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1.
Stem Cell Res ; 40: 101542, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31473565

RESUMO

CFTR encodes for a chloride ion channel expressed primarily in secretory epithelia in the airways, intestine, liver and other tissues. Mutations in the CFTR gene have been identified in people suffering from Cystic Fibrosis. Here, we established a CFTR knock-in reporter cell line from a human iPSC line (MHHi006-A) using TALEN technology. The reporter enables the monitoring and optimization of the differentiation of pluripotent stem cells into CFTR expressing epithelia on a single cell level, as well as the enrichment of CFTR positive cells, which represent an excellent tool for Cystic Fibrosis disease modelling, drug screening and ultimately cellular therapies.

2.
Stem Cell Res ; 39: 101492, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31299562

RESUMO

NK homeobox 1 (NKX2.1; also known as thyroid transcription factor 1, TTF-1) encodes for a transcription factor involved in the development of thyroid, lung and brain. Here, we established a NKX2.1 knock-in reporter cell line from human induced pluripotent stem cells (iPSCs) using TALEN technology. The reporter enables the optimization and monitoring of the differentiation of pluripotent stem cells (PSCs) into NKX2.1 expressing cells on a single cell level, as well as the enrichment of NKX2.1 positive cells.

3.
Nat Commun ; 10(1): 2031, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048683

RESUMO

Mutations in the nucleophosmin 1 (NPM1) gene are considered founder mutations in the pathogenesis of acute myeloid leukemia (AML). To characterize the genetic composition of NPM1 mutated (NPM1mut) AML, we assess mutation status of five recurrently mutated oncogenes in 129 paired NPM1mut samples obtained at diagnosis and relapse. We find a substantial shift in the genetic pattern from diagnosis to relapse including NPM1mut loss (n = 11). To better understand these NPM1mut loss cases, we perform whole exome sequencing (WES) and RNA-Seq. At the time of relapse, NPM1mut loss patients (pts) feature distinct mutational patterns that share almost no somatic mutation with the corresponding diagnosis sample and impact different signaling pathways. In contrast, profiles of pts with persistent NPM1mut are reflected by a high overlap of mutations between diagnosis and relapse. Our findings confirm that relapse often originates from persistent leukemic clones, though NPM1mut loss cases suggest a second "de novo" or treatment-associated AML (tAML) as alternative cause of relapse.


Assuntos
Evolução Clonal , Leucemia Mieloide Aguda/genética , Recidiva Local de Neoplasia/genética , Segunda Neoplasia Primária/genética , Proteínas Nucleares/genética , Adulto , Idoso , Análise Mutacional de DNA , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/patologia , Segunda Neoplasia Primária/patologia , Sequenciamento Completo do Exoma
4.
Stem Cell Res ; 37: 101428, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30959346

RESUMO

Variants in SCYL1 can cause a syndrome with low γ-glutamyl-transferase cholestasis, acute liver failure, and neurodegeneration (CALFAN). The encoded protein is involved in intracellular trafficking between Golgi and ER, specific mechanisms are still to be elucidated. We reprogrammed fibroblasts of a 2 years old male patient with CALFAN Syndrome due to a homozygous nonsense variant in SCYL1 (c.[1882C > T]; c.[1882C > T]/p.[Gln628*]; p.[Gln628*]) and generated DHMCi005-A using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Cells showed a normal karyotype. Pluripotency was proven using immunohistochemistry, RT-PCR, and flow cytometry. Differentiation into all germ layers was shown using the STEMdiff™ Trilineage Differentiation Kit (Stemcell Technologies).

