RESUMEN
Patients with smoldering multiple myeloma (SMM) are observed until progression, but early treatment may improve outcomes. We conducted a phase II trial of elotuzumab, lenalidomide, and dexamethasone (EloLenDex) in patients with high-risk SMM and performed single-cell RNA and T cell receptor (TCR) sequencing on 149 bone marrow (BM) and peripheral blood (PB) samples from patients and healthy donors (HDs). We find that early treatment with EloLenDex is safe and effective and provide a comprehensive characterization of alterations in immune cell composition and TCR repertoire diversity in patients. We show that the similarity of a patient's immune cell composition to that of HDs may have prognostic relevance at diagnosis and after treatment and that the abundance of granzyme K (GZMK)+ CD8+ effector memory T (TEM) cells may be associated with treatment response. Last, we uncover similarities between immune alterations observed in the BM and PB, suggesting that PB-based immune profiling may have diagnostic and prognostic utility.
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Mieloma Múltiple , Mieloma Múltiple Quiescente , Humanos , Biomarcadores , Progresión de la Enfermedad , Factores Inmunológicos , Inmunoterapia , Lenalidomida/efectos adversos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple Quiescente/terapia , Ensayos Clínicos Fase II como AsuntoRESUMEN
Multiple myeloma is a plasma cell malignancy almost always preceded by precursor conditions, but low tumor burden of these early stages has hindered the study of their molecular programs through bulk sequencing technologies. Here, we generate and analyze single cell RNA-sequencing of plasma cells from 26 patients at varying disease stages and 9 healthy donors. In silico dissection and comparison of normal and transformed plasma cells from the same bone marrow biopsy enables discovery of patient-specific transcriptional changes. Using Non-Negative Matrix Factorization, we discover 15 gene expression signatures which represent transcriptional modules relevant to myeloma biology, and identify a signature that is uniformly lost in abnormal cells across disease stages. Finally, we demonstrate that tumors contain heterogeneous subpopulations expressing distinct transcriptional patterns. Our findings characterize transcriptomic alterations present at the earliest stages of myeloma, providing insight into the molecular underpinnings of disease initiation.
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Mieloma Múltiple , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Carcinogénesis/genética , Carcinogénesis/patología , Transformación Celular Neoplásica/patología , Células Plasmáticas/patología , Médula Ósea/patologíaRESUMEN
Protein-coding and non-coding genes like miRNAs tightly control hematopoietic differentiation programs. Although miRNAs are frequently located within introns of protein-coding genes, the molecular interplay between intronic miRNAs and their host genes is unclear. By genomic integration site mapping of gamma-retroviral vectors in genetically corrected peripheral blood from gene therapy patients, we identified the EVL/MIR342 gene locus as a hotspot for therapeutic vector insertions indicating its accessibility and expression in human hematopoietic stem and progenitor cells. We therefore asked if and how EVL and its intronic miRNA-342 regulate hematopoiesis. Here we demonstrate that overexpression (OE) of Evl in murine primary Lin- Sca1+ cKit+ cells drives lymphopoiesis whereas miR-342 OE increases myeloid colony formation in vitro and in vivo, going along with a profound upregulation of canonical pathways essential for B-cell development or myelopoietic functions upon Evl or miR-342 OE, respectively. Strikingly, miR-342 counteracts its host gene by targeting lymphoid signaling pathways, resulting in reduced pre-B-cell output. Moreover, EVL overexpression is associated with lymphoid leukemia in patients. In summary, our data show that one common gene locus regulates distinct hematopoietic differentiation programs depending on the gene product expressed, and that the balance between both may determine hematopoietic cell fate decision.
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Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Hematopoyesis , Células Madre Hematopoyéticas/citología , MicroARNs/genética , Animales , Moléculas de Adhesión Celular/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Intrones , RatonesRESUMEN
Precursor states of Multiple Myeloma (MM) and its native tumor microenvironment need in-depth molecular characterization to better stratify and treat patients at risk. Using single-cell RNA sequencing of bone marrow cells from precursor stages, MGUS and smoldering myeloma (SMM), to full-blown MM alongside healthy donors, we demonstrate early immune changes during patient progression. We find NK cell abundance is frequently increased in early stages, and associated with altered chemokine receptor expression. As early as SMM, we show loss of GrK+ memory cytotoxic T-cells, and show their critical role in MM immunosurveillance in mouse models. Finally, we report MHC class II dysregulation in CD14+ monocytes, which results in T cell suppression in vitro. These results provide a comprehensive map of immune changes at play over the evolution of pre-malignant MM, which will help develop strategies for immune-based patient stratification.
