RESUMO
The analysis of cells frozen within the International Space Station (ISS) will provide crucial insights into the impact of the space environment on cellular functions and properties. The objective of this study was to develop a method for cryopreserving blood cells under the specific constraints of the ISS. In a ground experiment, mouse blood was directly mixed with a cryoprotectant and gradually frozen at -80 °C. Thawing the frozen blood sample resulted in the successful recovery of viable mononuclear cells when using a mixed solution of dimethylsulfoxide and hydroxyethyl starch as a cryoprotectant. In addition, we developed new freezing cases to minimize storage space utilization within the ISS freezer. Finally, we confirmed the recovery of major mononuclear immune cell subsets from the cryopreserved blood cells through a high dimensional analysis of flow cytometric data using 13 cell surface markers. Consequently, this ground study lays the foundation for the cryopreservation of viable blood cells on the ISS, enabling their analysis upon return to Earth. The application of this method in ISS studies will contribute to understanding the impact of space environments on human cells. Moreover, this method may find application in the cryopreservation of blood cells in situations where research facilities are inadequate.
RESUMO
Blastocyst complementation is an intriguing way of generating humanized animals for organ preparation in regenerative medicine and establishing novel models for drug development. Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4+ and CD8+ T cells proliferated and produced IFNγ, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFNγ-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs/cells in interspecific chimeras will be important in the near future.
Assuntos
Blastocisto , Linfócitos T CD8-Positivos , Animais , Blastocisto/metabolismo , Quimera/genética , Células-Tronco Embrionárias , Camundongos , Camundongos Endogâmicos C57BLRESUMO
We have previously established a concept of developing exogenic pancreas in a genetically modified pig fetus with an apancreatic trait, thereby proposing the possibility of in vivo generation of functional human organs in xenogenic large animals. In this study, we aimed to demonstrate a further proof-of-concept of the compensation for disabled organogeneses in pig, including pancreatogenesis, nephrogenesis, hepatogenesis, and vasculogenesis. These dysorganogenetic phenotypes could be efficiently induced via genome editing of the cloned pigs. Induced dysorganogenetic traits could also be compensated by allogenic blastocyst complementation, thereby proving the extended concept of organ regeneration from exogenous pluripotent cells in empty niches during various organogeneses. These results suggest that the feasibility of blastocyst complementation using genome-edited cloned embryos permits experimentation toward the in vivo organ generation in pigs from xenogenic pluripotent cells.
Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular , Organogênese , Animais , Animais Geneticamente Modificados , Biomarcadores , Diferenciação Celular/genética , Clonagem de Organismos , Feto , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Proteínas de Homeodomínio , Organogênese/genética , Pâncreas/embriologia , Fenótipo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Regeneração , Suínos , Transativadores/deficiênciaRESUMO
To study development of the conceptus in xenogeneic environments, we assessed interspecies chimera formation as well as tetraploid complementation between mouse and rat. Overall contribution of donor PSC-derived cells was lower in interspecies chimeras than in intraspecies chimeras, and high donor chimerism was associated with anomalies or embryonic death. Organ to organ variation in donor chimerism was greater in interspecies chimeras than in intraspecies chimeras, suggesting species-specific affinity differences among interacting molecules necessary for organogenesis. In interspecies tetraploid complementation, embryo development was near normal until the stage of placental formation, after which no embryos survived.
Assuntos
Proteínas do Sistema Complemento/imunologia , Desenvolvimento Embrionário , Organogênese , Tetraploidia , Quimeras de Transplante , Animais , Blastocisto/citologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Pluripotentes/citologia , Gravidez , Ratos , Ratos Wistar , Especificidade da Espécie , Quimeras de Transplante/crescimento & desenvolvimento , Quimeras de Transplante/imunologiaRESUMO
In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5-9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived.