5.
Genes Chromosomes Cancer ; 58(10): 689-697, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30994215

RESUMO

The karyotype is a strong independent prognostic factor in myelodysplastic syndromes (MDS). Since the implementation of the new comprehensive cytogenetic scoring system for MDS, chromosome 7 anomalies are no longer generally assigned to poor risk features but are thoroughly separated. However, der(1;7)(q10;p10), hereinafter der(1;7), is merged into the group labeled "any other single" and belongs to the intermediate risk group, just by definition due to lack of adequate clinical data. The aim of our international collaborative was to clarify the "real" prognostic impact of der(1;7) on a homogenous and well-documented data base. We performed detailed analysis of 63 MDS patients with isolated der(1;7) constituting the largest cohort hitherto reported. Furthermore, clinical data are compared with those of patients with isolated del(7q) and isolated monosomy 7. Median overall survival (OS) of patients with der(1;7) is 26 months (hazard ratio (HR) 0.91 for del(7q) vs der(1;7) and 2.53 for monosomy 7 vs der(1;7)). The der(1;7) is associated with profound thrombocytopenia most probably causing the reduced OS which is in striking contrast to the low risk for AML transformation (HR 3.89 for del(7q) vs der(1;7) and 5.88 for monosomy 7 vs der(1;7)). Molecular karyotyping indicates that der(1;7) is generated in a single step during mitosis and that a chromosomal imbalance rather than a single disrupted gene accounts for malignancy. Thus, the current cytogenetic scoring system assigning isolated der(1;7) to the intermediate risk group is now confirmed by a sufficient data set.

6.
Stem Cell Res ; 36: 101402, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30901742

RESUMO

Skin fibroblasts were isolated from a male patient with DNAJC12 deficiency and reprogrammed to iPSCs using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Two clones, DHMCi003-A and DHMCi003-B, were characterized for expression of pluripotency marker genes (Oct4, Nanog, Lin28, SSEA-4, TRA-1-60) and differentiated into all three germ layers using embryoid body (EB) formation. Karyotype of both clones was normal and presence of the homozygous mutation in the DNAJC12 gene was verified by PCR and Sanger sequencing. Both clones represent a useful tool to study the pathomechanisms underlying the deficiency.

7.
Stem Cell Res ; 35: 101394, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30772682

RESUMO

The utilization of human induced pluripotent stem cells (hiPSCs) for disease modeling and drug discovery is already reality, and several first-in-man-applications as cellular therapeutics have been initiated. Implementation of good manufacturing practice (GMP)-compliant protocols for the generation of hiPSC lines is crucial to increase the application safety as well as to fulfil the legal requirements for clinical trials approval. Here we describe the development of a GMP-compatible protocol for the reprogramming of CD34+ hematopoietic stem cells from peripheral blood (CD34+ PBHSC) into hiPSCs using Sendai virus-based reprogramming vectors. Three GMP-compatible hiPSC (GMP-hiPSC) lines were manufactured and characterized under these conditions.


Assuntos
Linhagem Celular , Técnicas de Reprogramação Celular , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo
8.
Stem Cell Res ; 35: 101398, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30772683

RESUMO

Fibroblasts of a patient with Infantile Liver Failure Syndrome 2 (OMIM #616483) due to a homozygous missense variant in the neuroblastoma amplified sequence gene (NBAS; c.[2708T>G]; c.[2708T>G]/p.[Leu903Arg]; p.[Leu903Arg]) were reprogrammed to iPSCs using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit (Invitrogen) delivering the reprogramming factors Oct3/4, Sox2, c-Myc and Klf4. Cells showed a normal karyotype. Pluripotency of DHMCi004-A was proven using immunohistochemistry, RT-PCR analysis, flow cytometry and differentiation into all three germ layers using the STEMdiff™ Trilineage Differentiation Kit (Stemcell Technologies). DHMCi004-A represents the first iPS-based cell model system to elucidate the pathomechanism underlying this disease.


Assuntos
Linhagem Celular , Doenças Genéticas Inatas , Células-Tronco Pluripotentes Induzidas , Falência Hepática , Mutação de Sentido Incorreto , Proteínas de Neoplasias , Pré-Escolar , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Doenças Genéticas Inatas/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Falência Hepática/genética , Falência Hepática/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Síndrome
9.
Genes Chromosomes Cancer ; 58(3): 139-148, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30614587

RESUMO

Chromosomal rearrangements involving one donor chromosome and two or more recipient chromosomes are called jumping translocations. To date only few cases of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with jumping translocations have been described and the underlying mechanisms remain unclear. Here, we analyzed 11 AML and 5 MDS cases with jumping translocations. The cases were analyzed by karyotyping, FISH, telomere length measurement, and next-generation sequencing with an AML/MDS gene panel. Cases with jumping translocations showed significantly (P < .01) shorter telomeres in comparison to healthy age-matched controls. Additional neo-telomeres were found in two cases. In total, eight cases showed recipient chromosomes with a breakpoint in the centromeric region all of them harboring a pathogenic variant in the TP53 gene (n = 6) and/or a loss of TP53 (n = 5). By contrast, no pathogenic variant or loss of TP53 was identified in the six cases showing recipient chromosomes with a breakpoint in the telomeric region. In conclusion, our results divide the cohort of AML and MDS cases with jumping translocations into two groups: the first group with a telomeric breakpoint of the recipient chromosome is characterized by short telomeres and a possibly telomere-based mechanism of chromosomal instability formation. The second group with a centromeric breakpoint of the recipient chromosome is defined by mutation and/or loss of TP53. We, therefore, assume that both critically short telomeres as well as pathogenic variants of TP53 influence jumping translocation formation.