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Gammopatía Monoclonal de Relevancia Indeterminada , Mieloma Múltiple , Mieloma Múltiple Quiescente , Animales , Humanos , Ratones , Gammopatía Monoclonal de Relevancia Indeterminada/genética , Mieloma Múltiple/genética , Análisis de Secuencia de ARN , Microambiente Tumoral/genéticaRESUMEN
Nitroxide-based organic-radical contrast agents (ORCAs) are promising as safe, next-generation magnetic resonance imaging (MRI) tools. Nevertheless, stimuli-responsive ORCAs that enable MRI monitoring of prodrug activation have not been reported; such systems could open new avenues for prodrug validation and image-guided drug delivery. Here, we introduce a novel "pro-ORCA" concept that addresses this challenge. By covalent conjugation of nitroxides and drug molecules (doxorubicin, DOX) to the same brush-arm star polymer (BASP) through chemically identical cleavable linkers, we demonstrate that pro-ORCA and prodrug activation, i.e., ORCA and DOX release, leads to significant changes in MRI contrast that correlate with cytotoxicity. This approach is shown to be general for a range of commonly used linker cleavage mechanisms (e.g., photolysis and hydrolysis) and release rates. Pro-ORCAs could find applications as research tools or clinically viable "reporter theranostics" for in vitro and in vivo MRI-correlated prodrug activation.
RESUMEN
PURPOSE: Ulocuplumab (BMS-936564) is a first-in-class fully human IgG4 monoclonal anti-CXCR4 antibody that inhibits the binding of CXCR4 to CXCL12. PATIENTS AND METHODS: This phase Ib/II study aimed to determine the safety and tolerability of ulocuplumab alone and in combination with lenalidomide and dexamethasone (Arm A), or bortezomib and dexamethasone (Arm B), in patients with relapsed/refractory multiple myeloma. RESULTS: Forty-six patients were evaluated (median age, 60 years; range, 53-67). The median number of prior therapies was 3 (range, 1-11), with 70% of subjects having received ≥3. This trial had a dose-escalation and a dose-expansion part. Using a 3+3 design on both arms of the trial, ulocuplumab's dose was escalated to a maximum of 10 mg/kg without reaching MTD. The most common treatment-related adverse events (AE) were neutropenia (13 patients, 43.3%) in Arm A and thrombocytopenia (6 patients, 37.5%) in Arm B. No deaths related to study drugs occurred. The combination of ulocuplumab with lenalidomide and dexamethasone showed a high response rate (PR or better) of 55.2% and a clinical benefit rate of 72.4%, even in patients who had been previously treated with immunomodulatory agents (IMiD). CONCLUSIONS: This study showed that blockade of the CXCR4-CXCL12 axis by ulocuplumab is safe with acceptable AEs and leads to a high response rate in combination with lenalidomide and dexamethasone in patients with relapsed/refractory myeloma, making CXCR4 inhibitors a promising class of antimyeloma drugs that should be further explored in clinical trials.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Receptores CXCR4/antagonistas & inhibidores , Anciano , Anticuerpos Monoclonales Humanizados/administración & dosificación , Bortezomib/administración & dosificación , Dexametasona/administración & dosificación , Humanos , Lenalidomida/administración & dosificación , Dosis Máxima Tolerada , Persona de Mediana Edad , Mieloma Múltiple/patología , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Seguridad del Paciente , Receptores CXCR4/inmunología , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
We tested the hypothesis that using CXCR4 inhibition to target the interaction between the tumor cells and the microenvironment leads to sensitization of the tumor cells to apoptosis. Eligibility criteria included multiple myeloma (MM) patients with 1-5 prior lines of therapy. The purposes of the phase I study were to evaluate the safety and maximal-tolerated dose (MTD) of the combination. The treatment-related adverse events and response rate of the combination were assessed in the phase II study. A total of 58 patients were enrolled in the study. The median age of the patients was 63 years (range, 43-85), and 78% of them received prior bortezomib. In the phase I study, the MTD was plerixafor 0.32 mg/kg, and bortezomib 1.3 mg/m2 . The overall response rate for the phase II study was 48.5%, and the clinical benefit rate 60.6%. The median disease-free survival was 12.6 months. The CyTOF analysis demonstrated significant mobilization of plasma cells, CD34+ stem cells, and immune T cells in response to plerixafor. This is an unprecedented study that examines therapeutic targeting of the bone marrow microenvironment and its interaction with the tumor clone to overcome resistance to therapy. Our results indicate that this novel combination is safe and that the objective response rate is high even in patients with relapsed/refractory MM. ClinicalTrials.gov, NCT00903968.