Assuntos
Blastocisto/citologia , Células Endoteliais/citologia , Células-Tronco Hematopoéticas/citologia , Envelhecimento/metabolismo , Animais , Blastocisto/metabolismo , Quimera , Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica , Pericitos/citologia , Pericitos/metabolismo , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Aims: The aim of the present study is to develop in vitro experimental analytical method for the electrophysiological properties of allogeneic induced pluripotent stem cell-derived cardiomyocytes (CMs) in cardiac conduction defect model. Methods and results: Cardiomyocytes were derived from rat induced pluripotent stem cells CMs (riPSC-CMs) using an embryoid body-based differentiation method with the serial application of growth factors including activin-A, bone morphogenetic protein 4 (BMP-4), and inhibitor of wnt production 2 (IWP-2). Flow cytometry analysis showed that 74.0 ± 2.7% of riPSC-CMs expressed cardiac troponin-T (n = 3). Immunostaining analysis revealed organized sarcomeric structure in riPSC-CMs and the expression of connexin 43 between riPSC-CMs and neonatal rat ventricular CMs (NRVMs). Ca2+ transient recordings revealed the simultaneous excitement of riPSC-CMs and NRVMs, and prolonged Ca2+ transient duration of riPSC-CMs as compared with NRVMs (731 ± 15.9 vs. 610 ± 7.72 ms, P < 0.01, n = 3). Isolated NRVMs were cultured in two discrete regions to mimic cardiac conduction defects on multi-electrode array dish, and riPSC-CMs were seeded in the channel between the two discrete regions. Membrane potential imaging with di-8-ANEPPS discerned the propagation of the electrical impulse from one NRVM region to the other through a riPSC-CM pathway. This pathway had significantly longer action potential duration as compared with NRVMs. Electrophysiological studies using a multi-electrode array platform demonstrated the longer conduction time and functional refractory period of the riPSC-CM pathway compared with the NRVM pathway. Conclusion: Using an in vitro experimental system to mimic cardiac conduction defect, transplanted allogeneic riPSC-CMs showed electrical coupling between two discrete regions of NRVMs. Electrophysiological testing using our platform will enable electrophysiological screening prior to transplantation of stem cell-derived CMs.
Assuntos
Potenciais de Ação/fisiologia , Doença do Sistema de Condução Cardíaco/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/fisiologia , Ativinas/farmacologia , Células Alógenas , Animais , Animais Recém-Nascidos , Benzotiazóis/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Proteínas de Ligação a Calmodulina/metabolismo , Diferenciação Celular , Conexina 43/metabolismo , Fenômenos Eletrofisiológicos , Citometria de Fluxo , Ventrículos do Coração/citologia , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/transplante , Ratos , Sarcômeros , Transplante HomólogoRESUMO
Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse-rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.
Assuntos
Diabetes Mellitus Experimental/terapia , Xenoenxertos/fisiologia , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/fisiologia , Organogênese , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Glicemia/metabolismo , Quimera , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Feminino , Xenoenxertos/imunologia , Proteínas de Homeodomínio , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/transplante , Ratos , Fatores de Tempo , Transativadores/deficiênciaRESUMO
Round spermatid injection (ROSI) into unfertilized oocytes enables a male with a severe spermatogenesis disorder to have children. One limitation of the application of this technique in the clinic is the identification and isolation of round spermatids from testis tissue. Here we developed an efficient and simple method to isolate rodent haploid round spermatids using flow cytometric cell sorting, based on DNA content (stained with Hoechst 33342 or Dye Cycle Violet) or by cell diameter and granularity (forward and side scatter). ROSI was performed with round spermatids selected by flow cytometry, and we obtained healthy offspring from unstained cells. This non-invasive method could therefore be an effective option for breeding domestic animals and human male infertility treatment. Mol. Reprod. Dev. 83: 488-496, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Citometria de Fluxo/métodos , Espermátides/citologia , Animais , Benzimidazóis/química , DNA/metabolismo , Masculino , Camundongos , Ratos , Ratos Wistar , Injeções de Esperma Intracitoplásmicas , Espermátides/metabolismoRESUMO
Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra- and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naïve-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs.
