RESUMEN
In pursuit of treating Parkinson's disease with cell replacement therapy, differentiated induced pluripotent stem cells (iPSC) are an ideal source of midbrain dopaminergic (mDA) cells. We previously established a protocol for differentiating iPSC-derived post-mitotic mDA neurons capable of reversing 6-hydroxydopamine-induced hemiparkinsonism in rats. In the present study, we transitioned the iPSC starting material and defined an adapted differentiation protocol for further translation into a clinical cell transplantation therapy. We examined the effects of cellular maturity on survival and efficacy of the transplants by engrafting mDA progenitors (cryopreserved at 17 days of differentiation, D17), immature neurons (D24), and post-mitotic neurons (D37) into immunocompromised hemiparkinsonian rats. We found that D17 progenitors were markedly superior to immature D24 or mature D37 neurons in terms of survival, fiber outgrowth and effects on motor deficits. Intranigral engraftment to the ventral midbrain demonstrated that D17 cells had a greater capacity than D24 cells to innervate over long distance to forebrain structures, including the striatum. When D17 cells were assessed across a wide dose range (7,500-450,000 injected cells per striatum), there was a clear dose response with regards to numbers of surviving neurons, innervation, and functional recovery. Importantly, although these grafts were derived from iPSCs, we did not observe teratoma formation or significant outgrowth of other cells in any animal. These data support the concept that human iPSC-derived D17 mDA progenitors are suitable for clinical development with the aim of transplantation trials in patients with Parkinson's disease.
RESUMEN
Nongenetic methodologies to reduce undesirable proliferation would be valuable when generating dopamine neurons from stem cells for transplantation in Parkinson's disease (PD). To this end, we modified an established method for controlled differentiation of human induced pluripotent stem cells (iPSCs) into midbrain dopamine neurons using two distinct methods: omission of FGF8 or the in-process use of the DNA cross-linker mitomycin-C (MMC). We transplanted the cells to athymic rats with unilateral 6-hydroxydopamine lesions and monitored long-term survival and function of the grafts. Transplants of cells manufactured using MMC had low proliferation while still permitting robust survival and function comparable to that seen with transplanted dopamine neurons derived using genetic drug selection. Conversely, cells manufactured without FGF8 survived transplantation but exhibited poor in vivo function. Our results suggest that MMC can be used to reduce the number of proliferative cells in stem cell-derived postmitotic neuron preparations for use in PD cell therapy.
Asunto(s)
Neuronas Dopaminérgicas , Células Madre Pluripotentes Inducidas , Mitomicina , Enfermedad de Parkinson , Animales , Diferenciación Celular , Proliferación Celular , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Mitomicina/farmacología , Enfermedad de Parkinson/terapia , Ratas , Trasplante de Células MadreRESUMEN
A major challenge for clinical application of pluripotent stem cell therapy for Parkinson's disease (PD) is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle, midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA) and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved, post-mitotic iPSC-mDA neurons retained high viability with gene, protein, and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy, cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth, supporting the translational development of pluripotent cell-based therapies in PD.
Asunto(s)
Criopreservación , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/trasplante , Células Madre Pluripotentes Inducidas/citología , Enfermedad de Parkinson/terapia , Animales , Línea Celular , Cuerpo Estriado/citología , Cuerpo Estriado/patología , Criopreservación/métodos , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Haplorrinos , Humanos , Mesencéfalo/citología , Mesencéfalo/patología , Neurogénesis , Enfermedad de Parkinson/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Few biological markers of immune function have been thoroughly validated for use in epidemiologic studies that involve delayed sample processing and analysis. Here, we report our validation results for flow cytometric detection of intracellular T-helper 1/T-helper 2 (Th1/Th2) cytokines using 500 microL of whole blood obtained from children and adults. The detection of Th1/Th2 cytokine profiles by flow cytometry is a practical and mechanistically relevant assay because dysregulated cytokine production has been observed in many immune-mediated disorders, including cancer. We evaluated the intraassay and intraindividual and interindividual variability and the effects of a 24- to 72-hour delayed analysis on Th1 and Th2 end points. We compared the distributions of %CD4 lymphocytes, %Th1, and %Th2 in young children (age 1 year, n = 50) and adults (age 25-52 years, n = 16). Subjects sampled monthly for up to 1 year showed minimal variation in CD4, Th1, and Th2 end points. Delayed analysis of samples (up to 24 hours) resulted in no significant differences in the expression of CD4, Th1, and Th2; however, at 48 and 72 hours, all end points differed significantly from baseline (P < 0.01). A random effects model confirmed that interindividual variability was much greater than intraindividual variability for CD4 and Th1. Compared with adults, children had marginally higher %CD4, similar %Th2, but significantly lower %Th1 (P < 0.01). These results show that flow cytometric detection of CD4, Th1, and Th2 markers using whole blood is reproducible and that these biomarkers can be effectively used in human population studies that involve transported samples, delayed processing and analysis, and limited blood volumes.