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Receptores CXCR4/antagonistas & inhibidores , Terapia Recuperativa , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Bencilaminas , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Bortezomib/administración & dosificación , Bortezomib/efectos adversos , Terapia Combinada , Ciclamas , Supervivencia sin Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Enfermedades Gastrointestinales/inducido químicamente , Enfermedades Hematológicas/inducido químicamente , Trasplante de Células Madre Hematopoyéticas , Compuestos Heterocíclicos/administración & dosificación , Compuestos Heterocíclicos/efectos adversos , Humanos , Estimación de Kaplan-Meier , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/efectos de los fármacos , Recurrencia , Microambiente Tumoral/efectos de los fármacosAsunto(s)
Bortezomib/administración & dosificación , Mutación , Receptores CXCR4/genética , Macroglobulinemia de Waldenström , Femenino , Estudios de Seguimiento , Humanos , Masculino , Macroglobulinemia de Waldenström/tratamiento farmacológico , Macroglobulinemia de Waldenström/genética , Macroglobulinemia de Waldenström/mortalidadRESUMEN
Myeloma bone disease is a devastating complication of multiple myeloma (MM) and is caused by dysregulation of bone remodeling processes in the bone marrow microenvironment. Previous studies showed that microRNA-138 (miR-138) is a negative regulator of osteogenic differentiation of mesenchymal stromal cells (MSCs) and that inhibiting its function enhances bone formation in vitro. In this study, we explored the role of miR-138 in myeloma bone disease and evaluated the potential of systemically delivered locked nucleic acid (LNA)-modified anti-miR-138 oligonucleotides in suppressing myeloma bone disease. We showed that expression of miR-138 was significantly increased in MSCs from MM patients (MM-MSCs) and myeloma cells compared to those from healthy subjects. Furthermore, inhibition of miR-138 resulted in enhanced osteogenic differentiation of MM-MSCs in vitro and increased the number of endosteal osteoblastic lineage cells (OBCs) and bone formation rate in mouse models of myeloma bone disease. RNA sequencing of the OBCs identified TRPS1 and SULF2 as potential miR-138 targets that were de-repressed in anti-miR-138-treated mice. In summary, these data indicate that inhibition of miR-138 enhances bone formation in MM and that pharmacological inhibition of miR-138 could represent a new therapeutic strategy for treatment of myeloma bone disease.
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Biomarcadores de Tumor/genética , Médula Ósea/patología , Células Madre Mesenquimatosas/patología , MicroARNs/antagonistas & inhibidores , Mieloma Múltiple/terapia , Osteoblastos/patología , Osteogénesis , Animales , Médula Ósea/metabolismo , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones SCID , MicroARNs/genética , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Osteoblastos/metabolismo , PronósticoRESUMEN
Despite significant advances in the treatment of multiple myeloma (MM), most patients succumb to disease progression. One of the major immunosuppressive mechanisms that is believed to play a role in myeloma progression is the expansion of regulatory T cells (Tregs). In this study, we demonstrate that myeloma cells drive Treg expansion and activation by secreting type 1 interferon (IFN). Blocking IFN α and ß receptor 1 (IFNAR1) on Tregs significantly decreases both myeloma-associated Treg immunosuppressive function and myeloma progression. Using syngeneic transplantable murine myeloma models and bone marrow (BM) aspirates of MM patients, we found that Tregs were expanded and activated in the BM microenvironment at early stages of myeloma development. Selective depletion of Tregs led to a complete remission and prolonged survival in mice injected with myeloma cells. Further analysis of the interaction between myeloma cells and Tregs using gene sequencing and enrichment analysis uncovered a feedback loop, wherein myeloma-cell-secreted type 1 IFN induced proliferation and expansion of Tregs. By using IFNAR1-blocking antibody treatment and IFNAR1-knockout Tregs, we demonstrated a significant decrease in myeloma-associated Treg proliferation, which was associated with longer survival of myeloma-injected mice. Our results thus suggest that blocking type 1 IFN signaling represents a potential strategy to target immunosuppressive Treg function in MM.