Assuntos
Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Encurtamento do Telômero , Translocação Genética , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Pontos de Quebra do Cromossomo , Feminino , Humanos , Lactente , Cariótipo , Masculino , Pessoa de Meia-Idade , Mutação
10.
Int J Mol Sci ; 19(10)2018 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-30347879

RESUMO

Myelodysplastic syndrome (MDS) can easily transform into acute myeloid leukemia (AML), a process which is often associated with clonal evolution and development of complex karyotypes. Deletion of 5q (del(5q)) is the most frequent aberration in complex karyotypes. This prompted us to analyze clonal evolution in MDS patients with del(5q). There were 1684 patients with low and intermediate-risk MDS and del(5q) with or without one additional cytogenetic abnormality, who were investigated cytogenetically in our department, involving standard karyotyping, fluorescence in situ hybridization (FISH) and multicolor FISH. We identified 134 patients (8%) with aspects of clonal evolution. There are two main routes of cytogenetic clonal evolution: a stepwise accumulation of cytogenetic events over time and a catastrophic event, which we defined as the occurrence of two or more aberrations present at the same time, leading to a sudden development of highly complex clones. Of the 134 patients, 61% underwent a stepwise accumulation of events whereas 39% displayed a catastrophic event. Patients with isolated del(5q) showed significantly more often a stepwise accumulation of events rather than a catastrophic event. The most frequent aberrations in the group of stepwise accumulation were trisomy 8 and trisomy 21 which were significantly more frequent in this group compared to the catastrophic event group. In the group with catastrophic events, del(7q)/-7 and del(17p)/-17 were the most common aberrations. A loss of 17p, containing the tumor suppressor gene TP53, was found significantly more frequent in this group compared to the group of stepwise accumulation. This leads to the assumption that the loss of TP53 is the driving force in patients with del(5q) who undergo a sudden catastrophic event and evolve into complex karyotypes.

12.
Blood ; 132(16): 1703-1713, 2018 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-30190321

RESUMO

Molecular measurable residual disease (MRD) assessment is not established in approximately 60% of acute myeloid leukemia (AML) patients because of the lack of suitable markers for quantitative real-time polymerase chain reaction. To overcome this limitation, we established an error-corrected next-generation sequencing (NGS) MRD approach that can be applied to any somatic gene mutation. The clinical significance of this approach was evaluated in 116 AML patients undergoing allogeneic hematopoietic cell transplantation (alloHCT) in complete morphologic remission (CR). Targeted resequencing at the time of diagnosis identified a suitable mutation in 93% of the patients, covering 24 different genes. MRD was measured in CR samples from peripheral blood or bone marrow before alloHCT and identified 12 patients with persistence of an ancestral clone (variant allele frequency [VAF] >5%). The remaining 96 patients formed the final cohort of which 45% were MRD+ (median VAF, 0.33%; range, 0.016%-4.91%). In competing risk analysis, cumulative incidence of relapse (CIR) was higher in MRD+ than in MRD- patients (hazard ratio [HR], 5.58; P < .001; 5-year CIR, 66% vs 17%), whereas nonrelapse mortality was not significantly different (HR, 0.60; P = .47). In multivariate analysis, MRD positivity was an independent negative predictor of CIR (HR, 5.68; P < .001), in addition to FLT3-ITD and NPM1 mutation status at the time of diagnosis, and of overall survival (HR, 3.0; P = .004), in addition to conditioning regimen and TP53 and KRAS mutation status. In conclusion, NGS-based MRD is widely applicable to AML patients, is highly predictive of relapse and survival, and may help refine transplantation and posttransplantation management in AML patients.