Asunto(s)
Citocinas/sangre , Citometría de Flujo/estadística & datos numéricos , Células TH1/inmunología , Células Th2/inmunología , Adulto , Recolección de Muestras de Sangre , Linfocitos T CD4-Positivos/inmunología , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Valores de Referencia , Reproducibilidad de los Resultados , Manejo de EspecímenesRESUMEN
DNA immunization in conjunction with antiretroviral therapy was evaluated in SIV-infected rhesus macaques treated with [R]-9-[2-phosphonylmethoxypropyl]adenine (PMPA). Macaques were immunized monthly with DNA vaccines expressing either SIV gag/tat or SIV gag/tat and 19 CD8+ T cell epitopes during 7 months of therapy. Half the animals from each group were additionally immunized before infection. Only 60% of the animals (4 controls, 20 vaccinated) responded to PMPA (ART responders). All 4 ART responder controls demonstrated viral rebound or CD4 decline after PMPA was withdrawn. In contrast, 17 of 20 vaccinated ART responders contained viral rebound for over 7 months after PMPA was withdrawn. Viral control correlated with stable CD4 counts, higher lymphoproliferation and an increase in the magnitude and breadth of the CD8+ T cell response. Immunizing before infection or with multi-epitopes enhanced these effects. These results demonstrate that DNA immunization during antiretroviral therapy may be an effective strategy to treat HIV infection.
Asunto(s)
Adenina/análogos & derivados , Organofosfonatos/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas de ADN/administración & dosificación , Viremia/prevención & control , Adenina/administración & dosificación , Adenina/uso terapéutico , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/uso terapéutico , Recuento de Linfocito CD4 , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Progresión de la Enfermedad , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Productos del Gen tat/genética , Productos del Gen tat/inmunología , Inmunoterapia Activa/métodos , Macaca mulatta , Organofosfonatos/uso terapéutico , ARN Viral/sangre , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Estadística como Asunto , Tenofovir , Vacunas de ADN/inmunología , Privación de TratamientoRESUMEN
This clinical delivery system bridging study evaluated the performance of a single-use disposable, commercial prototype device (designated ND 5.5) for particle-mediated epidermal delivery (PMED) of a nucleic acid vaccine against Hepatitis B virus (HBV). Healthy adults, previously immunized with licensed HBV vaccine, received a single boost vaccination of HBV nucleic acid vaccine administered by ND 5.5 or XR-1, the clinical research device used in previous clinical trials. Similar increases in anti-HBV surface antigen serum antibody titers and cell-mediated immune responses were produced by ND 5.5 and XR-1 when delivering comparable effective doses of the vaccine. The overall intensity of the immune response was lower in those subjects vaccinated with two, rather than 4 administrations of vaccine delivered by ND 5.5. Skin reactions at sites of vaccine administration were equivalent with both devices. This is the first clinical demonstration of the safe and effective PMED of a nucleic acid vaccine with the ND 5.5 device.