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Proliferación Celular , Tolerancia Inmunológica , Mieloma Múltiple/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/inmunología , Receptor de Interferón alfa y beta/inmunología , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Antineoplásicos/farmacología , Línea Celular , Ratones , Ratones Noqueados , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Receptor de Interferón alfa y beta/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
The tumor suppressors B-lymphocyte-induced maturation protein-1 (BLIMP-1) and p53 play a crucial role in B-cell lymphomas, and their inactivation contributes to the pathogenesis of a wide spectrum of lymphoid malignancies, including diffuse large B-cell lymphomas (DLBCLs). Patients with activated B-cell-like (ABC) DLBCL may present with loss of BLIMP-1, c-Myc over-expression, decreased p53, and poor prognosis. Nevertheless, there is a lack of in vivo models recapitulating the biology of high-grade ABC DLBCL. We therefore aimed to develop an in vivo model aiming to recapitulate the phenotype observed in this cohort of patients. A Cre-Lox approach was used to achieve inactivation of both p53 and BLIMP-1 in murine B-cells. Contextual ablation of BLIMP-1 and p53 led to development of IgM-positive B-cell lymphoma with an aggressive phenotype, supported by c-Myc up-regulation, and accumulation of somatic mutations, as demonstrated by whole exome sequencing. Sensitivity of B-tumor cells to BTK inhibition was demonstrated. This model mirrors what reported in patients with ABC DLBLC, and therefore represents a novel model for studying the biology of ABC-DLBCL harboring the dual loss of BLIMP-1/p53 and c-Myc over-expression.
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Linfocitos B/metabolismo , Linfocitos B/patología , Transformación Celular Neoplásica/genética , Proteínas Represoras/deficiencia , Proteína p53 Supresora de Tumor/deficiencia , Animales , Linfocitos B/efectos de los fármacos , Biomarcadores , Evolución Clonal/genética , Modelos Animales de Enfermedad , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes myc , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Ratones Transgénicos , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Selective killing of cancer cells while minimizing damage to healthy tissues is the goal of clinical radiation therapy. This therapeutic ratio can be improved by image-guided radiation delivery and selective radiosensitization of cancer cells. Here, we have designed and tested a novel trimodal theranostic nanoparticle made of bismuth and gadolinium for on-site radiosensitization and image contrast enhancement to improve the efficacy and accuracy of radiation therapy. We demonstrate in vivo magnetic resonance (MR), computed tomography (CT) contrast enhancement, and tumor suppression with prolonged survival in a non-small cell lung carcinoma model during clinical radiation therapy. Histological studies show minimal off-target toxicities due to the nanoparticles or radiation. By mimicking existing clinical workflows, we show that the bismuth-gadolinium nanoparticles are highly compatible with current CT-guided radiation therapy and emerging MR-guided approaches. This study reports the first in vivo proof-of-principle for image-guided radiation therapy with a new class of theranostic nanoparticles.
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Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/radioterapia , Bismuto/uso terapéutico , Medios de Contraste/uso terapéutico , Gadolinio/uso terapéutico , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/radioterapia , Nanopartículas/uso terapéutico , Dióxido de Silicio/uso terapéutico , Células A549 , Adenocarcinoma del Pulmón , Animales , Bismuto/química , Medios de Contraste/química , Gadolinio/química , Humanos , Imagen por Resonancia Magnética , Ratones , Nanopartículas/química , Radioterapia Guiada por Imagen , Dióxido de Silicio/química , Nanomedicina Teranóstica , Tomografía Computarizada por Rayos XRESUMEN
Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady-state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady-state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification-mediated-polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV-injected animals for up to 24 months, indicating stable marking of murine steady-state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long-term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short-lived progenitor cells. Myelosuppressive treatment of mice prior to LV-injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady-state hematopoiesis.
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Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Animales , Diferenciación Celular/fisiología , Linaje de la Célula , Técnicas de Transferencia de Gen , Vectores Genéticos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
Lentiviral vectors (LV) are widely used to stably transfer genes into target cells investigating or treating gene functions. In addition, gene transfer into early murine embryos may be improved to efficiently generate transgenic mice. We applied lentiviral gene transfer to generate a mouse model transgenic for SET binding protein-1 (Setbp1) and enhanced green fluorescent protein (eGFP). Neither transgenic founders nor their vector-positive offspring transcribed or expressed the transgenes. Bisulfite sequencing of the internal spleen focus-forming virus (SFFV) promoter demonstrated extensive methylation of all analyzed CpGs in the transgenic mice. To analyze the impact of Setbp1 on epigenetic silencing, embryonic stem cells (ESC) were differentiated into cardiomyocytes (CM) in vitro. In contrast to human promoters in LV, virally derived promoter sequences were strongly methylated during differentiation, independent of the transgene. Moreover, the commonly used SFFV promoter (SFFVp) was highly methylated with remarkable strength and frequency during hematopoietic differentiation in vivo in LV but less in γ-retroviral (γ-RV) backbones. In summary, we conclude that LV using an internal SFFVp are not suitable to generate transgenic mice or perform constitutive expression studies in differentiating cells. Choosing the appropriate promoter is also crucial to allow stable transgene expression in clinical gene therapy.