13.
Stem Cell Reports ; 11(3): 696-710, 2018 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-30100408

RESUMO

Induced pluripotent stem cell (iPSC)-derived hematopoietic cells represent a highly attractive source for cell and gene therapy. Given the longevity, plasticity, and self-renewal potential of distinct macrophage subpopulations, iPSC-derived macrophages (iPSC-Mφ) appear of particular interest in this context. We here evaluated the airway residence, plasticity, and therapeutic efficacy of iPSC-Mφ in a murine model of hereditary pulmonary alveolar proteinosis (herPAP). We demonstrate that single pulmonary macrophage transplantation (PMT) of 2.5-4 × 106 iPSC-Mφ yields efficient airway residence with conversion of iPSC-Mφ to an alveolar macrophage (AMφ) phenotype characterized by a distinct surface marker and gene expression profile within 2 months. Moreover, PMT significantly improves alveolar protein deposition and other critical herPAP disease parameters. Thus, our data indicate iPSC-Mφ as a source of functional macrophages displaying substantial plasticity and therapeutic potential that upon pulmonary transplantation will integrate into the lung microenvironment, adopt an AMφ phenotype and gene expression pattern, and profoundly ameliorate pulmonary disease phenotypes.

14.
Oncotarget ; 9(52): 29869-29876, 2018 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-30042819

RESUMO

TP53 deficiencies characterize myeloid malignancies with a dismal prognosis. To unravel the pathomechanism of TP53 mutations in the development of myeloid malignancies, we analyzed the functional properties of TP53 conformational and contact mutations and TP53 loss in human CD34+ cells. We show for the first time that the analyzed conformational mutations lead to higher cell viability in human hematopoietic stem progenitor cells. In contrast to these conformational mutations, contact mutations interfered with efficient erythropoiesis. These findings show that not only the detection of a TP53 mutation is important, but also the specific mutation may play a role in malignant transformation and progression.

15.
Stem Cell Res ; 30: 117-121, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29852468

RESUMO

Caudal-type homeobox 2 (CDX2) transcription factor is an important marker for early trophoblast lineages and intestinal epithelium. Due to its nuclear expression the immunostaining and sorting of viable CDX2pos cells is not possible. In this paper we report the generation and describe key characteristics of a CDX2Venus knock-in reporter hiPSC-cell line (MHHi007-A-1) which can serve as an in vitro tool to study human trophoblast and intestinal differentiation.

16.
Leukemia ; 32(7): 1657-1669, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29925908

RESUMO

A total of 156 patients (age range 1.3-18.0 years, median 13.2 years; 91 (58.3%) male) with newly diagnosed CML (N = 146 chronic phase (CML-CP), N = 3 accelerated phase (CML-AP), N = 7 blastic phase (CML-BP)) received imatinib up-front (300, 400, 500 mg/m2, respectively) within a prospective phase III trial. Therapy response, progression-free survival, causes of treatment failure, and side effects were analyzed in 148 children and adolescents with complete data. Event-free survival rate by 18 months for patients in CML-CP (median follow-up time 25 months, range: 1-120) was 97% (95% CI, 94.2-99.9%). According to the 2006 ELN-criteria complete hematologic response by month 3, complete cytogenetic response (CCyR) by month 12, and major molecular response (MMR) by month 18 were achieved in 98, 63, and 59% of the patients, respectively. By month 36, 86% of the patients achieved CCyR and 74% achieved MMR. Thirty-eight patients (27%) experienced imatinib failure because of unsatisfactory response or intolerance (N = 9). In all, 28/148 patients (19%) underwent stem cell transplantation (SCT). In the SCT sub-cohort 2/23 patients diagnosed in CML-CP, 0/1 in CML-AP, and 2/4 in CML-BP, respectively, died of relapse (N = 3) or SCT-related complications (N = 2). This large pediatric trial extends and confirms data from smaller series that first-line imatinib in children is highly effective.