Asunto(s)
Vacunas contra Hepatitis B/administración & dosificación , Hepatitis B/prevención & control , Inyecciones a Chorro/instrumentación , Vacunas de ADN/administración & dosificación , Adolescente , Adulto , ADN Viral/administración & dosificación , ADN Viral/inmunología , Femenino , Anticuerpos contra la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/efectos adversos , Vacunas contra Hepatitis B/inmunología , Humanos , Inmunidad Celular , Inmunización Secundaria , Inyecciones a Chorro/métodos , Interferón gamma/análisis , Interleucina-5/análisis , Masculino , Persona de Mediana Edad , Vacunas de ADN/efectos adversos , Vacunas de ADN/inmunologíaRESUMEN
Little is known concerning the stimulatory receptors responsible for tumor cell lysis by NK cells. We generated a monoclonal antibody specific for murine NKG2D in order to investigate its function. Blocking of NKG2D inhibited natural cytotoxicity of all tumor cells tested that express ligands for the receptor. Staining analysis showed that NKG2D is also expressed by activated CD8(+) T cells and macrophages, and subsets of TCRgammadelta(+) and NK1.1(+) T cells. Contradicting reports that NKG2D is solely a costimulatory receptor, we observed that cross-linking of NKG2D directly stimulates NK cells and activated macrophages. In contrast, NKG2D costimulates activated CD8(+) T cells. Thus, NKG2D engagement directly stimulates NK cells and macrophages, costimulates CD8(+) T cells, and plays a substantial role in natural killing.
Asunto(s)
Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Activación de Linfocitos , Activación de Macrófagos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Subfamilia K de Receptores Similares a Lectina de Células NK , Neoplasias/inmunología , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/inmunología , Receptores de Células Asesinas Naturales , Subgrupos de Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
NK cells express several families of receptors that play central roles in target cell recognition. These NK cell receptors are also expressed by certain memory phenotype CD8(+) T cells, and in some cases are up-regulated in T cells responding to viral infection. To determine how the profile of NK receptor expression changes in murine CD8(+) T cells as they respond to intracellular pathogens, we used class I tetramer reagents to directly examine Ag-specific T cells during lymphocytic choriomeningitis virus and Listeria monocytogenes infections. We found that the majority of pathogen-specific CD8(+) T cells initiated expression of the inhibitory CD94/NKG2A heterodimer, the KLRG1 receptor, and a novel murine NK cell marker (10D7); conversely, very few Ag-specific T cells expressed Ly49 family members. The up-regulation of these receptors was independent of IL-15 and persisted long after clearance of the pathogen. The expression of CD94/NKG2A was rapidly initiated in naive CD8(+) T cells responding to peptide Ags in vitro and on many of the naive T cells that proliferate when transferred into lymphopenic (Rag-1(-/-)) hosts. Thus, CD94/NKG2A expression is a common consequence of CD8(+) T cell activation. Binding of the CD94/NKG2A receptor by its ligand (Qa-1(b)) did not significantly inhibit CD8(+) T cell effector functions. However, expression of CD94 and NKG2A transgenes partially inhibited early events of T cell activation. These subtle effects suggest that CD94/NKG2A-mediated inhibition of T cells may be limited to particular circumstances or may synergize with other receptors that are similarly up-regulated.
Asunto(s)
Antígenos CD/biosíntesis , Antígenos Ly , Infecciones Bacterianas/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Asesinas Naturales/metabolismo , Lectinas Tipo C , Glicoproteínas de Membrana/biosíntesis , Receptores Inmunológicos/biosíntesis , Virosis/inmunología , Animales , Antígenos CD/química , Dimerización , Homeostasis , Interleucina-15/fisiología , Activación de Linfocitos , Glicoproteínas de Membrana/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Subfamília D de Receptores Similares a Lectina de las Células NK , Receptores Inmunológicos/química , Receptores Similares a Lectina de Células NKRESUMEN
As with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristic lifetime infection of its host. We compared infection of C57BL/6 mice with wild-type murine CMV (MCMV) and a virus lacking each of MCMV's three known VIPRs: m4, m6, and m152. During acute infection, there was very little difference between the two viruses with respect to the kinetics of viral replication and clearance, or in the size and kinetics of the virus-specific CD8 T cell response. During chronic infection, a large, effector memory, virus-specific CD8 T cell population (CD8(low)CD62L(-)CD11c(+)NKG2A(+)) was maintained in both infections; the size and phenotype of the CD8 T cell response to both viruses was remarkably similar. The characteristic effector memory phenotype of the CD8 T cells suggested that both wild-type and Deltam4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both Deltam4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host.