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1.
Cytotherapy ; 25(7): 750-762, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37086241

RESUMEN

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Receptores Quiméricos de Antígenos , Humanos , Animales , Ratones , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Receptores de Antígenos de Linfocitos T , Receptor de Muerte Celular Programada 1/metabolismo , Línea Celular Tumoral , Linfocitos T , Inmunoterapia Adoptiva
2.
J Cell Physiol ; 234(5): 7569-7578, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30368818

RESUMEN

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold's mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.


Asunto(s)
Diferenciación Celular/fisiología , Cresta Neural/citología , Células-Madre Neurales/citología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Hidrogeles/farmacología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Nanofibras/química , Cresta Neural/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Neuroglía/citología , Neuroglía/efectos de los fármacos , Polímeros/química , Ratas , Ingeniería de Tejidos/métodos , Andamios del Tejido
3.
Cancer Immunol Res ; 12(4): 462-477, 2024 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-38345397

RESUMEN

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Mieloma Múltiple , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Antígeno de Maduración de Linfocitos B/metabolismo , Antígenos HLA-E , Linfocitos T , Receptores de Antígenos de Linfocitos T , Inmunoterapia Adoptiva , Antígenos de Histocompatibilidad Clase I/metabolismo , Aloinjertos/patología
4.
Nat Commun ; 14(1): 3488, 2023 06 13.
Artículo en Inglés | MEDLINE | ID: mdl-37311756

RESUMEN

While the generation of many lineages from pluripotent stem cells has resulted in basic discoveries and clinical trials, the derivation of tissue-specific mesenchyme via directed differentiation has markedly lagged. The derivation of lung-specific mesenchyme is particularly important since this tissue plays crucial roles in lung development and disease. Here we generate a mouse induced pluripotent stem cell (iPSC) line carrying a lung-specific mesenchymal reporter/lineage tracer. We identify the pathways (RA and Shh) necessary to specify lung mesenchyme and find that mouse iPSC-derived lung mesenchyme (iLM) expresses key molecular and functional features of primary developing lung mesenchyme. iLM recombined with engineered lung epithelial progenitors self-organizes into 3D organoids with juxtaposed layers of epithelium and mesenchyme. Co-culture increases yield of lung epithelial progenitors and impacts epithelial and mesenchymal differentiation programs, suggesting functional crosstalk. Our iPSC-derived population thus provides an inexhaustible source of cells for studying lung development, modeling diseases, and developing therapeutics.


Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Animales , Ratones , Diferenciación Celular , Tórax , Mesodermo
5.
Stem Cells Dev ; 29(4): 249-256, 2020 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-31701817

RESUMEN

The neural crest stem cells derived from human induced pluripotent stem cells (iPSC-NCSCs) are a valuable autologous cell source for tissue engineering and regenerative medicine. In this study, we investigated how iPSC-NCSCs could be regulated to regenerate arteries by microenvironmental factors, including the physical factor of matrix stiffness, and the chemical factor of transforming growth factor beta-1 (TGF-ß1). We found that, compared to soft substrate, stiff substrate drove iPSC-NCSCs differentiation into smooth muscle cells, which was further enhanced by TGF-ß1. To investigate the regulatory role of TGF-ß1 in vivo, we fabricated vascular grafts composed of electrospun nanofibrous scaffolds, collagen gel, iPSC-NCSCs, and TGF-ß1, and implanted them into athymic rats. The results showed that TGF-ß1 significantly promoted extracellular matrix synthesis and increased mechanical strength of vascular grafts. This study presents a proof of concept that iPSC-NCSCs can be used as a promising autologous cell source for vascular regeneration when combined with physical and chemical engineering.


Asunto(s)
Prótesis Vascular , Arterias Carótidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Andamios del Tejido , Factor de Crecimiento Transformador beta1/farmacología , Animales , Fenómenos Biomecánicos , Arterias Carótidas/citología , Arterias Carótidas/fisiología , Diferenciación Celular/efectos de los fármacos , Colágeno/química , Colágeno/farmacología , Geles , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/fisiología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Nanofibras/química , Nanofibras/ultraestructura , Cresta Neural/citología , Cresta Neural/efectos de los fármacos , Cresta Neural/fisiología , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Poliésteres/química , Ratas , Ratas Desnudas , Regeneración/efectos de los fármacos , Regeneración/fisiología , Ingeniería de Tejidos/métodos
6.
Stem Cell Reports ; 13(3): 499-514, 2019 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-31422908

RESUMEN

Induced pluripotent stem cells (iPSCs) provide a potential source for the derivation of smooth muscle cells (SMCs); however, current approaches are limited by the production of heterogeneous cell types and a paucity of tools or markers for tracking and purifying candidate SMCs. Here, we develop murine and human iPSC lines carrying fluorochrome reporters (Acta2hrGFP and ACTA2eGFP, respectively) that identify Acta2+/ACTA2+ cells as they emerge in vitro in real time during iPSC-directed differentiation. We find that Acta2hrGFP+ and ACTA2eGFP+ cells can be sorted to purity and are enriched in markers characteristic of an immature or synthetic SMC. We characterize the resulting GFP+ populations through global transcriptomic profiling and functional studies, including the capacity to form engineered cell sheets. We conclude that these reporter lines allow for generation of sortable, live iPSC-derived Acta2+/ACTA2+ cells highly enriched in smooth muscle lineages for basic developmental studies, tissue engineering, or future clinical regenerative applications.


Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Miocitos del Músculo Liso/metabolismo , Ingeniería de Tejidos , Actinas/genética , Actinas/metabolismo , Animales , Aorta/metabolismo , Diferenciación Celular , Linaje de la Célula , Femenino , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocitos del Músculo Liso/citología , Cadenas Pesadas de Miosina/metabolismo , Análisis de Componente Principal
7.
Tissue Eng Part C Methods ; 17(7): 705-15, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21501089

RESUMEN

Trauma injuries often cause peripheral nerve damage and disability. A goal in neural tissue engineering is to develop synthetic nerve conduits for peripheral nerve regeneration having therapeutic efficacy comparable to that of autografts. Nanofibrous conduits with aligned nanofibers have been shown to promote nerve regeneration, but current fabrication methods rely on rolling a fibrous sheet into the shape of a conduit, which results in a graft with inconsistent size and a discontinuous joint or seam. In addition, the long-term effects of nanofibrous nerve conduits, in comparison with autografts, are still unknown. Here we developed a novel one-step electrospinning process and, for the first time, fabricated a seamless bi-layer nanofibrous nerve conduit: the luminal layer having longitudinally aligned nanofibers to promote nerve regeneration, and the outer layer having randomly organized nanofibers for mechanical support. Long-term in vivo studies demonstrated that bi-layer aligned nanofibrous nerve conduits were superior to random nanofibrous conduits and had comparable therapeutic effects to autografts for nerve regeneration. In summary, we showed that the engineered nanostructure had a significant impact on neural tissue regeneration in situ. The results from this study will also lead to the scalable fabrication of engineered nanofibrous nerve conduits with designed nanostructure. This technology platform can be combined with drug delivery and cell therapies for tissue engineering.


Asunto(s)
Regeneración Tisular Dirigida/métodos , Nanofibras/química , Regeneración Nerviosa/fisiología , Nervios Periféricos/fisiología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Axones/patología , Fenómenos Biomecánicos , Fenómenos Electrofisiológicos/fisiología , Femenino , Ensayo de Materiales , Vaina de Mielina/patología , Nervios Periféricos/patología , Ratas , Ratas Endogámicas Lew , Recuperación de la Función/fisiología
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