RESUMEN
Pluripotent stem (PS) cells enable the scalable production of tissue-specific derivatives with therapeutic potential for various clinical applications, including muscular dystrophies. Given the similarity to human counterparts, the non-human primate (NHP) is an ideal preclinical model to evaluate several questions, including delivery, biodistribution, and immune response. While the generation of human-induced PS (iPS)-cell-derived myogenic progenitors is well established, there have been no data for NHP counterparts, probably due to the lack of an efficient system to differentiate NHP iPS cells towards the skeletal muscle lineage. Here, we report the generation of three independent Macaca fascicularis iPS cell lines and their myogenic differentiation using PAX7 conditional expression. The whole-transcriptome analysis confirmed the successful sequential induction of mesoderm, paraxial mesoderm, and myogenic lineages. NHP myogenic progenitors efficiently gave rise to myotubes under appropriate in vitro differentiation conditions and engrafted in vivo into the TA muscles of NSG and FKRP-NSG mice. Lastly, we explored the preclinical potential of these NHP myogenic progenitors in a single wild-type NHP recipient, demonstrating engraftment and characterizing the interaction with the host immune response. These studies establish an NHP model system through which iPS-cell-derived myogenic progenitors can be studied.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Animales , Ratones , Células Madre Pluripotentes Inducidas/metabolismo , Distribución Tisular , Células Madre Pluripotentes/metabolismo , Músculo Esquelético/metabolismo , Primates , Pentosiltransferasa/metabolismoRESUMEN
Adoptive therapies with genetically modified somatic T cells rendered HIV resistance have shown promise for AIDS therapy. A renewable source of HIV-resistant human T cells from induced pluripotent stem cells (iPSCs) would further facilitate and broaden the applicability of these therapies. Here, we report successful targeting of the CCR5 locus in iPSCs generated from T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian cynomolgus macaques (MCM), using CRISPR-Cas9 technology. We found that CCR5 editing does not affect hematopoietic and T cell differentiation potentials of fib-iPSCs. However, T-iPSCs with edited CCR5 lost their capacity to differentiate into CD4+CD8+ T cells while maintaining myeloid differentiation potential. T cells and macrophages produced from CCR5-edited MCM iPSCs did not support replication of the CCR5-tropic simian immunodeficiency viruses SIVmac239 (T cell tropic) and SIVmac316 (macrophage-tropic). Overall, these studies provide a platform for further exploration of AIDS therapies based on gene-edited iPSCs in a nonhuman primate model.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida , Células Madre Pluripotentes Inducidas , Animales , Linfocitos T CD8-positivos , Macaca fascicularis , Macrófagos , Receptores CCR5/genéticaRESUMEN
Administration of ex vivo expanded somatic myeloid progenitors has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival after myeloablation. Recent advances in induced pluripotent stem cell (iPSC) technologies have created alternative platforms for supplying off-the-shelf immunologically compatible myeloid progenitors, including cellular products derived from major histocompatibility complex (MHC) homozygous superdonors, potentially increasing the availability of MHC-matching cells and maximizing the utility of stem cell banking. However, the teratogenic and tumorigenic potential of iPSC-derived progenitor cells and whether they will induce alloreactive antibodies upon transfer remain unclear. We evaluated the safety and efficacy of using CD34+CD45+ hematopoietic progenitors derived from MHC homozygous iPSCs (iHPs) to treat cytopenia after myeloablative hematopoietic stem cell (HSC) transplantation in a Mauritian cynomolgus macaque (MCM) nonhuman primate (NHP) model. We demonstrated that infusion of iHPs was well tolerated and safe, observing no teratomas or tumors in the MCMs up to 1 year after HSC transplantation and iHP infusion. Importantly, the iHPs also did not induce significant levels of alloantibodies in MHC-matched or -mismatched immunocompetent MCMs, even after increasing MHC expression on iHPs with interferon-γ. These results support the feasibility of iHP use in the setting of myeloablation and suggest that iHP products pose a low risk of inducing alloreactive antibodies.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Pluripotentes Inducidas , Animales , Antígenos CD34 , Interferón gamma , Isoanticuerpos , Macaca fascicularis , Complejo Mayor de HistocompatibilidadRESUMEN
Background Arterial bypass and interposition grafts are used routinely across multiple surgical subspecialties. Current options include both autologous and synthetic materials; however, each graft presents specific limitations. Engineering artificial small-diameter arteries with vascular cells derived from induced pluripotent stem cells could provide a useful therapeutic solution. Banking induced pluripotent stem cells from rare individuals who are homozygous for human leukocyte antigen alleles has been proposed as a strategy to facilitate economy of scale while reducing the potential for rejection of induced pluripotent stem cell-derived transplanted tissues. Currently, there is no standardized model to study transplantation of small-diameter arteries in major histocompatibility complex-defined backgrounds. Methods and Results In this study, we developed a limb-sparing nonhuman primate model to study arterial allotransplantation in the absence of immunosuppression. Our model was used to compare degrees of major histocompatibility complex matching between arterial grafts and recipient animals with long-term maintenance of patency and function. Unexpectedly, we (1) found that major histocompatibility complex partial haplomatched allografts perform as well as autologous control grafts; (2) detected little long-term immune response in even completely major histocompatibility complex mismatched allografts; and (3) observed that arterial grafts become almost completely replaced over time with recipient cells. Conclusions Given these findings, induced pluripotent stem cell-derived tissue-engineered blood vessels may prove to be promising and customizable grafts for future use by cardiac, vascular, and plastic surgeons.