17.
Artigo em Inglês | MEDLINE | ID: mdl-29720704

RESUMO

Conventional analysis of host chimerism (HC) frequently fails to detect relapse before its clinical manifestation in patients with hematological malignancies after allogeneic stem cell transplantation (allo-SCT). Quantitative PCR (qPCR)-based highly-sensitive chimerism analysis extends the detection limit of conventional (short tandem repeats-based) chimerism analysis from 1 to 0.01% host cells in whole blood. To date, the diagnostic value of highly-sensitive chimerism analysis is hardly defined. Here, we applied qPCR-based chimerism analysis to 901 blood samples of 71 out-patients with hematological malignancies after allo-SCT. Receiver operating characteristics (ROC) curves were calculated for absolute HC values and for the increments of HC before relapse. Using the best cut-offs, relapse was detected with sensitivities of 74 or 85% and specificities of 69 or 75%, respectively. Positive predictive values (PPVs) were only 12 or 18%, but the respective negative predictive values were 98 or 99%. Relapse was detected median 38 or 45 days prior to clinical diagnosis, respectively. Considering also durations of steadily increasing HC of more than 28 days improved PPVs to more than 28 or 59%, respectively. Overall, highly-sensitive chimerism analysis excludes relapses with high certainty and predicts relapses with high sensitivity and specificity more than a month prior to clinical diagnosis.

18.
Leukemia ; 32(7): 1621-1630, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29720733

RESUMO

In this phase Ib/IIa study (ClinicalTrials.gov Identifier: NCT00850382) of the German-Austrian AML Study Group (AMLSG) the multikinase inhibitor dasatinib was added to intensive induction and consolidation chemotherapy and administered as single agent for 1-year maintenance in first-line treatment of adult patients with core-binding factor (CBF) acute myeloid leukemia (AML). The primary combined end point in this study was safety and feasibility, and included the rates of early (ED) and hypoplastic (HD) deaths, pleural/pericardial effusion 3°/4° and liver toxicity 3°/4°, and the rate of refractory disease. Secondary end points were cumulative incidence of relapse (CIR) and death in complete remission (CID), and overall survival (OS). Eighty-nine pts [median age 49.5 years, range: 19-73 years; t(8;21), n = 37; inv (16), n = 52] were included. No unexpected excess in toxicity was observed. The rates of ED/HD and CR/CRi were 4.5% (4/89) and 94% (84/89), respectively. The 4-year estimated CIR, CID, and OS were 33.1% [95%-CI (confidence interval), 22.7-43.4%], 6.0% (95% CI, 0.9-11.2%), and 74.7% (95% CI, 66.1-84.5%), respectively. On the basis of the acceptable toxicity profile and favorable outcome in the AMLSG 11-08 trial, a confirmatory randomized phase III trial with dasatinib in adults with CBF-AML is ongoing (ClinicalTrials.gov Identifier: NCT02013648).

19.
Stem Cell Reports ; 10(5): 1657-1672, 2018 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-29681541

RESUMO

Endothelial cells (ECs) are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor) cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs) represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability.

20.
Am J Respir Crit Care Med ; 198(3): 350-360, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29652170

RESUMO

RATIONALE: Although the transplantation of induced pluripotent stem cell (iPSC)-derived cells harbors enormous potential for the treatment of pulmonary diseases, in vivo data demonstrating clear therapeutic benefits of human iPSC-derived cells in lung disease models are missing. OBJECTIVES: We have tested the therapeutic potential of iPSC-derived macrophages in a humanized disease model of hereditary pulmonary alveolar proteinosis (PAP). Hereditary PAP is caused by a genetic defect of the GM-CSF (granulocyte-macrophage colony-stimulating factor) receptor, which leads to disturbed macrophage differentiation and protein/surfactant degradation in the lungs, subsequently resulting in severe respiratory insufficiency. METHODS: Macrophages derived from human iPSCs underwent intrapulmonary transplantation into humanized PAP mice, and engraftment, in vivo differentiation, and therapeutic efficacy of the transplanted cells were analyzed. MEASUREMENTS AND MAIN RESULTS: On intratracheal application, iPSC-derived macrophages engrafted in the lungs of humanized PAP mice. After 2 months, transplanted cells displayed the typical morphology, surface markers, functionality, and transcription profile of primary human alveolar macrophages. Alveolar proteinosis was significantly reduced as demonstrated by diminished protein content and surfactant protein D levels, decreased turbidity of the BAL fluid, and reduced surfactant deposition in the lungs of transplanted mice. CONCLUSIONS: We here demonstrate for the first time that pulmonary transplantation of human iPSC-derived macrophages leads to pulmonary engraftment, their in situ differentiation to an alveolar macrophage phenotype, and a reduction of alveolar proteinosis in a humanized PAP model. To our knowledge, this finding presents the first proof-of-concept for the therapeutic potential of human iPSC-derived cells in a pulmonary disease and may have profound implications beyond the rare disease of PAP.

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