Induced Pluripotent Stem Cells to Model Juvenile Myelomonocytic Leukemia: New Perspectives for Preclinical Research.

Juvenile myelomonocytic leukemia (JMML) is a malignant myeloproliferative disorder arising in infants and young children. The origin of this neoplasm is attributed to an early deregulation of the Ras signaling pathway in multipotent hematopoietic stem/progenitor cells. Since JMML is notoriously refractory to conventional cytostatic therapy, allogeneic hematopoietic stem cell transplantation remains the mainstay of curative therapy for most cases. However, alternative therapeutic approaches with small epigenetic molecules have recently entered the stage and show surprising efficacy at least in specific subsets of patients. Hence, the establishment of preclinical models to test novel agents is a priority. Induced pluripotent stem cells (IPSCs) offer an opportunity to imitate JMML ex vivo, after attempts to generate immortalized cell lines from primary JMML material have largely failed in the past. Several research groups have previously generated patient-derived JMML IPSCs and successfully differentiated these into myeloid cells with extensive phenotypic similarities to primary JMML cells. With infinite self-renewal and the capability to differentiate into multiple cell types, JMML IPSCs are a promising resource to advance the development of treatment modalities targeting specific vulnerabilities. This review discusses current reprogramming techniques for JMML stem/progenitor cells, related clinical applications, and the challenges involved.

Author Correction: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis.

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Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis.

Urothelial carcinoma (UC), the most common cancer of the urinary bladder causes severe morbidity and mortality, e.g. about 40.000 deaths in the EU annually, and incurs considerable costs for the health system due to the need for prolonged treatments and long-term monitoring. Extensive aberrant  DNA methylation is described to prevail in urothelial carcinoma and is thought to contribute to genetic instability, altered gene expression and tumor progression. However, it is unknown how this epigenetic alteration arises during carcinogenesis. Intact methyl group metabolism is required to ensure maintenance of cell-type specific methylomes and thereby genetic integrity and proper cellular function. Here, using two independent techniques for detecting DNA methylation, we observed DNA hypermethylation of the 5'-regulatory regions of the key methyl group metabolism genes ODC1, AHCY and MTHFR in early urothelial carcinoma. These hypermethylation events are associated with genome-wide DNA hypomethylation which is commonly associated with genetic instability. We therefore infer that hypermethylation of methyl group metabolism genes acts in a feed-forward cycle to promote additional DNA methylation changes and suggest a new hypothesis on the molecular etiology of urothelial carcinoma.

Protective vaccination and blood-stage malaria modify DNA methylation of gene promoters in the liver of Balb/c mice.

Epigenetic mechanisms such as DNA methylation are increasingly recognized to be critical for vaccination efficacy and outcome of different infectious diseases, but corresponding information is scarcely available for host defense against malaria. In the experimental blood-stage malaria Plasmodium chabaudi, we investigate the possible effects of a blood-stage vaccine on DNA methylation of gene promoters in the liver, known as effector against blood-stage malaria, using DNA methylation microarrays. Naturally susceptible Balb/c mice acquire, by protective vaccination, the potency to survive P. chabaudi malaria and, concomitantly, modifications of constitutive DNA methylation of promoters of numerous genes in the liver; specifically, promoters of 256 genes are hyper(=up)- and 345 genes are hypo(=down)-methylated (p < 0.05). Protective vaccination also leads to changes in promoter DNA methylation upon challenge with P. chabaudi at peak parasitemia on day 8 post infection (p.i.), when 571 and 1013 gene promoters are up- and down-methylated, respectively, in relation to constitutive DNA methylation (p < 0.05). Gene set enrichment analyses reveal that both vaccination and P. chabaudi infections mainly modify promoters of those genes which are most statistically enriched with functions relating to regulation of transcription. Genes with down-methylated promoters encompass those encoding CX3CL1, GP130, and GATA2, known to be involved in monocyte recruitment, IL-6 trans-signaling, and onset of erythropoiesis, respectively. Our data suggest that vaccination may epigenetically improve parts of several effector functions of the liver against blood-stage malaria, as, e.g., recruitment of monocyte/macrophage to the liver accelerated liver regeneration and extramedullary hepatic erythropoiesis, thus leading to self-healing of otherwise lethal P. chabaudi blood-stage malaria.

IDLN-MSP: Idiolocal normalization of real-time methylation-specific PCR for genetic imbalanced DNA specimens.