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Proteínas Portadoras/genética , Vectores Genéticos , Lentivirus/genética , Ratones Transgénicos/genética , Virus Formadores de Foco en el Bazo/genética , Células Madre/metabolismo , Animales , Diferenciación Celular , Islas de CpG/genética , Metilación de ADN , Epigénesis Genética , Efecto Fundador , Silenciador del Gen , Genes Esenciales , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Células Madre/citología , TransgenesRESUMEN
Human colon cancer harbors a small subfraction of tumor-initiating cells (TICs) that is assumed to be a functionally homogeneous stem-cell-like population driving tumor maintenance and metastasis formation. We found unexpected cellular heterogeneity within the TIC compartment, which contains three types of TICs. Extensively self-renewing long-term TICs (LT-TICs) maintained tumor formation in serial xenotransplants. Tumor transient amplifying cells (T-TACs) with limited or no self-renewal capacity contributed to tumor formation only in primary mice. Rare delayed contributing TICs (DC-TICs) were exclusively active in secondary or tertiary mice. Bone marrow was identified as an important reservoir of LT-TICs. Metastasis formation was almost exclusively driven by self-renewing LT-TICs. Our results demonstrate that tumor initiation, self-renewal, and metastasis formation are limited to particular subpopulations of TICs in primary human colon cancer. We identify LT-TICs as a quantifiable target for therapies aimed toward eradication of self-renewing tumorigenic and metastatic colon cancer cells.
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Neoplasias del Colon/patología , Células Madre Neoplásicas/patología , Anciano , Animales , Médula Ósea/patología , Proliferación Celular , Femenino , Humanos , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Metástasis de la Neoplasia , Células Madre Neoplásicas/clasificación , Esferoides Celulares/patología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Kinetics of hematopoietic recovery driven by different types of human stem and progenitor cells after transplantation are not fully understood. Short-term repopulating cells (STRCs) dominate early hematopoiesis after transplantation. STRCs are highly enriched in adult mobilized peripheral blood compared with cord blood, but the length of their contribution to hematopoiesis remains unclear. To understand posttransplantation durability and lineage contribution of STRCs, we compared repopulation kinetics of mobilized peripheral blood (high STRC content) with cord blood transplants (low STRC content) in long-lived NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (IL2RG(-/-)) mice. This comparison demonstrates that quantitative contribution of human STRCs to hematopoiesis is restricted to the first 5 months after transplantation. The ratio of STRCs to long-term repopulating cells dramatically changes during ontogeny. This model enables to precisely determine early and late engraftment kinetics of defined human repopulating cell types and to preclinically assess the engraftment kinetics of engineered stem cell transplants.
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Trasplante de Células Madre de Sangre del Cordón Umbilical , Supervivencia de Injerto/fisiología , Hematopoyesis/fisiología , Movilización de Célula Madre Hematopoyética , Trasplante de Células Madre Hematopoyéticas , Animales , División Celular/fisiología , Linaje de la Célula/fisiología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de Interleucina-2/genética , Recuperación de la Función/fisiología , Trasplante HeterólogoRESUMEN
Herpes simplex virus 1 (HSV-1) strain ANG and ANGpath were cloned as bacterial artificial chromosome (BAC). Two different types of BAC genomes were obtained. BAC genomes of type I contained the BAC replicon at the intended target region between the genes of UL48 and UL49. In BAC genomes of type II, the BAC sequences were found to be aberrantly fused between the termini of the HSV-1 genome. Both the BAC types were used to establish a conditional gene expression system for HSV-1 by Flp recombinase-mediated insertion of expression vectors that were modified to respond to the T-REx tetracycline (Tet)-inducible transcription switch. During BAC cloning and mutagenesis in E. coli, not only deletions but also defined mutations of the HSV-1 genome were observed. Successful virus reconstitution from BACs with large inserts demonstrated that HSV-1 has a packaging capacity for foreign sequences of at least 8.1% of its genome size. Targets for Tet-regulated gene expression were the viral DNA polymerase gene (pol) and a reporter gene of glycoprotein B fused to enhanced green fluorescent protein (gBGFP). Results with the pol gene as target showed that virus plaque production could not be significantly controlled by the T-REx gene switch using vectors encoding one copy of the tetR gene. In contrast, an efficient Tet-response was achieved with the gBGFP reporter, which was optimal in a Tet repressor (TetR)-producing cell line, demonstrating that the TetR concentration provided by the virus was not sufficient for a tight control of Tet-regulated gene expression.