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 (â¼57%-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 â¼6%-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 , TropismoRESUMEN
Scarless genome editing in human pluripotent stem cells (hPSCs) represents a goal for both precise research applications and clinical translation of hPSC-derived therapies. Here we established a versatile and efficient method that combines CRISPR-Cas9-mediated homologous recombination with positive-negative selection of edited clones to generate scarless genetic changes in hPSCs.
Asunto(s)
Sistemas CRISPR-Cas , Células Madre Embrionarias/metabolismo , Edición Génica , Genoma Humano , Recombinación Homóloga , Células Madre Pluripotentes/metabolismo , ARN Interferente Pequeño/genética , Células Madre Embrionarias/citología , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes/citologíaRESUMEN
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/metabolismoRESUMEN
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.
RESUMEN
The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (â¼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Células Madre Hematopoyéticas , Células Madre Pluripotentes , Animales , Humanos , Ratones , Células Endoteliales/metabolismo , Células Endoteliales/citología , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismoRESUMEN
The discovery and development of induced pluripotent stem cells (iPSCs) opened a novel venue for disease modeling, drug discovery, and personalized medicine. Additionally, iPSCs have been utilized for a wide variety of research and clinical applications without immunological and ethical concerns that arise from using embryonic stem cells. Understanding the in vivo behavior of iPSCs, as well as their derivatives, requires the monitoring of their localization, proliferation, and viability after transplantation. Bioluminescence imaging (BLI) gives investigators a non-invasive and sensitive means for spatio-temporal tracking in vivo. For scientists working within the field of iPSCs, this protocol provides a walk-through on how to conduct in vitro and in vivo experiments with an iPSCs constitutively expressing luciferase.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Embrionarias , Humanos , Luciferasas/genéticaRESUMEN
Despite their rapidly-expanding therapeutic potential, human pluripotent stem cell (hPSC)-derived cell therapies continue to have serious safety risks. Transplantation of hPSC-derived cell populations into preclinical models has generated teratomas (tumors arising from undifferentiated hPSCs), unwanted tissues, and other types of adverse events. Mitigating these risks is important to increase the safety of such therapies. Here we use genome editing to engineer a general platform to improve the safety of future hPSC-derived cell transplantation therapies. Specifically, we develop hPSC lines bearing two drug-inducible safeguards, which have distinct functionalities and address separate safety concerns. In vitro administration of one small molecule depletes undifferentiated hPSCs >106-fold, thus preventing teratoma formation in vivo. Administration of a second small molecule kills all hPSC-derived cell-types, thus providing an option to eliminate the entire hPSC-derived cell product in vivo if adverse events arise. These orthogonal safety switches address major safety concerns with pluripotent cell-derived therapies.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/genética , Edición Génica/métodos , Células Madre Pluripotentes/metabolismo , Trasplante de Células Madre/métodos , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Tacrolimus/análogos & derivados , Tacrolimus/farmacología , Teratoma/genética , Teratoma/metabolismo , Teratoma/prevención & controlRESUMEN
Human induced pluripotent stem cells (iPSCs) are a potential source of blood cells for transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. The development of widely varying reprogramming methods has enabled us nowadays to obtain iPSCs even from a small number of antigen-specific T cells from patients. As these T-cell-derived iPSCs (T-iPSCs) carry TCR gene rearrangements in their genomic DNA, they are likely useful for producing antigen-specific T cells and for studying T-cell development. T-cell immunotherapy is potentially an effective therapeutic strategy against many types of cancers and viral infections. If antigen-specific T cells tailored against diseases and for patients can be easily obtained, T-cell immunotherapy should become a popular choice of therapy. Here, we show the in vitro way to guide T-iPSCs sequentially to yield hematopoietic stem/progenitor cells (HSPCs), T-lineage cells, and mature CD8 single-positive T cells. These in vitro-generated CD8+ T cells display antigen-specific cytotoxity and perform general T-cell functions. This novel protocol thus provides means to generate antigen-specific T cells as well as chances to study normal human lymphopoiesis. It may help identify, and then clear away, barriers to T-cell immunotherapy such as immunological tolerance and cell exhaustion. T-iPSCs can confer their juvenile status upon their descendant T cells during pluripotency reprogramming and redifferentiation. This phenomenon should help to eliminate T-cell exhaustion.