Asunto(s)
Arterias/trasplante , Células Madre Pluripotentes Inducidas/trasplante , Complejo Mayor de Histocompatibilidad , Grado de Desobstrucción Vascular , Animales , Autoinjertos , Femenino , Macaca , Masculino , Modelos AnimalesRESUMEN
Advances in the scalable production of blood cells from induced pluripotent stem cells (iPSCs) open prospects for the clinical translation of de novo generated blood products, and evoke the need for preclinical evaluation of their efficacy, safety, and immunogenicity in large animal models. Due to substantial similarities with humans, the outcomes of cellular therapies in non-human primate (NHP) models can be readily extrapolated to a clinical setting. However, the use of this model is hampered by relatively low efficiency of blood generation and lack of lymphoid potential in NHP-iPSC differentiation cultures. Here, we generated transgene-free iPSCs from different NHP species and showed the efficient induction of mesoderm, myeloid, and lymphoid cells from these iPSCs using a GSK3ß inhibitor. Overall, our studies enable scalable production of hematopoietic progenitors from NHP-iPSCs, and lay the foundation for preclinical testing of iPSC-based therapies for blood and immune system diseases in an NHP model.
Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Hematopoyesis/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Linfocitos/citología , Células Mieloides/citología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Línea Celular , Linaje de la Célula/efectos de los fármacos , Técnicas de Cocultivo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Linfocitos/efectos de los fármacos , Mesodermo/citología , Ratones , Células Mieloides/efectos de los fármacos , PrimatesRESUMEN
The derivation of genetically modified induced pluripotent stem (iPS) cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating "seamless" single base-pair changes.
Asunto(s)
Sistemas CRISPR-Cas , Reprogramación Celular , Fibroblastos/metabolismo , Marcación de Gen/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Adulto , Secuencia de Bases , Línea Celular , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasas/genética , Electroporación/métodos , Fibroblastos/citología , Vectores Genéticos/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Datos de Secuencia Molecular , Factor 3 de Transcripción de Unión a Octámeros/genética , ADN Metiltransferasa 3BRESUMEN
In this study, we demonstrate a newly derived mouse model that supports engraftment of human hematopoietic stem cells (HSCs) in the absence of irradiation. We cross the NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) strain with the C57BL/6J-Kit(W-41J)/J (C57BL/6.Kit(W41)) strain and engraft, without irradiation, the resulting NBSGW strain with human cord blood CD34+ cells. At 12-weeks postengraftment in NBSGW mice, we observe human cell chimerism in marrow (97% ± 0.4%), peripheral blood (61% ± 2%), and spleen (94% ± 2%) at levels observed with irradiation in NSG mice. We also detected a significant number of glycophorin-A-positive expressing cells in the developing NBSGW marrow. Further, the observed levels of human hematopoietic chimerism mimic those reported for both irradiated NSG and NSG-transgenic strains. This mouse model permits HSC engraftment while avoiding the complicating hematopoietic, gastrointestinal, and neurological side effects associated with irradiation and allows investigators without access to radiation to pursue engraftment studies with human HSCs.
Asunto(s)
Diferenciación Celular , Supervivencia de Injerto , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Subunidad gamma Común de Receptores de Interleucina/genética , Proteínas Proto-Oncogénicas c-kit/genética , Animales , Linaje de la Célula , Genotipo , Xenoinjertos , Humanos , Inmunofenotipificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Fenotipo , Factores de Tiempo , Quimera por TrasplanteRESUMEN
BACKGROUND: DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification. METHODOLOGY: We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit(+) hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX).