Assuntos
Técnicas de Cultura de Células/métodos , Quimera/embriologia , Desenvolvimento Embrionário/fisiologia , Técnicas In Vitro/métodos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Animais , Linhagem Celular , Haplorrinos , Humanos , Camundongos , Análise em Microsséries , Microinjeções , Ratos , Especificidade da EspécieRESUMO
This chapter describes a newly developed method for generating and maintaining rat induced pluripotent stem cells (riPSCs). We first provide a detailed protocol for the generation of lentiviral vector carrying three reprogramming factors to produce high-quality riPSCs. This technique allows reprogramming of rat somatic cells to ground state with germ-line competence. Subsequently, we elaborate a detailed protocol for the generation of riPSCs from rat embryonic fibroblast (REF). Finally, the protocols for the optimal culture conditions of riPSCs and preparation of frozen stock are described. We also outline the advantages of generating riPSCs.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Diferenciação Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Lentivirus/genética , Ratos , Transdução Genética , TransgenesRESUMO
Primordial germ cells (PGCs) are germ cell progenitors in the fetal genital ridge; female PGCs give rise to definitive oocytes that contribute to the next generation. Artificial PGCs have been induced in vitro from pluripotent stem cells and gonad-like tissue has been induced in vivo by cotransplantation of PGCs with PGC-free gonadal cells. To apply these technologies to human infertility treatment or conservation of rare species, PGC transplantation must be established in xenogenic animals. Here, we established a xenogeneic transplantation model by inducing ovary-like tissue from PGCs in xenogenic animals. We transplanted enzymatically dispersed PGCs with PGC-free gonadal cells under the kidney capsule of xenogenic immunodeficient animals. The transplanted cells formed ovary-like tissues under the kidney capsule. These tissues were histologically similar to the normal gonad and expressed the oocyte markers Vasa and Stella. In addition, mouse germinal vesicle-stage oocyte-like cells collected from ovary-like tissue in rats matured to metaphase II via in vitro maturation and gave rise to offspring by intracytoplasmic sperm injection. Our studies show that rat/mouse female PGCs and PGC-free gonadal cells can develop and reconstruct ovary-like tissue containing functional oocytes in an ectopic xenogenic microenvironment.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/transplante , Oócitos/fisiologia , Animais , Benzofuranos , Feminino , Células Germinativas , Xenoenxertos , Rim/citologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Oogênese/fisiologia , Quinolinas , Ratos , Ratos Endogâmicos , Transplante de Células-TroncoRESUMO
Consensus holds that hematopoietic stem cells (HSCs) give rise to multipotent progenitors (MPPs) of reduced self-renewal potential and that MPPs eventually produce lineage-committed progenitor cells in a stepwise manner. Using a single-cell transplantation system and marker mice, we unexpectedly found myeloid-restricted progenitors with long-term repopulating activity (MyRPs), which are lineage-committed to megakaryocytes, megakaryocyte-erythroid cells, or common myeloid cells (MkRPs, MERPs, or CMRPs, respectively) in the phenotypically defined HSC compartment together with HSCs. Paired daughter cell assays combined with transplantation revealed that HSCs can give rise to HSCs via symmetric division or directly differentiate into MyRPs via asymmetric division (yielding HSC-MkRP or HSC-CMRP pairs). These myeloid bypass pathways could be essential for fast responses to ablation stress. Our results show that loss of self-renewal and stepwise progression through specific differentiation stages are not essential for lineage commitment of HSCs and suggest a revised model of hematopoietic differentiation.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células Progenitoras Mieloides/citologia , Animais , Antígenos CD34 , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Progenitoras Mieloides/metabolismoRESUMO
Fluorescent-protein transgenic mice are useful for obtaining marked somatic cells to study kinetics of development or differentiation. Fluorescence-marked hematopoietic stem cells in particular are commonly used for studying hematopoiesis. However, as far as we know, no transgenic mouse line is described in which a fluorescent protein is stably and constitutively expressed in all hematopoietic cells, including erythrocytes and platelets. Using the random segregation of provirus (RSP) method, we generated from retrovirally transduced mouse embryonic stem cells a transgenic mouse line expressing a red/orange fluorescent protein, Kusabira Orange (KuO). KuO transgenic mouse line cells carry only one proviral integration site and stably express KuO in all hematopoietic-lineage elements, including erythrocytes and platelets. Moreover, bone-marrow transplantation in KuO transgenic mice demonstrated normal hematopoieisis. KuO transgenic mice likely will prove useful for study of hematopoiesis that includes erythropoiesis and megakaryopoiesis.
Assuntos
Eritrócitos/metabolismo , Engenharia Genética/métodos , Proteínas Luminescentes/metabolismo , Camundongos Transgênicos/metabolismo , Camundongos Transgênicos/virologia , Provírus/genética , Transfecção/métodos , Animais , Proteínas Luminescentes/genética , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética , Especificidade de Órgãos , Distribuição Tecidual , Proteína Vermelha FluorescenteRESUMO
Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Lentivirus/genética , Animais , Diferenciação Celular , Proliferação de Células , Doxiciclina/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Técnicas Genéticas , Vetores Genéticos , Células-Tronco Hematopoéticas/citologia , Hepatócitos/citologia , Humanos , Fator 4 Semelhante a Kruppel , Lentivirus/metabolismo , Camundongos , Modelos Genéticos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Fatores de TempoRESUMO
The question of whether a single hematopoietic stem cell (HSC) gives rise to all of the B-cell subsets [B-1a, B-1b, B-2, and marginal zone (MZ) B cells] in the mouse has been discussed for many years without resolution. Studies here finally demonstrate that individual HSCs sorted from adult bone marrow and transferred to lethally irradiated recipients clearly give rise to B-2, MZ B, and B-1b, but does not detectably reconstitute B-1a cells. These findings place B-2, MZ, and B-1b in a single adult developmental lineage and place B-1a in a separate lineage derived from HSCs that are rare or missing in adults. We discuss these findings with respect to known developmental heterogeneity in other HSC-derived lymphoid, myeloid, and erythroid lineages, and how HSC developmental heterogeneity conforms to the layered model of the evolution of the immune system that we proposed some years ago. In addition, of importance to contemporary medicine, we consider the implications that HSC developmental heterogeneity may have for selecting HSC sources for human transplantation.
Assuntos
Linfócitos B/citologia , Células da Medula Óssea/citologia , Linhagem da Célula , Células-Tronco Hematopoéticas/citologia , Animais , Citometria de Fluxo , CamundongosRESUMO
BACKGROUND: Recent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs. METHODOLOGY/PRINCIPAL FINDINGS: We generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines. CONCLUSIONS/SIGNIFICANCE: Our data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras.
Assuntos
Técnicas de Cultura de Células/métodos , Células Germinativas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Quimera/metabolismo , Quimerismo , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Células Germinativas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Camundongos , Ratos , Ratos Wistar , Especificidade da EspécieRESUMO
The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment.
Assuntos
Blastocisto , Quimera/embriologia , Pâncreas/citologia , Pâncreas/embriologia , Células-Tronco Pluripotentes , Animais , Diabetes Mellitus/induzido quimicamente , Diabetes Mellitus/terapia , Desenvolvimento Embrionário , Técnicas de Introdução de Genes , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos , Organogênese , Ratos , Ratos Wistar , Transativadores/genéticaRESUMO
Gene transfer vectors based on retroviruses are commonly used in gene therapy applications because of their unique ability to integrate efficiently into host genomes. This ability also forms the basis of a transformation event that can be induced in transduced cells by transactivation of proto-oncogenes near the vector integration sites. Here, we report on the development of lymphoma in mice generated from embryonic stem cells transduced with an enhanced green fluorescent protein. The cells expressed B220, CD5, Mac1, and IgM on their surfaces and expressed transcription factors characteristic of B-cell lymphoma. Importantly, each mouse had a single copy of the provirus in its genome; the copy was integrated into the second intron of the dopamine receptor 3 (D3) gene, and high-level expression of D3 was detected only in the lymphoma cells. Ectopic expression of D3 in murine marrow cells resulted in preferential proliferation of cells at the pre-B-cell stage in response to a D3-specific agonist, but this proliferation was not observed in vivo. Cells cotransduced with D3 and Bcl-x(L) genes had a phenotype similar to that of lymphoma in vivo, suggesting that the leukemogenesis induced by retroviral integration required "second hit" mutations of additional genes.
Assuntos
Células-Tronco Embrionárias/metabolismo , Linfoma de Células B/genética , Receptores de Dopamina D3/genética , Retroviridae/genética , Transdução Genética , Integração Viral , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Northern Blotting , Southern Blotting , Western Blotting , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação TranscricionalRESUMO
BACKGROUND: Genetic marking of hematopoietic stem cells (HSCs) with multiple fluorescent proteins (FPs) would allow analysis of their features, including interaction with adjacent cells. However, there are few red FPs that are comparable to green FPs in terms of low toxicity and high fluorescent intensity. This study has evaluated the usefulness of Kusabira Orange (KO) originated from the coral stone Fungia concinna as a red FP for marking of HSCs METHODS: A vector used was the MSCV-type retroviral vector, D Delta Nsap that has the PCC4 cell-passaged myeloproliferative sarcoma virus derived long terminal repeat devoid of a binding site for YY1 and the primer-binding site derived from the dl587rev, respectively. The vector was cloned with the codon-optimized KO cDNA for higher expression in mammalian cells (huKO) and converted to the corresponding retroviruses pseudotyped with the vesicular stomatitis virus G envelope protein, then transduced into c-KIT(+)Sca-1(+)Lineage(-) cells obtained from C57BL/6 (Ly5.1) mice followed by transplantation into lethally irradiated Ly5.2 mice. RESULTS: Approximately 70% of donor-derived cells highly expressed huKO at 16 weeks post-transplantation. Furthermore, the high expression of huKO was also detected in serially transplanted mice, suggesting that expression of huKO per se had little deleterious effect on murine hematopoiesis. In double marking experiments, huKO-expressing hematopoietic cells were easily distinguished from those expressing EGFP by flow cytometry and fluorescent microscope analysis. CONCLUSIONS: Overall, the results obtained from the present study suggest that huKO can be used as a valuable and versatile red fluorescent marker for HSCs.
Assuntos
Corantes Fluorescentes/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Proteínas Luminescentes/metabolismo , Animais , Linhagem da Célula , Células Cultivadas , DNA Complementar/metabolismo , Citometria de Fluxo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/química , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Retroviridae/genética , Retroviridae/metabolismo , Transdução Genética , Proteína Vermelha FluorescenteRESUMO
Silencing of transduced genes hampers production of transgenic mice using retroviral vectors. We show stable expression of the enhanced green fluorescent protein (EGFP) gene in chimeric mice generated from retrovirally transduced embryonic stem cells. The vector was a murine stem cell virus-typed retroviral vector (GCDsap) in which the long terminal repeat and primer-binding site were derived from a PCC4 cell-passaged myeloproliferative sarcoma virus and the endogenous retrovirus dl587rev, respectively. To increase the viral titer, the vector was packaged with vesicular stomatitis virus G protein, which allowed concentration of the virus into pellets followed by resuspension in serum-free medium. In chimeric mice, EGFP was detected in various tissues including hematopoietic cells, neurons, cardiac muscle, and intestine. Furthermore, high expression was maintained in the progeny of these mice, suggesting successful germline transmission of active proviruses. Although the proportion of EGFP-expressing cells and the mean intensity of EGFP expression varied among tissues and mice, 100% of peripheral blood leukocytes expressed EGFP in mice carrying a single provirus copy, as well as in their progeny. Therefore, the gene transfer system described here provides a useful tool not only to generate transgenic animals but also to manipulate human embryonic stem cells..