Asunto(s)
Antígenos CD8/inmunología , Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/citología , Linfocitos T/citología , Humanos , Inmunoterapia , Células Madre Pluripotentes Inducidas/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunologíaRESUMEN
The discovery and development of induced pluripotent stem cells (iPSCs) opened a novel venue for disease modeling, drug discovery, and personalized medicine. Additionally, iPSCs have been utilized for a wide variety of research and clinical applications without immunological and ethical concerns that encounter embryonic stem cells. Adoptive T cell immunotherapy is a form of cellular immunotherapy that involves transfusion of functional T cells. However, this approach requires T cell expansion and the process causes T cell exhaustion. As a result, highly expanded T cells have not proven to be particularly effective for treatments. This exhaustion issue could be overcome due to rejuvenation of T cells by reprogramming to pluripotency and redifferentiation to T cells. This is a potential therapeutic strategy for combating various types of cancer.
Asunto(s)
Reprogramación Celular/inmunología , Células Madre Pluripotentes Inducidas/fisiología , Cultivo Primario de Células/métodos , Linfocitos T Citotóxicos/fisiología , Reprogramación Celular/genética , Vectores Genéticos/genética , Humanos , Inmunoterapia Adoptiva/métodos , Neoplasias/inmunología , Neoplasias/terapia , Proteínas Recombinantes/genética , Virus Sendai/genética , Factores de Transcripción/genética , Transducción Genética/métodosRESUMEN
For scientists working within the field of induced pluripotent stem cells (iPSCs), this protocol will provide a thorough walk-through on how to conduct in vitro and in vivo experiments that validate the function of a particular safeguard system technology. In short, we provide instructions on how to generate inducible Caspase-9 (iC9) safeguard system with human iPSCs that act as normal or abnormal models of the cells for therapeutics to be tried after differentiation. These iC9-iPSCs should be modified prior to beginning this protocol by constitutively expressing luciferase, an enzyme capable of generating bioluminescent signals through the oxidation of the substrate luciferin. Monitoring the bioluminescent signal over time provides the information on whether a safeguard system is working or not.
Asunto(s)
Genes Transgénicos Suicidas , Microscopía Intravital/métodos , Mediciones Luminiscentes/métodos , Teratoma/diagnóstico por imagen , Animales , Benzotiazoles/administración & dosificación , Benzotiazoles/química , Caspasa 9/genética , Caspasa 9/metabolismo , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Línea Celular , Medios de Cultivo/metabolismo , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Genes Reporteros/genética , Humanos , Inmunoterapia/efectos adversos , Inmunoterapia/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Inyecciones Intraperitoneales , Microscopía Intravital/instrumentación , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Mediciones Luminiscentes/instrumentación , Ratones Endogámicos NOD , Ratones SCID , Tacrolimus/administración & dosificación , Tacrolimus/análogos & derivados , Teratoma/inmunología , Teratoma/patología , Teratoma/terapia , Carga TumoralRESUMEN
Serum albumin has long been an essential supplement for ex vivo hematopoietic and immune cell cultures. However, serum albumin medium supplements represent a major source of biological contamination in cell cultures and often cause loss of cellular function. As serum albumin exhibits significant batch-to-batch variability, it has also been blamed for causing major issues in experimental reproducibility. We recently discovered the synthetic polymer polyvinyl alcohol (PVA) as an inexpensive, Good Manufacturing Practice-compatible, and biologically inert serum albumin replacement for ex vivo hematopoietic stem cell cultures. Importantly, PVA is free of the biological contaminants that have plagued serum albumin-based media. Here, we describe that PVA can replace serum albumin in a range of blood and immune cell cultures including cell lines, primary leukemia samples, and human T lymphocytes. PVA can even replace human serum in the generation and expansion of functional chimeric antigen receptor (CAR) T cells, offering a potentially safer and more cost-efficient approach for this clinical cell therapy. In summary, PVA represents a chemically defined, biologically inert, and inexpensive alternative to serum albumin for a range of cell cultures in hematology and immunology.