RESUMEN
Transfer of autologous tumor infiltrating lymphocytes (TIL) to patients with refractory melanoma has shown clinical efficacy in a number of trials. However, extending the clinical benefit to patients with other cancers poses a challenge. Inefficient costimulation in the tumor microenvironment can lead to T cell anergy and exhaustion resulting in poor anti-tumor activity. Here, we describe a chimeric costimulatory antigen receptor (CoStAR) comprised of FRα-specific scFv linked to CD28 and CD40 intracellular signaling domains. CoStAR signaling alone does not activate T cells, while the combination of TCR and CoStAR signaling enhances T cell activity resulting in less differentiated T cells, and augmentation of T cell effector functions, including cytokine secretion and cytotoxicity. CoStAR activity resulted in superior T cell proliferation, even in the absence of exogenous IL-2. Using an in vivo transplantable tumor model, CoStAR was shown to improve T cell survival after transfer, enhanced control of tumor growth, and improved host survival. CoStAR could be reliably engineered into TIL from multiple tumor indications and augmented TIL activity against autologous tumor targets both in vitro and in vivo. CoStAR thus represents a general approach to improving TIL therapy with synthetic costimulation.
Asunto(s)
Melanoma , Receptores Quiméricos de Antígenos , Humanos , Linfocitos T , Antígenos CD28 , Linfocitos Infiltrantes de Tumor , Receptor 1 de Folato , Receptores Quiméricos de Antígenos/genética , Antígenos CD40 , Microambiente TumoralRESUMEN
BACKGROUND: Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs. METHODS: Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1ß, tumor necrosis factor and CD107a and by lysis of target tumor cells. RESULTS: CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro. CONCLUSIONS: These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Mitogénicos/metabolismo , Trombomodulina/metabolismo , Animales , Femenino , Voluntarios Sanos , Humanos , RatonesRESUMEN
PURPOSE: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. EXPERIMENTAL DESIGN: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. RESULTS: TCR genetically modified and ex vivo-cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. CONCLUSIONS: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471.
Asunto(s)
Terapia Genética/métodos , Células Madre Hematopoyéticas/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/genética , Células Cultivadas , Ensayos Clínicos como Asunto , Drogas en Investigación/uso terapéutico , Antígeno HLA-A2/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/genética , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCR-αß+ single-positive CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vß expression, consistent with allelic exclusion of Vß-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.
Asunto(s)
Órganos Artificiales , Diferenciación Celular , Células Madre Hematopoyéticas/citología , Organoides/citología , Linfocitos T/citología , Timo/citología , Biotecnología/métodos , Células Madre Hematopoyéticas/inmunología , Humanos , Organoides/inmunología , Células Madre/citología , Células Madre/inmunología , Linfocitos T/inmunología , Timo/inmunologíaRESUMEN
T cells engineered to express a tumor-specific αß T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αß chains with endogenous αß chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and ß chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αß TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy.
Asunto(s)
Genes Codificadores de los Receptores de Linfocitos T , Terapia Genética/efectos adversos , Terapia Genética/métodos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Animales , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped , Ratones , Dominios Proteicos , Recombinación GenéticaRESUMEN
Autologous human induced pluripotent stem cells (hiPSCs) should allow cellular therapeutics without an associated immune response. This concept has been controversial since the original report that syngeneic mouse iPSCs elicited an immune response after transplantation. However, an investigative analysis of any potential acute immune responses in hiPSCs and their derivatives has yet to be conducted. In the present study, we used correlative gene expression analysis of two putative mouse "immunogenicity" genes, ZG16 and HORMAD1, to assay their human homologous expression levels in human pluripotent stem cells and their derivatives. We found that ZG16 expression is heterogeneous across multiple human embryonic stem cell and hiPSC-derived cell types. Additionally, ectopic expression of ZG16 in antigen-presenting cells is insufficient to trigger a detectable response in a peripheral blood mononuclear cell coculture assay. Neither of the previous immunogenicity-associated genes in the mouse currently appears to be relevant in a human context.
Asunto(s)
Células Madre Embrionarias/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Células Madre Pluripotentes/inmunología , Animales , Línea Celular , Células Madre Embrionarias/citología , Humanos , Lectinas/inmunología , Proteínas de la Membrana/inmunología , Ratones , Células Madre Pluripotentes/citología , Especificidad de la EspecieRESUMEN
Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors.