Asunto(s)
Aptámeros de Nucleótidos , Técnica SELEX de Producción de Aptámeros , Animales , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Biología Computacional/métodos , Citometría de Flujo , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/metabolismoRESUMEN
A subset of the mucosotropic human papillomaviruses (HPV), including HPV16, are etiologic agents for the vast majority of cervical cancers, other anogenital cancers, and a subset of head and neck squamous cell carcinomas. HPV16 encodes three oncogenes: E5, E6, and E7. Although E6 and E7 have been well-studied and clearly shown to be important contributors to these cancers, less is known about E5. In this study, we used E5 transgenic mice to investigate the role of E5 in cervical cancer. When treated for 6 months with estrogen, a cofactor for cervical carcinogenesis, E5 transgenic mice developed more severe neoplastic cervical disease than similarly treated nontransgenic mice, although no frank cancers were detected. In addition, E5 when combined with either E6 or E7 induced more severe neoplastic disease than seen in mice expressing only one viral oncogene. Prolonged treatment of E5 transgenic mice with exogenous estrogen uncovered an ability of E5 to cause frank cancer. These data indicate that E5 acts as an oncogene in the reproductive tracts of female mice.
Asunto(s)
Transformación Celular Viral/fisiología , Proteínas Oncogénicas Virales/fisiología , Neoplasias del Cuello Uterino/virología , Animales , Ciclo Celular/fisiología , Estradiol/administración & dosificación , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Transgénicos , Proteínas Oncogénicas Virales/genética , Oncogenes , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/fisiología , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Neoplasias del Cuello Uterino/inducido químicamente , Neoplasias del Cuello Uterino/genéticaRESUMEN
The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) serves as a prototype for a range of environmental toxicants and as a pharmacologic probe to study signal transduction by the aryl hydrocarbon receptor (AHR). Despite a detailed understanding of how TCDD exposure leads to the transcriptional up-regulation of cytochrome P450-dependent monooxygenases, we know little about how compounds like TCDD lead to a variety of AHR-dependent toxic end points such as liver pathology, terata, thymic involution, and cancer. Using an acute exposure protocol and the toxic response of the mouse liver as a model system, we have begun a detailed microarray analysis to describe the transcriptional changes that occur after various TCDD doses and treatment times. Through correlation analysis of time- and dose-dependent toxicological end points, we are able to identify coordinately responsive transcriptional events that can be defined as primary transcriptional events and downstream events that may represent mechanistically linked sequelae or that have potential as biomarkers of toxicity.
Asunto(s)
Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Animales , Biomarcadores , Citocromo P-450 CYP1A1/fisiología , Citocromo P-450 CYP1A2/fisiología , Relación Dosis-Respuesta a Droga , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Elementos de Respuesta/fisiologíaRESUMEN
High-risk human papillomaviruses (HPVs), which cause the vast majority of cervical cancer, other anogenital cancers, and a subset of head and neck squamous cell carcinomas, encode three oncogenes: E5, E6, and E7. To determine the oncogenic properties of HPV16 E5 in vivo, we previously generated K14E5 transgenic mice, in which expression of E5 was directed to the basal compartment of stratified squamous epithelia. In these mice, E5 induced epidermal hyperplasia and spontaneous skin tumors. In the current study, we determined how E5 contributes to tumor formation in the skin using a multistage model for skin carcinogenesis that specifies the role of genes in three stages: initiation, promotion, and malignant progression. Both initiation and promotion are required steps for papilloma formation. K14E5 mice treated with the initiating agent 7,12-dimethylbenz(a)anthracene (DMBA) developed more papillomas than like-treated nontransgenic mice, whereas neither K14E5 nor nontransgenic mice treated with the promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) developed papillomas. K14E5 mice treated with both DMBA and TPA to induce large numbers of papillomas had a higher incidence and earlier onset of carcinoma progression compared with like-treated nontransgenic mice. Thus, HPV16 E5 contributes to two stages of skin carcinogenesis: promotion and progression. The progressive neoplastic disease in K14E5 mice differed from that in nontransgenic mice in that benign tumors converted from exophytic to endophytic papillomas before progressing to carcinomas. Initial genetic and immunohistopathologic analyses did not determine the underlying basis for this distinct morphology, which correlates with a highly penetrant neoplastic phenotype.