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1.
N Engl J Med ; 387(23): 2113-2125, 2022 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-36477031

RESUMEN

BACKGROUND: Immune checkpoint inhibitors and targeted therapies have dramatically improved outcomes in patients with advanced melanoma, but approximately half these patients will not have a durable benefit. Phase 1-2 trials of adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) have shown promising responses, but data from phase 3 trials are lacking to determine the role of TILs in treating advanced melanoma. METHODS: In this phase 3, multicenter, open-label trial, we randomly assigned patients with unresectable stage IIIC or IV melanoma in a 1:1 ratio to receive TIL or anti-cytotoxic T-lymphocyte antigen 4 therapy (ipilimumab at 3 mg per kilogram of body weight). Infusion of at least 5×109 TILs was preceded by nonmyeloablative, lymphodepleting chemotherapy (cyclophosphamide plus fludarabine) and followed by high-dose interleukin-2. The primary end point was progression-free survival. RESULTS: A total of 168 patients (86% with disease refractory to anti-programmed death 1 treatment) were assigned to receive TILs (84 patients) or ipilimumab (84 patients). In the intention-to-treat population, median progression-free survival was 7.2 months (95% confidence interval [CI], 4.2 to 13.1) in the TIL group and 3.1 months (95% CI, 3.0 to 4.3) in the ipilimumab group (hazard ratio for progression or death, 0.50; 95% CI, 0.35 to 0.72; P<0.001); 49% (95% CI, 38 to 60) and 21% (95% CI, 13 to 32) of the patients, respectively, had an objective response. Median overall survival was 25.8 months (95% CI, 18.2 to not reached) in the TIL group and 18.9 months (95% CI, 13.8 to 32.6) in the ipilimumab group. Treatment-related adverse events of grade 3 or higher occurred in all patients who received TILs and in 57% of those who received ipilimumab; in the TIL group, these events were mainly chemotherapy-related myelosuppression. CONCLUSIONS: In patients with advanced melanoma, progression-free survival was significantly longer among those who received TIL therapy than among those who received ipilimumab. (Funded by the Dutch Cancer Society and others; ClinicalTrials.gov number, NCT02278887.).


Asunto(s)
Inmunoterapia Adoptiva , Linfocitos Infiltrantes de Tumor , Melanoma , Humanos , Tratamiento Basado en Trasplante de Células y Tejidos , Ipilimumab/efectos adversos , Melanoma/tratamiento farmacológico
2.
Haematologica ; 2024 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-38961746

RESUMEN

Differentiation of induced pluripotent stem cells (iPSCs) into hematopoietic lineages offers great therapeutic potential. During embryogenesis, hemogenic endothelium (HE) gives rise to hematopoietic stem and progenitor cells through the endothelial-to-hematopoietic transition (EHT). Understanding this process using iPSCs is key to generating functional hematopoietic stem cells (HSCs), a currently unmet challenge. In this study, we examined the role of the transcriptional factor GFI1B and its co-factor LSD1/KDM1A in EHT. To this end, we employed patient-derived iPSC lines with a dominant negative dysfunctional GFI1BQ287* and irreversible pharmacological LSD1/KDM1A inhibition in healthy iPSC lines. The formation of HE remained unaffected; however, hematopoietic output was severely reduced in both conditions. Single-cell RNA sequencing (scRNAseq) performed on the CD144+/CD31+ population derived from healthy iPSCs revealed similar expression dynamics of genes associated with in vivo EHT. Interestingly, LSD1/KDM1A inhibition in healthy lines before EHT resulted in a complete absence of hematopoietic output. However, uncommitted HE cells did not display GFI1B expression, suggesting a timed transcriptional program. To test this hypothesis, we ectopically expressed GFI1B in uncommitted HE cells, leading to downregulation of endothelial genes and upregulation of hematopoietic genes, including GATA2, KIT, RUNX1, and SPI1. Thus, we demonstrate that LSD1/KDM1A and GFI1B can function at distinct temporal points in different cellular subsets during EHT. Although GFI1B is not detected in uncommitted HE cells, its ectopic expression allows for partial hematopoietic specification. These data indicate that precisely timed expression of specific transcriptional regulators during EHT is crucial to the eventual outcome of EHT.

3.
Int J Mol Sci ; 21(3)2020 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-32024018

RESUMEN

Megakaryopoiesis is the process during which megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.


Asunto(s)
Ataxina-2/genética , Perfilación de la Expresión Génica/métodos , Células Progenitoras de Megacariocitos/citología , Animales , Antígenos CD34/genética , Ataxina-2/metabolismo , Diferenciación Celular , Línea Celular , ARN Helicasas DEAD-box/genética , Regulación de la Expresión Génica , Humanos , Ratones , Glicoproteína IIb de Membrana Plaquetaria/genética , Proteínas Proto-Oncogénicas/genética
4.
Haematologica ; 104(7): 1460-1472, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30655368

RESUMEN

Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 - REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.


Asunto(s)
Trastornos de la Coagulación Sanguínea/patología , Plaquetas/patología , Regulación de la Expresión Génica , Células Madre Pluripotentes Inducidas/patología , Megacariocitos/patología , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Trastornos de la Coagulación Sanguínea/genética , Trastornos de la Coagulación Sanguínea/metabolismo , Plaquetas/metabolismo , Diferenciación Celular , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Megacariocitos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Mapas de Interacción de Proteínas , Proteoma/análisis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo
5.
EJNMMI Res ; 11(1): 81, 2021 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-34424429

RESUMEN

BACKGROUND: The therapeutic use of [131I]meta-iodobenzylguanidine ([131I]MIBG) is often accompanied by hematological toxicity, primarily consisting of severe and persistent thrombocytopenia. We hypothesize that this is caused by selective uptake of MIBG via the serotonin transporter (SERT) located on platelets and megakaryocytes. In this study, we have investigated whether in vitro cultured human megakaryocytes are capable of selective plasma membrane transport of MIBG and whether pharmacological intervention with selective serotonin reuptake inhibitors (SSRIs) may prevent this radiotoxic MIBG uptake. METHODS: Peripheral blood CD34+ cells were differentiated to human megakaryocytic cells using a standardized culture protocol. Prior to [3H]serotonin and [125I]MIBG uptake experiments, the differentiation status of megakaryocyte cultures was assessed by flow cytometry. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess SERT and NET (norepinephrine transporter) mRNA expression. On day 10 of differentiation, [3H]serotonin and [125I]MIBG uptake assays were conducted. Part of the samples were co-incubated with the SSRI citalopram to assess SERT-specific uptake. HEK293 cells transfected with SERT, NET, and empty vector served as controls. RESULTS: In vitro cultured human megakaryocytes are capable of selective plasma membrane transport of MIBG. After 10 days of differentiation, megakaryocytic cell culture batches from three different hematopoietic stem and progenitor cell donors showed on average 9.2 ± 2.4 nmol of MIBG uptake per milligram protein per hour after incubation with 10-7 M MIBG (range: 6.6 ± 1.0 to 11.2 ± 1.0 nmol/mg/h). Co-incubation with the SSRI citalopram led to a significant reduction (30.1%-41.5%) in MIBG uptake, implying SERT-specific uptake of MIBG. A strong correlation between the number of mature megakaryocytes and SERT-specific MIBG uptake was observed. CONCLUSION: Our study demonstrates that human megakaryocytes cultured in vitro are capable of MIBG uptake. Moreover, the SSRI citalopram selectively inhibits MIBG uptake via the serotonin transporter. The concomitant administration of citalopram to neuroblastoma patients during [131I]MIBG therapy might be a promising strategy to prevent the onset of thrombocytopenia.

6.
FEBS Lett ; 593(23): 3288-3303, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31520530

RESUMEN

In vitro cultured blood cells for transfusion purposes provide a safe alternative to donor blood, particularly for patients who require recurrent transfusions, and can be used as carriers of therapeutic molecules. In vitro derivation of hematopoietic cell types from human-induced pluripotent stem cells (iPSCs) allows for a constant, well-defined production pipeline for such advanced therapeutic and medicinal products. Application of selected iPSC-derived hematopoietic stem cells and hematopoietic effector cells in transplantation/transfusions would avoid the risk of alloimmunization and blood-borne diseases, as well as enable the production of enhanced blood cells expressing molecules that enforce blood cell function or endow novel therapeutic properties. Here, we discuss the state of the art approaches to produce erythroid, megakaryoid and myeloid cells from iPSCs and the biological and technical hurdles that we need to overcome prior to therapeutic application.


Asunto(s)
Células Eritroides/citología , Células Madre Hematopoyéticas/citología , Células Madre Pluripotentes Inducidas/citología , Células Mieloides/citología , Células Sanguíneas/citología , Células Sanguíneas/trasplante , Donantes de Sangre , Transfusión Sanguínea/métodos , Diferenciación Celular/genética , Humanos , Trombopoyesis/genética
7.
Blood Cancer J ; 9(3): 33, 2019 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-30850577

RESUMEN

The inv(16) acute myeloid leukemia-associated CBFß-MYH11 fusion is proposed to block normal myeloid differentiation, but whether this subtype of leukemia cells is poised for a unique cell lineage remains unclear. Here, we surveyed the functional consequences of CBFß-MYH11 in primary inv(16) patient blasts, upon expression during hematopoietic differentiation in vitro and upon knockdown in cell lines by multi-omics profiling. Our results reveal that primary inv(16) AML cells share common transcriptomic signatures and epigenetic determiners with megakaryocytes and erythrocytes. Using in vitro differentiation systems, we reveal that CBFß-MYH11 knockdown interferes with normal megakaryocyte maturation. Two pivotal regulators, GATA2 and KLF1, are identified to complementally occupy RUNX1-binding sites upon fusion protein knockdown, and overexpression of GATA2 partly induces a gene program involved in megakaryocyte-directed differentiation. Together, our findings suggest that in inv(16) leukemia, the CBFß-MYH11 fusion inhibits primed megakaryopoiesis by attenuating expression of GATA2/KLF1 and interfering with a balanced transcriptional program involving these two factors.


Asunto(s)
Factor de Transcripción GATA2/metabolismo , Regulación Leucémica de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Megacariocitos/metabolismo , Proteínas de Fusión Oncogénica/genética , Sitios de Unión , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Epigénesis Genética , Células Eritroides/citología , Células Eritroides/metabolismo , Eritropoyesis/genética , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Megacariocitos/citología , Proteínas de Fusión Oncogénica/metabolismo , Unión Proteica , Trombopoyesis , Transcripción Genética
8.
Stem Cell Res ; 29: 232-244, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29751281

RESUMEN

Hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) provide opportunities not only for fundamental research and disease modelling/drug testing but also for large-scale production of blood effector cells for future clinical application. Although there are multiple ways to differentiate human iPSCs towards hematopoietic lineages, there is a need to develop reproducible and robust protocols. Here we introduce an efficient way to produce three major blood cell types using a standardized differentiation protocol that starts with a single hematopoietic initiation step. This system is feeder-free, avoids EB-formation, starts with a hematopoietic initiation step based on a novel single cell-derived iPSC colony differentiation and produces multi-potential progenitors within 8-10 days. Followed by lineage-specific growth factor supplementation these cells can be matured into well characterized erythroid, megakaryocytic and myeloid cells with high-purity, without transcription factor overexpression or any kind of pre-purification step. This standardized differentiation system provides a simple platform to produce specific blood cells in a reproducible manner for hematopoietic development studies, disease modelling, drug testing and the potential for future therapeutic applications.


Asunto(s)
Técnicas de Cultivo de Célula , Diferenciación Celular , Células Eritroides , Células Madre Pluripotentes Inducidas , Megacariocitos , Células Mieloides , Células Eritroides/citología , Células Eritroides/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Células Mieloides/citología , Células Mieloides/metabolismo
9.
Stem Cell Res ; 25: 30-33, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29040913

RESUMEN

Peripheral blood mononuclear cells were isolated and cultured to a pure pro-EBL population and reprogrammed using episomal plasmids. The pluripotency of transgene-free induced pluripotent stem cell (iPSC) line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. Peripheral blood is a non-invasive easy accessible cell source and combined with EBL outgrowth in vitro, a routine process obtaining sufficient amount of homogenous cells can be obtained within a week. Using episomal delivery, pro-EBLs can be reprogrammed in a transgene-free, cost effective system.


Asunto(s)
Eritroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Humanos , Cariotipificación
10.
Stem Cell Res ; 25: 34-37, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29055225

RESUMEN

Peripheral blood mononuclear cells were isolated from an individual harboring a heterozygous c.859C→T p.Q287* mutation in GFI1B, causing an autosomal dominant bleeding disorder, platelet type, 17 (BDPLT17). PBMCs were differentiated to erythroblasts and reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. Pluripotency of iPSC line was confirmed by expression of associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Normal karyotype confirmed the genomic integrity of iPSCs and the presence of disease causing mutation was shown by Sanger sequencing. The generated iPSCs can be used to study BDPLT17 pathophysiology and basic functions of GFI1B.


Asunto(s)
Plaquetas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Heterocigoto , Humanos , Cariotipo , Mutación
11.
Stem Cell Res ; 25: 42-45, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29055227

RESUMEN

Mobilized peripheral blood (MPB) CD34+ cells were differentiated to CD34+/CD41+ megakaryoblasts. Cells were sorted to obtain a pure megakaryoblast population which was reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector. Resulting iPSC showed normal karyotype and expression of pluripotency associated markers and in vitro spontaneous differentiation towards the 3 germ layers confirmed pluripotency of iPSC lines. Besides normal iPSC applications, these lines can be used as a control line for other megakaryoid origin iPSC and could be applied for epigenetic based research.


Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Progenitoras de Megacariocitos/citología , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Inmunohistoquímica , Cariotipificación
12.
Stem Cell Res ; 18: 26-28, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-28395797

RESUMEN

Mobilized peripheral blood (MPB) CD34+ cells were cultured to CD41+/CD34+ megakaryoblasts. Cells were sorted to obtain a pure megakaryoblast population that was reprogramed by a hOKSM self-silencing polycistronic vector using lentiviral delivery. The generated induced pluripotent stem cell (iPSC) lines were tested for silencing of the reprogramming construct by flow cytometry. Pluripotency of MML-6838-Cl2 iPSC line was confirmed by expression of associated markers and by in vivo spontaneous differentiation towards the 3 germ layers. The genomic integrity of iPSC line was shown by karyotyping. The MML-6838-Cl2 iPSC is, to our knowledge, the first to be generated from megakaryoblasts.


Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Células Progenitoras de Megacariocitos/citología , Animales , Antígenos CD34/metabolismo , Línea Celular , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Cariotipo , Lentivirus/genética , Masculino , Células Progenitoras de Megacariocitos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Teratoma/metabolismo , Teratoma/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
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