 system usage. Our results suggest that careful use of the iC9 system will serve as an important countermeasure against posttransplantation adverse events in stem cell transplantation therapies.


Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Animales , Apoptosis , Caspasa 9/genética , Caspasa 9/metabolismo , Diferenciación Celular , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células Madre Pluripotentes/metabolismo
3.
J Biol Chem ; 289(29): 20333-44, 2014 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-24847081

RESUMEN

Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[(3)H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.


Asunto(s)
Receptores sigma/química , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Reactivos de Enlaces Cruzados , Cobayas , Haloperidol/metabolismo , Humanos , Ligandos , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pentazocina/metabolismo , Multimerización de Proteína , Estabilidad Proteica , Receptores sigma/genética , Receptores sigma/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Receptor Sigma-1
4.
Nat Biotechnol ; 2024 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-38589662

RESUMEN

CRISPR-Cas9 paired with adeno-associated virus serotype 6 (AAV6) is among the most efficient tools for producing targeted gene knockins. Here, we report that this system can lead to frequent concatemeric insertions of the viral vector genome at the target site that are difficult to detect. Such errors can cause adverse and unreliable phenotypes that are antithetical to the goal of precision genome engineering. The concatemeric knockins occurred regardless of locus, vector concentration, cell line or cell type, including human pluripotent and hematopoietic stem cells. Although these highly abundant errors were found in more than half of the edited cells, they could not be readily detected by common analytical methods. We describe strategies to detect and thoroughly characterize the concatemeric viral vector insertions, and we highlight analytical pitfalls that mask their prevalence. We then describe strategies to prevent the concatemeric inserts by cutting the vector genome after transduction. This approach is compatible with established gene editing pipelines, enabling robust genetic knockins that are safer, more reliable and more reproducible.

5.
Protein Expr Purif ; 89(2): 203-9, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23562661

RESUMEN

Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling.


Asunto(s)
Clonación Molecular , Escherichia coli/genética , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/aislamiento & purificación , Receptores sigma/genética , Receptores sigma/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Vectores Genéticos/genética , Cobayas , Proteínas de Unión a Maltosa/química , Datos de Secuencia Molecular , Plásmidos/genética , Receptores sigma/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Receptor Sigma-1
6.
Sci Rep ; 12(1): 10223, 2022 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-35715477

RESUMEN

Animal chimeras are widely used for biomedical discoveries, from developmental biology to cancer research. However, the accurate quantitation of mixed cell types in chimeric and mosaic tissues is complicated by sample preparation bias, transgenic silencing, phenotypic similarity, and low-throughput analytical pipelines. Here, we have developed and characterized a droplet digital PCR single-nucleotide discrimination assay to detect chimerism among common albino and non-albino mouse strains. In addition, we validated that this assay is compatible with crude lysate from all solid organs, drastically streamlining sample preparation. This chimerism detection assay has many additional advantages over existing methods including its robust nature, minimal technical bias, and ability to report the total number of cells in a prepared sample. Moreover, the concepts discussed here are readily adapted to other genomic loci to accurately measure mixed cell populations in any tissue.


Asunto(s)
Quimerismo , Trasplante de Células Madre Hematopoyéticas , Animales , Reacción en Cadena de la Polimerasa/métodos
7.
Cell Rep ; 40(9): 111264, 2022 08 30.
Artículo en Inglés | MEDLINE | ID: mdl-36044843

RESUMEN

As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research.


Asunto(s)
Células Madre Pluripotentes Inducidas , Pan troglodytes , Animales , Macaca mulatta , Macaca nemestrina/genética , Proteómica
8.
Cell Stem Cell ; 28(1): 141-149.e3, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33373620

RESUMEN

Interspecies organ generation via blastocyst complementation has succeeded in rodents, but not yet in evolutionally more distant species. Early developmental arrest hinders the formation of highly chimeric fetuses. We demonstrate that the deletion of insulin-like growth factor 1 receptor (Igf1r) in mouse embryos creates a permissive "cell-competitive niche" in several organs, significantly augmenting both mouse intraspecies and mouse/rat interspecies donor chimerism that continuously increases from embryonic day 11 onward, sometimes even taking over entire organs within intraspecies chimeras. Since Igf1r deletion allows the evasion of early developmental arrest, interspecies fetuses with high levels of organ chimerism can be generated via blastocyst complementation. This observation should facilitate donor cell contribution to host tissues, resulting in whole-organ generation via blastocyst complementation across wide evolutionary distances.


Asunto(s)
Quimera , Células Madre Pluripotentes , Animales , Blastocisto , Quimerismo , Ratones , Ratas , Roedores
9.
PLoS One ; 9(6): e97198, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24937088

RESUMEN

Human rhinovirus strains differ greatly in their virulence, and this has been correlated with the differing substrate specificity of the respective 2A protease (2Apro). Rhinoviruses use their 2Apro to cleave a spectrum of cellular proteins important to virus replication and anti-host activities. These enzymes share a chymotrypsin-like fold stabilized by a tetra-coordinated zinc ion. The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues. We used a semi-automated NMR protocol developed at NMRFAM to determine the solution structure of 2Apro (C105A variant) from an isolate of the clinically important rhinovirus C species (RV-C). The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%. Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.


Asunto(s)
Cisteína Endopeptidasas/química , Rhinovirus/enzimología , Proteínas Virales/química , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína
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