Asunto(s)
Vectores Genéticos , Intrones , Lentivirus/genética , Regiones Promotoras Genéticas , Ubiquitina C/genética , Elementos de Facilitación Genéticos , Exones , Expresión Génica , Células HEK293 , Humanos , Células K562 , Factor 1 de Elongación Peptídica/genética , Empalme del ARNRESUMEN
Engineering immunity against cancer by the adoptive transfer of hematopoietic stem cells (HSC) modified to express antigen-specific T-cell receptors (TCR) or chimeric antigen receptors generates a continual supply of effector T cells, potentially providing superior anticancer efficacy compared with the infusion of terminally differentiated T cells. Here, we demonstrate the in vivo generation of functional effector T cells from CD34-enriched human peripheral blood stem cells modified with a lentiviral vector designed for clinical use encoding a TCR recognizing the cancer/testes antigen NY-ESO-1, coexpressing the PET/suicide gene sr39TK. Ex vivo analysis of T cells showed antigen- and HLA-restricted effector function against melanoma. Robust engraftment of gene-modified human cells was demonstrated with PET reporter imaging in hematopoietic niches such as femurs, humeri, vertebrae, and the thymus. Safety was demonstrated by the in vivo ablation of PET signal, NY-ESO-1-TCR-bearing cells, and integrated lentiviral vector genomes upon treatment with ganciclovir, but not with vehicle control. Our study provides support for the efficacy and safety of gene-modified HSCs as a therapeutic modality for engineered cancer immunotherapy. Cancer Res; 74(18); 5173-83. ©2014 AACR.
Asunto(s)
Genes Transgénicos Suicidas , Células Madre Hematopoyéticas/fisiología , Herpesvirus Humano 1/genética , Inmunoterapia/métodos , Tomografía de Emisión de Positrones/métodos , Linfocitos T/inmunología , Animales , Antígenos CD34/sangre , Antígenos CD34/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos/genética , Células Madre Hematopoyéticas/diagnóstico por imagen , Células Madre Hematopoyéticas/inmunología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Transducción GenéticaRESUMEN
Hematopoietic stem cells (HSCs) provide an attractive target for immunotherapy of cancer and leukemia by the introduction of genes encoding T-cell receptors (TCRs) or chimeric antigen receptors (CARs) directed against tumor-associated antigens. HSCs engraft for long-term blood cell production and could provide a continuous source of targeted anti-cancer effector cells to sustain remissions. T cells produced de novo from HSCs may continuously replenish anti-tumor T cells that have become anergic or exhausted from ex vivo expansion or exposure to the intratumoral microenvironment. In addition, transgenic T cells produced in vivo undergo allelic exclusion, preventing co-expression of an endogenous TCR that could mis-pair with the introduced TCR chains and blunt activity or even cause off-target reactivity. CAR-engineered HSCs may produce myeloid and natural killer cells in addition to T cells expressing the CAR, providing broader anti-tumor activity that arises quickly after transplant and does not solely require de novo thymopoiesis. Use of TCR- or CAR-engineered HSCs would likely require cytoreductive conditioning to achieve long-term engraftment, and this approach may be used in clinical settings where autologous HSC transplant is being performed to add a graft-versus-tumor effect. Results of experimental and preclinical studies performed to date are reviewed.
Asunto(s)
Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , Animales , Técnicas de Transferencia de Gen , Genes Transgénicos Suicidas , Ingeniería Genética , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunoterapia/métodos , Inmunoterapia Adoptiva/métodos , Neoplasias/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34(+) HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8(+) and CD4(+) T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4-5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8(+) and CD4(+) T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34(+) HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-ß genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.
Asunto(s)
Alelos , Células Madre Hematopoyéticas/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Subgrupos de Linfocitos T/metabolismo , Animales , Antígenos CD34/metabolismo , Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Epítopos de Linfocito T/inmunología , Femenino , Orden Génico , Vectores Genéticos/genética , Supervivencia de Injerto , Trasplante de Células Madre Hematopoyéticas , Humanos , Interferón gamma/biosíntesis , Lentivirus/genética , Masculino , Melanoma/genética , Melanoma/inmunología , Melanoma/terapia , Ratones , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Timocitos/citología , Timocitos/metabolismo , Transducción Genética , Trasplante HeterólogoRESUMEN
Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-ß-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.
Asunto(s)
Desoxicitidina Quinasa/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Animales , Arabinofuranosil Uracilo/análogos & derivados , Arabinofuranosil Uracilo/química , Arabinofuranosil Uracilo/metabolismo , Western Blotting , Línea Celular Tumoral , Desoxicitidina Quinasa/genética , Femenino , Radioisótopos de Flúor/química , Células Madre Hematopoyéticas/metabolismo , Inmunohistoquímica , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Estimación de Kaplan-Meier , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Endogámicos , Ratones Noqueados , Ratones SCID , Mutación , Timo/diagnóstico por imagen , Timo/metabolismo , Factores de Tiempo , Trasplante HeterólogoRESUMEN
We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.
