RESUMEN
Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.
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Neoplasias Hepáticas , Humanos , Ratones , Animales , Neoplasias Hepáticas/metabolismo , Hepatocitos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Matriz Extracelular/metabolismo , Comunicación CelularRESUMEN
BACKGROUND: Base editing via CRISPR-Cas9 has garnered attention as a method for correcting disease-specific mutations without causing double-strand breaks, thereby avoiding large deletions and translocations in the host chromosome. However, its reliance on the protospacer adjacent motif (PAM) can limit its use. We aimed to restore a disease mutation in a patient with severe hemophilia B using base editing with SpCas9-NG, a modified Cas9 with the board PAM flexibility. METHODS: We generated induced pluripotent stem cells (iPSCs) from a patient with hemophilia B (c.947T>C; I316T) and established HEK293 cells and knock-in mice expressing the patient's F9 cDNA. We transduced the cytidine base editor (C>T), including the nickase version of Cas9 (wild-type SpCas9 or SpCas9-NG), into the HEK293 cells and knock-in mice through plasmid transfection and an adeno-associated virus vector, respectively. RESULTS: Here we demonstrate the broad PAM flexibility of SpCas9-NG near the mutation site. The base-editing approach using SpCas9-NG but not wild-type SpCas9 successfully converts C to T at the mutation in the iPSCs. Gene-corrected iPSCs differentiate into hepatocyte-like cells in vitro and express substantial levels of F9 mRNA after subrenal capsule transplantation into immunodeficient mice. Additionally, SpCas9-NG-mediated base editing corrects the mutation in both HEK293 cells and knock-in mice, thereby restoring the production of the coagulation factor. CONCLUSION: A base-editing approach utilizing the broad PAM flexibility of SpCas9-NG can provide a solution for the treatment of genetic diseases, including hemophilia B.
In patients with hemophilia B, the blood does not clot properly, leading to excessive bruising and bleeding. Hemophilia B is caused by an error in a gene called coagulation factor IX (F9). To treat patients with hemophilia B, we might be able to use a technology called CRISPR-Cas9 to edit and correct this genetic error, restoring factor IX function and improving clotting. Here, we test a specific CRISPR-Cas9 approach in cells and animals. We show that we are able to correct the genetic error in F9 in cells isolated from a patient with severe hemophilia B. We also show that we can fix the error in mice and that this increases levels of factor IX in the blood of the mice. With further testing, this gene-editing approach may be a viable therapy for patients with hemophilia B or similar genetic disorders.
RESUMEN
Knock-in therapy, in which an insertion site can be controlled, would be more suitable for the treatment of genetic blood disorders as compared to conventional gene therapy with lentivirus vectors that introduce genes into the genome randomly. Recent advancements in genome editing technology have substantially improved the knock-in efficiency, making it a reality. We present the details of a virus-free CRISPR/Cas9-based genome editing method for bona fide mouse hematopoietic stem cells.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Ratones , Animales , Edición Génica/métodos , Terapia Genética/métodos , Lentivirus/genéticaRESUMEN
A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.
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Sistemas CRISPR-Cas , Técnicas de Sustitución del Gen/métodos , Células Madre Pluripotentes Inducidas/citología , Animales , Células Cultivadas , Células Clonales , Medios de Cultivo , Cartilla de ADN , Resistencia a Medicamentos/genética , Electroporación , Células Madre Embrionarias/citología , Edición Génica/métodos , Genes Reporteros , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Puromicina/farmacología , ARN Guía de Kinetoplastida/genética , Reparación del ADN por Recombinación/genéticaRESUMEN
Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins - α-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) - and one M-line protein - Myomesin-2 (Myom2) - were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.
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Colorantes Fluorescentes/metabolismo , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Sarcómeros/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Dependovirus/metabolismo , Cuerpos Embrioides/citología , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Miocitos Cardíacos/citología , Coloración y Etiquetado , Imagen de Lapso de TiempoRESUMEN
We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b (Itga2b) with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain (Il2rg) into intron 1 of Il2rg via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.
RESUMEN
We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.
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Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes Inducidas/trasplante , Ovinos/embriología , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Linaje de la Célula , Movimiento Celular , Microambiente Celular , Ensayo de Unidades Formadoras de Colonias , Trasplante de Células Madre de Sangre del Cordón Umbilical , Femenino , Técnicas Genéticas , Supervivencia de Injerto , Hemangioblastos/citología , Xenoinjertos , Humanos , Células Madre Pluripotentes Inducidas/citología , Antígenos Comunes de Leucocito/análisis , Hígado/embriología , Subgrupos Linfocitarios , Embarazo , Especificidad de la EspecieRESUMEN
Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) hold great promise for disease modeling and drug discovery. However, PSC-CMs exhibit immature phenotypes in culture, and the lack of maturity limits their broad applications. While physical and functional analyses are generally used to determine the status of cardiomyocyte maturation, they could be time-consuming and often present challenges in comparing maturation-enhancing strategies. Therefore, there is a demand for a method to assess cardiomyocyte maturation rapidly and reproducibly. In this study, we found that Myomesin-2 (Myom2), encoding M-protein, is upregulated postnatally, and based on this, we targeted TagRFP to the Myom2 locus in mouse embryonic stem cells. Myom2-RFP+ PSC-CMs exhibited more mature phenotypes than RFP- cells in morphology, function and transcriptionally, conductive to sarcomere shortening assays. Using this system, we screened extracellular matrices (ECMs) and identified laminin-511/521 as potent enhancers of cardiomyocyte maturation. Together, we developed and characterized a novel fluorescent reporter system for the assessment of cardiomyocyte maturation and identified potent maturation-enhancing ECMs through this simple and rapid assay. This system is expected to facilitate use of PSC-CMs in a variety of scientific and medical investigations.
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Biomarcadores , Diferenciación Celular , Expresión Génica , Genes Reporteros , Laminina/genética , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Calcio , Diferenciación Celular/genética , Biología Computacional/métodos , Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Laminina/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Sarcómeros/metabolismo , Transcripción Genética , TranscriptomaRESUMEN
X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.
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Proteína 9 Asociada a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Animales , Modelos Animales de Enfermedad , Edición Génica , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones , Ratones Noqueados , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma XRESUMEN
Human induced pluripotent stem cells (hiPSCs) are creating great expectations for regenerative medicine. However, safety strategies must be put in place to guard against teratoma formation after transplantation of hiPSC-derived cells into patients. Recent studies indicate that epigenetic regulators act at the initial step of tumorigenesis. Using gain-of-function and loss-of-function approaches, we show here that the expression and function of lysine-specific demethylase 1 (LSD1) are tightly regulated in hiPSCs, and their deregulation underlies the development of teratomas. Consistent with these results, we demonstrate that an LSD1 inhibitor, S2157, prevented teratoma formation from hiPSCs transplanted into immunodeficient mice. This novel action of LSD1 and the effects of its inhibition potentially allow for the development of new clinical applications and therapeutic strategies using hiPSCs.
RESUMEN
Genetically engineered pigs play an indispensable role in the study of rare monogenic diseases. Pigs harboring a gene responsible for a specific disease can be efficiently generated via somatic cell cloning. The generation of somatic cell-cloned pigs from male cells with mutation(s) in an X chromosomal gene is a reliable and straightforward method for reproducing X-linked genetic diseases (XLGDs) in pigs. However, the severe symptoms of XLGDs are often accompanied by impaired growth and reproductive disorders, which hinder the reproduction of these valuable model animals. Here, we generated unique chimeric boars composed of mutant cells harboring a lethal XLGD and normal cells. The chimeric boars exhibited the cured phenotype with fertility while carrying and transmitting the genotype of the XLGD. This unique reproduction system permits routine production of XLGD model pigs through the male-based breeding, thereby opening an avenue for translational research using disease model pigs.
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Técnicas de Cultivo de Embriones/métodos , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Reproducción/genética , Animales , Animales Modificados Genéticamente/genética , Cruzamiento , Quimera , Clonación de Organismos/métodos , Modelos Animales de Enfermedad , Fertilidad , Técnicas de Inactivación de Genes/métodos , Ingeniería Genética/métodos , Masculino , Técnicas de Transferencia Nuclear , Porcinos/genéticaRESUMEN
Center for Development of Advanced Medical Technology (CDAMTec) in Jichi Medical University was established in 2009. It is the first educational research facility specialized for medical research and training using swine in Japan. Preclinical studies on large animals are essential prior to clinical trials to develop regenerative medical products and medical equipment. We have continued comprehensively considering using miniature swine for experiments to develop advanced medical technologies and train physicians with advanced clinical abilities, while paying attention to animal welfare. The center plays a pioneering role in this field by accumulating know-how such as (1) Construction and effective utilization of research facilities, (2) Procurement of quality animal resources, (3) Education and training of technical staff, (4) Establishment of support system for physicians and researchers. We now open up widely these expertise and foundation for medical research and training not only within our university but also outside the university, so as to move faster to practical use of advanced medical technology and contribute to human health and welfare.
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Bienestar del Animal , Modelos Animales , Facultades de Medicina , Porcinos Enanos , Investigación Biomédica Traslacional/educación , Investigación Biomédica Traslacional/ética , Animales , Arquitectura y Construcción de Instituciones de Salud , Humanos , Japón , Porcinos , Investigación Biomédica Traslacional/tendenciasRESUMEN
Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20-100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=-0.97, P<0.05). Therefore, it is critical to deliver piglets within 5 min to achieve a high resuscitation rate (82% estimated from regression analysis). In a fifth trial with an IL2RG+/- pig, four piglets were delivered within 4.2 min of uterus excision and three were alive (75%). One of the live born piglets was genotypically and phenotypically determined to be X-SCID and was reared for 12 weeks. The X-SCID piglet was free from both bacteria and fungi at all time points tested by microbial culture and grew without any abnormal signs or symptoms. This study showed successful production and rearing of germ-free pigs, enabling experiments involving long-term follow-up of X-SCID pigs.
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Modelos Animales de Enfermedad , Organismos Libres de Patógenos Específicos , Porcinos , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X , Crianza de Animales Domésticos , Animales , Susceptibilidad a Enfermedades , Femenino , Histerectomía , Infecciones , Subunidad gamma Común de Receptores de Interleucina/genética , Mutación , Embarazo , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/genéticaRESUMEN
We and others have reported that human hematopoietic stem cells (HSCs) are also present in the CD34-negative (CD34-) fraction of human cord blood (CB). Here, we examined the hematopoietic engraftment potential of 13 or 18 lineage-negative (13Lin- or 18Lin-) CD34+/- cells from human CB in mice and sheep. Both 13Lin- and 18Lin- CD34+ cells efficiently engrafted in mice irrespective of transplantation route, be it by tail-vein injection (TVI) or by intra-bone marrow injection (IBMI). These cells also engrafted in sheep after in utero fetal intra-hepatic injection (IHI). In contrast, neither 13Lin- nor 18Lin- CD34- cells engrafted in either mice or sheep when transplanted by regular routes (i.e., TVI and fetal IHI, respectively), although both 13Lin- and 18Lin- CD34- cells engrafted in mice when transplanted by IBMI and exhibited multilineage reconstitution ability. Thus, the homing ability of CD34- HSCs is significantly more limited than that of CD34+ HSCs. As for 18Lin-, CD34- HSCs are characterized by low expression of the tetraspanin CD9, which promotes homing, and high expression of the peptidase CD26, which inhibits homing. This unique expression pattern homing-related molecules on CD34- HSCs could thus explain in part their reduced ability to home to the BM niche.
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Dipeptidil Peptidasa 4/biosíntesis , Regulación de la Expresión Génica/fisiología , Supervivencia de Injerto , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Tetraspanina 29/biosíntesis , Animales , Antígenos CD34 , Femenino , Células Madre Hematopoyéticas/citología , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , OvinosRESUMEN
Haemophilia B, a congenital haemorrhagic disease caused by mutations in coagulation factor IX gene (F9), is considered an appropriate target for genome editing technology. Here, we describe treatment strategies for haemophilia B mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Administration of adeno-associated virus (AAV) 8 vector harbouring Staphylococcus aureus Cas9 (SaCas9) and single guide RNA (sgRNA) to wild-type adult mice induced a double-strand break (DSB) at the target site of F9 in hepatocytes, sufficiently developing haemophilia B. Mutation-specific gene editing by simultaneous induction of homology-directed repair (HDR) sufficiently increased FIX levels to correct the disease phenotype. Insertion of F9 cDNA into the intron more efficiently restored haemostasis via both processes of non-homologous end-joining (NHEJ) and HDR following DSB. Notably, these therapies also cured neonate mice with haemophilia, which cannot be achieved with conventional gene therapy with AAV vector. Ongoing haemophilia therapy targeting the antithrombin gene with antisense oligonucleotide could be replaced by SaCas9/sgRNA-expressing AAV8 vector. Our results suggest that CRISPR/Cas9-mediated genome editing using an AAV8 vector provides a flexible approach to induce DSB at target genes in hepatocytes and could be a good strategy for haemophilia gene therapy.
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Proteína 9 Asociada a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Dependovirus/genética , Edición Génica , Vectores Genéticos/administración & dosificación , Hemofilia B/terapia , Animales , Animales Recién Nacidos , Antitrombina III/metabolismo , Secuencia de Bases , ADN Complementario/genética , Exones/genética , Factor IX/metabolismo , Intrones/genética , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Fenotipo , Reparación del ADN por Recombinación/genéticaRESUMEN
Animal models of thrombocytopenia are indispensable for evaluating the in vivo efficacy of hemostatic agents, cryopreserved platelets, and artificial platelets, but no large animal models are available. In this study, we generated a swine model of acute thrombocytopenia with prolonged bleeding times by administering the chemotherapeutic drug busulfan. First, we tested multiple doses of busulfan (4, 6, and 8 mg/kg) in pigs, and found that 6 mg/kg of busulfan is an optimal dose for producing a safe and moderate thrombocytopenia, with a platelet count of less than 30,000/µl. The pigs administered 6 mg/kg of busulfan (n=8) reached half their initial counts at day 7, counts below 30,000/µl at day 12, and their nadirs at day 15 (on average). The minimal platelet count was 14,000/µl. With this dose of busulfan (6 mg/kg), bleeding times were significantly prolonged in addition to the decrease in platelet counts (r=-0.63, P<0.01), while there were no cases of apparent hemorrhage. White blood cell counts were maintained at over 5,000/µl, and there were no infections or other adverse events including anemia or appetite or body weight loss. All pigs were sacrificed on day 16, with subsequent examination showing a significant reduction in cellularity and colony-forming units in the bone marrow, indicating that thrombocytopenia was the result of myelosuppression. In summary, administration with 6 mg/kg of busulfan induces safe and moderate thrombocytopenia with a prolonged bleeding time in swine.
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Antineoplásicos Alquilantes/efectos adversos , Busulfano/efectos adversos , Modelos Animales de Enfermedad , Trombocitopenia/etiología , Enfermedad Aguda , Animales , Tiempo de Sangría , Relación Dosis-Respuesta a Droga , Femenino , Pruebas Hematológicas , Humanos , Masculino , Porcinos , Porcinos EnanosRESUMEN
DNA methylation is an epigenetic modification important for cell fate determination and cell type-specific gene expression. Transcriptional regulatory regions of the mammalian genome contain a large number of tissue/cell type-dependent differentially methylated regions (T-DMRs) with DNA methylation patterns crucial for transcription of the corresponding genes. In general, tissues consist of multiple cell types in various proportions, making it difficult to detect T-DMRs of minor cell types in tissues. The present study attempts to detect T-DMRs of minor cell types in tissues by ultra-deep bisulfite sequencing of cell type-restricted genes and to assume proportions of minor cell types based on DNA methylation patterns of sequenced reads. For this purpose, we focused on transcriptionally active hypomethylated alleles (Hypo-alleles), which can be recognized by the high ratio of unmethylated CpGs in each sequenced read (allele). The pituitary gland contains multiple cell types including five hormone-expressing cell types and stem/progenitor cells, each of which is a minor cell type in the pituitary tissue. By ultra-deep sequencing of more than 100 reads for detection of Hypo-alleles in pituitary cell type-specific genes, we identified T-DMRs specific to hormone-expressing cells and stem/progenitor cells and used them to estimate the proportions of each cell type based on the Hypo-allele ratio in pituitary tissue. Therefore, introduction of the novel Hypo-allele concept enabled us to detect T-DMRs of minor cell types with estimation of their proportions in the tissue by ultra-deep bisulfite sequencing.
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Metilación de ADN/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , Hipófisis/metabolismo , Alelos , Animales , Islas de CpG/genética , Metilación de ADN/genética , PorcinosRESUMEN
Xenograft models of human hematopoiesis are essential to the study of the engraftment and proliferative potential of human hematopoietic stem cells (HSCs) in vivo. Immunodeficient mice and fetal sheep are often used as xenogeneic recipients because they are immunologically naive. In this study, we transplanted human HSCs into fetal sheep and assessed the long-term engraftment of transplanted human HSCs after birth. Fourteen sheep were used in this study. In 4 fetal sheep, HSCs were transduced with homeo-box B4 (HOXB4) gene before transplantation, which promoted the expansion of HSCs. Another 4 fetal sheep were subjected to non-myeloablative conditioning with busulfan. Seven of these 8 sheep showed successful engraftment of human HSCs (1-3% of colony-forming units) as assessed after the birth of fetal sheep (5 months post-transplantation), although HOXB4-transduced HSCs showed sustained engraftment for up to 40 months. Intact HSCs were transplanted into six non-conditioned fetal sheep, and human colony-forming units were not detected in the sheep after birth. These results suggest that, as compared with mouse models, where the short lifespan of mice limits long-term follow-up of HSC engraftment, the fetal sheep model provides a unique perspective for evaluating long-term engraftment and proliferation of human HSCs.
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Proliferación Celular , Feto , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Ovinos/embriología , Animales , Busulfano , Femenino , Estudios de Seguimiento , Xenoinjertos , Proteínas de Homeodominio , Humanos , Ratones , Modelos Animales , Embarazo , Factores de Tiempo , Factores de Transcripción , Transducción Genética , Acondicionamiento PretrasplanteRESUMEN
Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (nâ=â4) and ovary (nâ=â1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (nâ=â10) than in NK-competent NOD/SCID (T-B-NK+) mice (nâ=â8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.