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Alcohol Polivinílico/farmacología , Linfocitos T/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , División Celular/efectos de los fármacos , Células Cultivadas , Citotoxicidad Inmunológica , Humanos , Células K562 , Leucemia Mieloide Aguda/patología , Ratones , Receptores Quiméricos de Antígenos , Albúmina Sérica , Linfocitos T/citología , Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Genotipo , Células Madre Pluripotentes/fisiología , Proteína 9 Asociada a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Reparación del ADN , Edición Génica/métodos , Frecuencia de los Genes , Ingeniería Genética , Vectores Genéticos/genética , Recombinación Homóloga , Humanos , Patología Molecular , Donantes de TejidosRESUMEN
Thalidomide, which was formerly available commercially to control the symptoms of morning sickness, is a strong teratogen that causes fetal abnormalities. However, the mechanism of thalidomide teratogenicity is not fully understood; thalidomide toxicity is not apparent in rodents, and the use of human embryos is ethically and technically untenable. In this study, we designed an experimental system featuring human-induced pluripotent stem cells (hiPSCs) to investigate the effects of thalidomide. These cells exhibit the same characteristics as those of epiblasts originating from implanted fertilized ova, which give rise to the fetus. Therefore, theoretically, thalidomide exposure during hiPSC differentiation is equivalent to that in the human fetus. We examined the effects of thalidomide on undifferentiated hiPSCs and early-differentiated hiPSCs cultured in media containing bone morphogenetic protein-4, which correspond, respectively, to epiblast (future fetus) and trophoblast (future extra-embryonic tissue). We found that only the number of undifferentiated cells was reduced. In undifferentiated cells, application of thalidomide increased the number of apoptotic and dead cells at day 2 but not day 4. Application of thalidomide did not affect the cell cycle. Furthermore, immunostaining and flow cytometric analysis revealed that thalidomide exposure had no effect on the expression of specific markers of undifferentiated and early trophectodermal differentiated cells. These results suggest that the effect of thalidomide was successfully detected in our experimental system and that thalidomide eliminated a subpopulation of undifferentiated hiPSCs. This study may help to elucidate the mechanisms underlying thalidomide teratogenicity and reveal potential strategies for safely prescribing this drug to pregnant women.
Asunto(s)
Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Talidomida/farmacología , Biomarcadores/metabolismo , Recuento de Células , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ectodermo/citología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Antígeno Ki-67/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Trofoblastos/citologíaRESUMEN
Although it is important to clarify the pathogenic functions of T cells in human samples, their examination is often limited due to difficulty in obtaining sufficient numbers of dendritic cells (DCs), used as antigen-presenting cells, especially in autoimmune diseases. We describe the generation of DCs from induced pluripotent stem cells derived from T cells (T-iPSCs). We reprogrammed CD4+ T cell clones from a patient with Sjögren's syndrome (SS) into iPSCs, which were differentiated into DCs (T-iPS-DCs). T-iPS-DCs had dendritic cell-like morphology, and expressed CD11c, HLA-DR, CD80, CD86, and also BDCA-3. Compared with monocyte-derived DCs, the capacity for antigen processing was similar, and T-iPS-DCs induced the proliferative response of autoreactive CD4+ T cells. Moreover, we could evaluate T cell functions of the patient with SS. In conclusion, we obtained adequate numbers of DCs from T-iPSCs, which could be used to characterize pathogenic T cells in autoimmune diseases such as SS.