Asunto(s)
Agammaglobulinemia/terapia , Trasplante de Médula Ósea/métodos , Terapia Genética/métodos , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas/métodos , Inmunodeficiencia Combinada Grave/terapia , Adenosina Desaminasa/deficiencia , Adolescente , Antígenos CD34/metabolismo , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Retroviridae/genética , Transducción Genética , Acondicionamiento Pretrasplante , Adulto JovenRESUMEN
MicroRNAs (miRs) play an important role in cell differentiation and maintenance of cell identity, but relatively little is known of their functional role in modulating human hematopoietic lineage differentiation. Human embryonic stem cells (hESCs) provide a model system to study early human hematopoiesis. We differentiated hESCs by embryoid body (EB) formation and compared the miR expression profile of undifferentiated hESCs to CD34(+) EB cells. miRs-126/126* were the most enriched of the 7 miRs that were up-regulated in CD34(+) cells, and their expression paralleled the kinetics of hematopoietic transcription factors RUNX1, SCL, and PU.1. To define the role of miRs-126/126* in hematopoiesis, we created hESCs overexpressing doxycycline-regulated miRs-126/126* and analyzed their hematopoietic differentiation. Induction of miRs-126/126* during both EB differentiation and colony formation reduced the number of erythroid colonies, suggesting an inhibitory role of miRs-126/126* in erythropoiesis. Protein tyrosine phosphatase, nonreceptor type 9 (PTPN9), a protein tyrosine phosphatase that is required for growth and expansion of erythroid cells, is one target of miR-126. PTPN9 restoration partially relieved the suppressed erythropoiesis caused by miRs-126/126*. Our results define an important function of miRs-126/126* in negative regulation of erythropoiesis, providing the first evidence for a role of miR in hematopoietic differentiation of hESCs.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Eritropoyesis/genética , Eritropoyesis/fisiología , MicroARNs/genética , Antígenos CD34/metabolismo , Secuencia de Bases , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Cartilla de ADN/genética , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Cinética , MicroARNs/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Tirosina Fosfatasas no Receptoras/fisiologíaRESUMEN
Microfluidic image cytometry (MIC) has been developed to study phenotypes of various hPSC lines by screening several chemically defined serum/feeder-free conditions. A chemically defined hPSC culture was established using 20 ng mL(-1) of bFGF on 20 microg mL(-1) of Matrigel to grow hPSCs over a week in an undifferentiated state. Following hPSC culture, we conducted quantitative MIC to perform a single cell profiling of simultaneously detected protein expression (OCT4 and SSEA1). Using clustering analysis, we were able to systematically compare the characteristics of various hPSC lines in different conditions.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Separación Celular/instrumentación , Citometría de Flujo/instrumentación , Perfilación de la Expresión Génica/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Microscopía/instrumentación , Células Madre Pluripotentes/citología , Células Cultivadas , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We have successfully designed and fabricated an integrated microfluidic platform, the hESC-microChip, which is capable of reproducible and quantitative culture and analysis of individual hESC colonies in a semi-automated fashion. In this device, a serpentine microchannel allows pre-screening of dissociated hESC clusters, and six individually addressable cell culture chambers enable parallel hESC culture, as well as multiparameter analyses in sequence. In order to quantitatively monitor hESC proliferation and pluripotency status in real time, knock-in hESC lines with EGFP driven by the endogenous OCT4 promoter were constructed. On-chip immunoassays of several pluripotency markers were carried out to confirm that the hESC colonies maintained their pluripotency. For the first time, our studies demonstrated well characterized hESC culture and analysis in a microfluidic setting, as well as a proof-of-concept demonstration of parallel/multiparameter/real-time/automated examination of self-renewal and differentiation in the same device.
Asunto(s)
Células Madre Embrionarias/citología , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas de Cultivo de Célula , Diferenciación Celular , Procesos de Crecimiento Celular , Proliferación Celular , Diseño de Equipo , Humanos , Inmunohistoquímica , Técnicas Analíticas Microfluídicas/métodos , Factores de TiempoRESUMEN
With their unique ability to differentiate into all cell types, embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation, we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100-300 microm) are the most proliferative, hold the greatest differentiation potential, and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation, we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates, we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system, similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation.