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Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.
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Desenvolvimento Ósseo/fisiologia , Osso e Ossos/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Osso e Ossos/metabolismo , Cartilagem/citologia , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Análise de Célula Única/métodos , Células-Tronco/citologia , Células Estromais/citologia , Transcriptoma/genéticaRESUMO
The hierarchy of human hemopoietic progenitor cells that produce lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but they remain incompletely characterized. Here we demonstrated that lympho-myeloid progenitor populations in cord blood - lymphoid-primed multi-potential progenitors (LMPPs), granulocyte-macrophage progenitors (GMPs) and multi-lymphoid progenitors (MLPs) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Although most progenitors had the potential to develop into only one mature cell type ('uni-lineage potential'), bi- and rarer multi-lineage progenitors were present among LMPPs, GMPs and MLPs. Those findings, coupled with single-cell expression analyses, suggest that a continuum of progenitors execute lymphoid and myeloid differentiation, rather than only uni-lineage progenitors' being present downstream of stem cells.
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Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Mieloides/metabolismo , Análise de Célula Única/métodos , Animais , Linhagem da Célula/genética , Separação Celular/métodos , Células Cultivadas , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Camundongos , Transplante HeterólogoRESUMO
Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance owing to a lack of effective and nontoxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation and show transient, programmable gene regulation in primary cells, including human CD34+ hematopoietic stem and progenitor cells (HSPCs) and human CD3+ T cells. We show multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.
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Sistemas CRISPR-Cas , Endonucleases , Endonucleases/genética , Regulação da Expressão Gênica , Genoma , RNA Guia de Cinetoplastídeos/genética , Ativação TranscricionalRESUMO
Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co-expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK-STAT signalling, TPO-independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR-dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.
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Calreticulina , Transtornos Mieloproliferativos , Animais , Anticorpos Monoclonais , Calreticulina/genética , Calreticulina/metabolismo , Humanos , Janus Quinase 2/metabolismo , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , RatosRESUMO
TP53 aberrations are found in approximately 10% of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and are considered early driver events affecting leukemia stem cells. In this study, we compared features of a total of 84 patients with these disorders seen at a tertiary cancer center. Clinical and cytogenetic characteristics as well as immunophenotypes of immature blast cells were similar between AML and MDS patients. Median overall survival (OS) was 226 days (95% confidence interval [CI], 131-300) for the entire cohort with an estimated 3-year OS rate of 11% (95% CI, 6-22). OS showed a significant difference between MDS (median, 345 days; 95% CI, 235-590) and AML patients (median, 91 days; 95% CI, 64-226) which is likely due to a different co-mutational pattern as revealed by next-generation sequencing. Transformation of TP53 aberrant MDS occurred in 60.5% of cases and substantially reduced their survival probability. Cox regression analysis revealed treatment class and TP53 variant allele frequency as prognostically relevant parameters but not the TP53-specific prognostic scores EAp53 and RFS. These data emphasize similarities between TP53 aberrant AML and MDS and support previous notions that they should be classified and treated as a distinct disorder.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Citogenética , Humanos , Imunofenotipagem , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mutação , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The ß-haemoglobinopathies, such as sickle cell disease and ß-thalassaemia, are caused by mutations in the ß-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure ß-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult ß-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for ß-haemoglobinopathies.
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Anemia Falciforme/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Marcação de Genes , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Globinas beta/genética , Alelos , Anemia Falciforme/patologia , Anemia Falciforme/terapia , Animais , Antígenos CD34/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Diferenciação Celular , Linhagem da Célula , Separação Celular , Dependovirus/genética , Eritrócitos , Feminino , Citometria de Fluxo , Genes Reporter , Recombinação Homóloga , Humanos , Imãs , Camundongos Endogâmicos NOD , Camundongos SCID , Microesferas , Mutação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Talassemia beta/genética , Talassemia beta/terapiaRESUMO
Persistent measurable residual disease (MRD) is an increasingly important prognostic marker in acute myeloid leukemia (AML). Currently, MRD is determined by multi-parameter flow cytometry (MFC) or PCR-based methods detecting leukemia-specific fusion transcripts and mutations. However, while MFC is highly operator-dependent and difficult to standardize, PCR-based methods are only available for a minority of AML patients. Here we describe a novel, highly sensitive and broadly applicable method for MRD detection by combining MFC-based leukemic cell enrichment using an optimized combinatorial antibody panel targeting CLL-1, TIM-3, CD123 and CD117, followed by mutational analysis of recurrently mutated genes in AML. In dilution experiments this method showed a sensitivity of 10-4 to 10-5 for residual disease detection. In prospectively collected remission samples this marker combination allowed for a median 67-fold cell enrichment with sufficient DNA quality for mutational analysis using next generation sequencing (NGS) or digital PCR in 39 out of 41 patients. Twenty-one samples (53.8%) tested MRD positive, whereas 18 (46.2%) were negative. With a median follow-up of 559 days, 71.4% of MRD positive (15/21) and 27.8% (5/18) of MRD negative patients relapsed (P = .007). The cumulative incidence of relapse (CIR) was higher for MRD positive patients (5-year CIR: 90.5% vs 28%, P < .001). In multivariate analysis, MRD positivity was a prominent factor for CIR. Thus, MFC-based leukemic cell enrichment using antibodies against CLL-1, TIM-3, CD123 and CD117 followed by mutational analysis allows high sensitive MRD detection and is informative on relapse risk in the majority of AML patients.
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Artificial intelligence (AI) has potential to improve the accuracy of screening for valvular and congenital heart disease by auscultation. However, despite recent advances in signal processing and classification algorithms focused on heart sounds, clinical acceptance of this technology has been limited, in part due to lack of objective performance data. We hypothesized that a heart murmur detection algorithm could be quantitatively and objectively evaluated by virtual clinical trial. All cases from the Johns Hopkins Cardiac Auscultatory Recording Database (CARD) with either a pathologic murmur, an innocent murmur or no murmur were selected. The test algorithm, developed independently of CARD, analyzed each recording using an automated batch processing protocol. 3180 heart sound recordings from 603 outpatient visits were selected from CARD. Algorithm estimation of heart rate was similar to gold standard. Sensitivity and specificity for detection of pathologic cases were 93% (CI 90-95%) and 81% (CI 75-85%), respectively, with accuracy 88% (CI 85-91%). Performance varied according to algorithm certainty measure, age of patient, heart rate, murmur intensity, location of recording on the chest and pathologic diagnosis. This is the first reported comprehensive and objective evaluation of an AI-based murmur detection algorithm to our knowledge. The test algorithm performed well in this virtual clinical trial. This strategy can be used to efficiently compare performance of other algorithms against the same dataset and improve understanding of the potential clinical usefulness of AI-assisted auscultation.
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Inteligência Artificial/estatística & dados numéricos , Diagnóstico por Computador/métodos , Auscultação Cardíaca/métodos , Cardiopatias Congênitas/diagnóstico , Sopros Cardíacos/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Criança , Pré-Escolar , Bases de Dados Factuais , Humanos , Lactente , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
Acute myeloid leukemia (AML) is associated with deregulation of DNA methylation; however, many cases do not bear mutations in known regulators of cytosine guanine dinucleotide (CpG) methylation. We found that mutations in WT1, IDH2, and CEBPA were strongly linked to DNA hypermethylation in AML using a novel integrative analysis of The Cancer Genome Atlas data based on Boolean implications, if-then rules that identify all individual CpG sites that are hypermethylated in the presence of a mutation. Introduction of mutant WT1 (WT1mut) into wild-type AML cells induced DNA hypermethylation, confirming mutant WT1 to be causally associated with DNA hypermethylation. Methylated genes in WT1mut primary patient samples were highly enriched for polycomb repressor complex 2 (PRC2) targets, implicating PRC2 dysregulation in WT1mut leukemogenesis. We found that PRC2 target genes were aberrantly repressed in WT1mut AML, and that expression of mutant WT1 in CD34(+) cord blood cells induced myeloid differentiation block. Treatment of WT1mut AML cells with short hairpin RNA or pharmacologic PRC2/enhancer of zeste homolog 2 (EZH2) inhibitors promoted myeloid differentiation, suggesting EZH2 inhibitors may be active in this AML subtype. Our results highlight a strong association between mutant WT1 and DNA hypermethylation in AML and demonstrate that Boolean implications can be used to decipher mutation-specific methylation patterns that may lead to therapeutic insights.
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Metilação de DNA/genética , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica/genética , Genes do Tumor de Wilms , Leucemia Mieloide Aguda/genética , Complexo Repressor Polycomb 2/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Mutação , Análise de Sequência com Séries de OligonucleotídeosRESUMO
In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype, and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription, and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord, and skin cultured in humanized media. Surprisingly, only BM-derived MSCs spontaneously formed a BM cavity through a vascularized cartilage intermediate in vivo that was progressively replaced by hematopoietic tissue and bone. Only BM-derived MSCs exhibited a chondrogenic transcriptional program with hypomethylation and increased expression of RUNX3, RUNX2, BGLAP, MMP13, and ITGA10 consistent with a latent and primed skeletal developmental potential. The humanized MSC-derived microenvironment permitted homing and maintenance of long-term murine SLAM(+) hematopoietic stem cells (HSCs), as well as human CD34(+)/CD38(-)/CD90(+)/CD45RA(+) HSCs after cord blood transplantation. These studies underscore the profound differences in developmental potential between MSC sources independent of donor age, with implications for their clinical use. We also demonstrate a tractable human niche model for studying homing and engraftment of human hematopoietic cells in normal and neoplastic states.
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Linhagem da Célula , Epigênese Genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , Nicho de Células-Tronco , Western Blotting , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Condrogênese/fisiologia , Citometria de Fluxo , Humanos , Osteogênese/fisiologiaRESUMO
BACKGROUND AIMS: Autologous endothelial cells are promising alternative angiogenic cell sources in trials of therapeutic vasculogenesis, in the treatment of vascular diseases and in the field of tissue engineering. A population of endothelial cells (ECs) with long-term proliferative capability, endothelial colony-forming cells (ECFCs), can be isolated from human peripheral blood. ECFCs are considered an endothelial precursor population. They can be expanded in cell factories in sufficient numbers for clinical applications, but because the number of isolated primary ECs is low, the culture period required may be long. Another EC population that is easily available in the autologous setting and may be expanded in vitro through several population doublings are ECs from adipose tissue (AT-ECs). METHODS: Through extensive comparisons using whole-genome microarray analysis, morphology, phenotype and functional assays, we wanted to evaluate the potential of these EC populations for use in clinical neovascularization. RESULTS: Global gene expression profiling of ECFCs, AT-ECs and the classical EC population, human umbilical vein ECs, showed that the EC populations clustered as unique populations, but very close to each other. By cell surface phenotype and vasculogenic potential in vitro and in vivo, we also found the ECFCs to be extremely similar to AT-ECs. CONCLUSIONS: These properties, together with easy access in the autologous setting, suggest that both AT-ECs and ECFCs may be useful in trials of therapeutic neovascularization. However, AT-ECs may be a more practical alternative for obtaining large quantities of autologous ECs.
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Tecido Adiposo/citologia , Células Endoteliais da Veia Umbilical Humana/citologia , Neovascularização Fisiológica/fisiologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Diferenciação Celular , Células Cultivadas , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide/genética , MicroRNAs/genética , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , Decitabina/farmacologia , Decitabina/uso terapêutico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide/tratamento farmacológico , Células THP-1RESUMO
This article describes a new acquisition and reconstruction concept for positive contrast imaging of cells labeled with superparamagnetic iron oxides (SPIOs). Overcoming the limitations of a negative contrast representation as gained with gradient echo and fully balanced steady state (bSSFP), the proposed method delivers a spatially localized contrast with high cellular sensitivity not accomplished by other positive contrast methods. Employing a 3D radial bSSFP pulse sequence with half-echo sampling, positive cellular contrast is gained by adding artificial global frequency offsets to each half-echo before image reconstruction. The new contrast regime is highlighted with numerical intravoxel simulations including the point-spread function for 3D half-echo acquisitions. Furthermore, the new method is validated on the basis of in vitro cell phantom measurements on a clinical MRI platform, where the measured contrast-to-noise ratio (CNR) of the new approach exceeds even the negative contrast of bSSFP. Finally, an in vivo proof of principle study based on a mouse model with a clear depiction of labeled cells within a subcutaneous cell islet containing a cell density as low as 7 cells/mm(3) is presented. The resultant isotropic images show robustness to motion and a high CNR, in addition to an enhanced specificity due to the positive contrast of SPIO-labeled cells.
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Meios de Contraste , Dextranos/metabolismo , Imageamento Tridimensional , Imageamento por Ressonância Magnética/métodos , Coloração e Rotulagem , Animais , Simulação por Computador , Humanos , Injeções Subcutâneas , Nanopartículas de Magnetita , Camundongos , Imagens de Fantasmas , Processamento de Sinais Assistido por ComputadorAssuntos
Transplante de Medula Óssea , Leucemia Mieloide Aguda , Mutação , Transplante de Neoplasias , Neoplasias Experimentais , Proteína Supressora de Tumor p53 , Animais , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1α was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ± 6%; P = .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment.
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Células da Medula Óssea/citologia , Transplante de Medula Óssea/métodos , Microambiente Celular/fisiologia , Hematopoese Extramedular/fisiologia , Transplante Heterotópico , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Transplante de Medula Óssea/fisiologia , Células Cultivadas , Microambiente Celular/genética , Hematopoese Extramedular/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Modelos Animais , Osteogênese/genética , Osteogênese/fisiologia , Especificidade da EspécieRESUMO
INTRODUCTION: Gene therapy is an emerging topic in medicine. The first products have already been licensed in the European Union for the treatment of immune deficiency, spinal muscular atrophy, hemophilia, retinal dystrophy, a rare neurotransmitter disorder and some hematological cancers, while many more are being assessed in preclinical and clinical trials. OBJECTIVE: The purpose of this review is to provide an overview of the core principles of gene therapy along with information on challenges and risks. Benefits, adverse effects and potential risks are illustrated based on the examples of hemophilia and spinal muscular atrophy. RESULTS: At present, in-vitro and in-vivo gene addition or gene augmentation is the most commonly established type of gene therapy. More recently, more sophisticated and precise approaches such as in situ gene editing have moved into focus. However, all types of gene therapy require long-term observation of treated patients to ensure safety, efficacy, predictability and durability. Important safety concerns include immune reactions to the vector, the foreign DNA or the new protein resulting from gene therapy, and a remaining low cancer risk based on insertional mutagenesis. Ethical and regulatory issues need to be addressed, and new reimbursement models are called for to ease the financial burden that this new treatment poses for the health care system. CONCLUSION: Gene therapy holds great promise for considerable improvement or even cure of genetic diseases with serious clinical consequences. However, a number of questions and issues need to be clarified to ensure broad accessibility of safe and efficacious products.
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Acute myeloid leukemia (AML) is an aggressive malignant disease with a high relapse rate due to the persistence of chemoresistant cells. To some extent, these residual cells can be traced by sensitive flow cytometry and molecular methods resulting in the establishment of measurable residual disease (MRD). The detection of MRD after therapy represents a significant prognostic factor for predicting patients' individual risk of relapse. However, due to the heterogeneity of the disease, a single sensitive method for MRD detection applicable to all AML patients is lacking. This review will highlight the advantages and limitations of the currently available detection methods-PCR, multiparameter flow cytometry, and next generation sequencing-and will discuss emerging clinical implications of MRD test results in tailoring treatment of AML patients.
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OBJECTIVES: Secondary hemophagocytic lymphohistiocytosis (sHLH) is a cytokine-driven inflammatory syndrome that is associated with substantial morbidity and mortality and frequently leads to ICU admission. Overall survival in adults with sHLH remains poor, especially in those requiring intensive care. Classical chemotherapeutic treatment exhibits myelosuppression and toxicity. Recently, inhibition of Janus kinase signaling by ruxolitinib has shown efficacy in pediatric HLH. We therefore aimed to determine the activity and safety of a ruxolitinib-based regimen, in critically ill adults with sHLH. DESIGN: Observational pilot study. SETTING: Single-center tertiary academic ICU. PATIENTS: Nine adults (≥ 18 yr) who fulfilled at least five of the eight HLH-2004 criteria. INTERVENTION: Triplet regimen combining: 1) ruxolitinib, 2) polyvalent human IV immunoglobulins (IVIG) at a dose of 1 g/kg bodyweight for 5 days, and 3) high-dose corticosteroids (CSs, dexamethasone 10 mg/m² body surface area, or methylprednisolone equivalent) with subsequent tapering according to the HLH-2004 protocol. MEASUREMENT AND MAIN RESULTS: Nine patients (median age: 42 yr [25th-75th percentile: 32-54]; male: n = 6 males, median H-score: 299 [255-304]) were treated with the triplet regimen. The median Sequential Organ Failure Assessment score at HLH diagnosis was 9 (median; 25th-75th percentile: 7-12), indicating multiple-organ dysfunction in all patients. Within 10 days a significant decrease of the inflammatory parameters soluble interleukin-2 receptor and ferritin as well as a stabilization of the blood count could be shown. All patients were alive at ICU discharge (100% ICU survival), 1 patient died after ICU discharge because of traumatic intracerebral hemorrhage that might be related to HLH or treatment, corresponding to an overall survival of 86% in a 6 months follow-up period. CONCLUSION: In this small case series, a triplet regimen of ruxolitinib in combination with IVIG and CS was highly effective and save for treating critically ill adults with sHLH.
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BACKGROUND: Evaluation of novel cellular therapies in large-animal models and patients is currently hampered by the lack of imaging approaches that allow for long-term monitoring of viable transplanted cells. In this study, sodium iodide symporter (NIS) transgene imaging was evaluated as an approach to follow in vivo survival, engraftment, and distribution of human-induced pluripotent stem cell (hiPSC) derivatives in a pig model of myocardial infarction. METHODS AND RESULTS: Transgenic hiPSC lines stably expressing a fluorescent reporter and NIS (NIS(pos)-hiPSCs) were established. Iodide uptake, efflux, and viability of NIS(pos)-hiPSCs were assessed in vitro. Ten (±2) days after induction of myocardial infarction by transient occlusion of the left anterior descending artery, catheter-based intramyocardial injection of NIS(pos)-hiPSCs guided by 3-dimensional NOGA mapping was performed. Dual-isotope single photon emission computed tomographic/computed tomographic imaging was applied with the use of (123)I to follow donor cell survival and distribution and with the use of (99m)TC-tetrofosmin for perfusion imaging. In vitro, iodide uptake in NIS(pos)-hiPSCs was increased 100-fold above that of nontransgenic controls. In vivo, viable NIS(pos)-hiPSCs could be visualized for up to 15 weeks. Immunohistochemistry demonstrated that hiPSC-derived endothelial cells contributed to vascularization. Up to 12 to 15 weeks after transplantation, no teratomas were detected. CONCLUSIONS: This study describes for the first time the feasibility of repeated long-term in vivo imaging of viability and tissue distribution of cellular grafts in large animals. Moreover, this is the first report demonstrating vascular differentiation and long-term engraftment of hiPSCs in a large-animal model of myocardial infarction. NIS(pos)-hiPSCs represent a valuable tool to monitor and improve current cellular treatment strategies in clinically relevant animal models.
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Sobrevivência de Enxerto , Imagem Multimodal , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/terapia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplante , Tomografia por Emissão de Pósitrons , Transplante de Células-Tronco , Simportadores/metabolismo , Tomografia Computadorizada por Raios X , Animais , Diferenciação Celular , Sobrevivência Celular , Modelos Animais de Doenças , Estudos de Viabilidade , Expressão Gênica , Coração/diagnóstico por imagem , Humanos , Técnicas In Vitro , Injeções , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Transplante de Células-Tronco/métodos , Suínos , Simportadores/genética , Transgenes , Resultado do TratamentoRESUMO
Langerhans cell histiocytosis (LCH) is a rare and clinically heterogeneous hematological disease characterized by the accumulation of mononuclear phagocytes in various tissues and organs. LCH is often characterized by activating mutations of the mitogen-activated protein kinase (MAPK) pathway with BRAFV600E being the most recurrent mutation. Although this discovery has greatly helped in understanding the disease and in developing better investigational tools, the process of malignant transformation and the cell of origin are still not fully understood. In this review, we focus on the newest updates regarding the molecular pathogenesis of LCH and novel suggested pathways with treatment potential.