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Linfocitos T CD4-Positivos/citología , Reprogramación Celular , Células Dendríticas/citología , Células Madre Pluripotentes Inducidas/citología , Síndrome de Sjögren/patología , Presentación de Antígeno , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Antígenos CD11/genética , Antígenos CD11/metabolismo , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Células Cultivadas , Células Dendríticas/inmunología , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , TrombomodulinaRESUMEN
Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Proteínas Recombinantes/farmacología , Animales , Proteínas Sanguíneas/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Células Madre Hematopoyéticas/metabolismo , Hemopexina/farmacología , Interleucina-1alfa/farmacología , RatonesRESUMEN
Understanding the relative contributions of genetic and epigenetic abnormalities to acute myeloid leukemia (AML) should assist integrated design of targeted therapies. In this study, we generated induced pluripotent stem cells (iPSCs) from AML patient samples harboring MLL rearrangements and found that they retained leukemic mutations but reset leukemic DNA methylation/gene expression patterns. AML-iPSCs lacked leukemic potential, but when differentiated into hematopoietic cells, they reacquired the ability to give rise to leukemia in vivo and reestablished leukemic DNA methylation/gene expression patterns, including an aberrant MLL signature. Epigenetic reprogramming was therefore not sufficient to eliminate leukemic behavior. This approach also allowed us to study the properties of distinct AML subclones, including differential drug susceptibilities of KRAS mutant and wild-type cells, and predict relapse based on increased cytarabine resistance of a KRAS wild-type subclone. Overall, our findings illustrate the value of AML-iPSCs for investigating the mechanistic basis and clonal properties of human AML.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Leucemia Mieloide Aguda/patología , Modelos Biológicos , Crisis Blástica/patología , Línea Celular Tumoral , Linaje de la Célula , Forma de la Célula , Reprogramación Celular , Aberraciones Cromosómicas , Células Clonales , Metilación de ADN/genética , Epigénesis Genética , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células HEK293 , Hematopoyesis/genética , Humanos , Leucemia Mieloide Aguda/genética , Terapia Molecular Dirigida , Mutación/genética , Invasividad Neoplásica , FenotipoRESUMEN
It is not known whether atypical Mycobacterium (AM) causes peritonitis in humans. We described a case of tuberculosis-like peritonitis caused by an AM. Genetic analysis of the biopsy specimens suggested an AM infection. Thus, we concluded that peritonitis in humans can be caused by some AM species as well as by Mycobacterium tuberculosis complex.
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Micobacterias no Tuberculosas/aislamiento & purificación , Peritonitis Tuberculosa/microbiología , Adulto , Proteínas Bacterianas/genética , Biopsia , ARN Polimerasas Dirigidas por ADN , Femenino , Humanos , Japón , Micobacterias no Tuberculosas/genética , Peritonitis Tuberculosa/patología , FilogeniaRESUMEN
Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17+ endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17+ endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.
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Apoptosis , Quimera/metabolismo , Embrión de Mamíferos/citología , Animales , Células Madre Embrionarias/citología , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Transcripción SOX/metabolismoRESUMEN
The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.
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Apoptosis , Caspasa 9/genética , Células Madre Pluripotentes Inducidas/citología , Neoplasias/terapia , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/trasplante , Animales , Diferenciación Celular , Células Cultivadas , Terapia Genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Ratones SCID , Neoplasias/genética , Neoplasias/patología , Linfocitos T Citotóxicos/metabolismoRESUMEN
Adoptive immunotherapy with functional T cells is potentially an effective therapeutic strategy for combating many types of cancer and viral infection. However, exhaustion of antigen-specific T cells represents a major challenge to this type of approach. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8(+) T cells from an HIV-1-infected patient to pluripotency. The T cell-derived induced pluripotent stem cells were then redifferentiated into CD8(+) T cells that had a high proliferative capacity and elongated telomeres. These "rejuvenated" cells possessed antigen-specific killing activity and exhibited T cell receptor gene-rearrangement patterns identical to those of the original T cell clone from the patient. We also found that this method can be effective for generating specific T cells for other pathology-associated antigens. Thus, this type of approach may have broad applications in the field of adoptive immunotherapy.