Sensitive, accurate, and reliable measurements of tumor cell-specific DNA methylation changes are of fundamental importance in cancer diagnosis, prognosis, and monitoring. Real-time methylation-specific PCR (MSP) using intercalating dyes is an established method of choice for this purpose. Here we present a simple but crucial adaptation of this widely applied method that overcomes a major obstacle: genetic abnormalities in the DNA samples, such as aneuploidy or copy number variations, that could result in inaccurate results due to improper normalization if the copy numbers of the target and reference sequences are not the same. In our idiolocal normalization (IDLN) method, the locus for the normalizing, methylation-independent reference amplification is chosen close to the locus of the methylation-dependent target amplification. This ensures that the copy numbers of both the target and reference sequences will be identical in most cases if they are close enough to each other, resulting in accurate normalization and reliable comparative measurements of DNA methylation in clinical samples when using real-time MSP.

Effect on Multipotency and Phenotypic Transition of Unrestricted Somatic Stem Cells from Human Umbilical Cord Blood after Treatment with Epigenetic Agents.

The epigenetic mechanism of DNA methylation is of central importance for cellular differentiation processes. Unrestricted somatic stem cells (USSCs) from human umbilical cord blood, which have a broad differentiation spectrum, reside in an uncommitted epigenetic state with partial methylation of the regulatory region of the gene coding for the pluripotency master regulator OCT4. Thus we hypothesized that further opening of this "poised" epigenetic state could broaden the differentiation potential of USSCs. Here we document that USSCs drastically change their phenotype after treatment by a new elaborated cultivation protocol which utilizes the DNA hypomethylating compound 5'-aza-2-deoxycytidine (5-Aza-CdR) and the histone deacetylase inhibitor trichostatin A (TSA). This treatment leads to a new stable, spheroid-forming cell type which we have named SpheUSSC. These cells can be stably propagated over at least 150 cell divisions, express OCT4, retain the potential to undergo osteogenic differentiation, and have additionally acquired the ability to uniformly differentiate into adipocytes, unlike the source USSC population. Here we describe our treatment protocol and provide evidence that it induces a dedifferentiation step and concomitantly the acquisition of an extended differentiation capability of the new SpheUSSC type.

Epigenetic modifications of gene promoter DNA in the liver of adult female mice masculinized by testosterone.

Testosterone (T) is known to masculinize the female phenotype of the liver, evidenced as up- and down-regulated expressions of male- and female-predominant genes, respectively, involved in hepatic metabolism. This study is aimed at identifying epigenetic modifications of promoters of these differently expressed genes in the liver after masculinization by T of adult female C57BL/6 mice using methylated DNA immunoprecipitation and NimbleGen microarrays. Among the 17,354 promoters examined, 82 promoters in the liver have been identified to be significantly changed by T (p<0.05), with 47 and 35 promoters exhibiting increased and decreased DNA methylation, respectively. Most of these promoters display the changes of DNA methylation in their Ups-regions, which are between +500 and +2000 bp upstream from the transcription start site (TSS) of the genes. Less T-induced modifications have been detected in the Cor-regions of the promoters, i.e., +500 to -500 bp around the TSS. Only 13 and 7 Cor-promoters are hyper- and hypo-methylated, respectively, among which are 10 hyper- and 5 hypo-methylated promoters of genes with annotated functions. Surprisingly, the promoters are largely unmethylated in those genes whose expression has been previously found to be permanently deregulated by T in the liver, as e.g. the T-upregulated male-predominant genes Cyp7b1, Cyp2d9, Cyp4a10, Ugt2b1, Ugt2b38, Hsd3b5, Slco1a1 as well as the T-downregulated female-predominant genes Cyp2b9, Cyp2b13, Cyp3a41, Cyp3a44, Fmo3, Sult2a2, respectively. Though methylatable, the promoter DNA of Ar, Esr1, and Esr2 remained unaffected by T. However, T decreases DNA-methylation of the Cor-promoter region of Ddc encoding the AR-coactivator dopa decarboxylase. Among the identified 15 Cor-promoters of genes with annotated functions are also those of Defb43, Cst11, and Sele involved in innate immunity. Our data support the view that T may exert long-lasting epigenetic effects on functions of the liver-inherent immune system.

Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

Origin-dependent neural cell identities in differentiated human iPSCs in vitro and after transplantation into the mouse brain.

The differentiation capability of induced pluripotent stem cells (iPSCs) toward certain cell types for disease modeling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from fetal neural stem cells (fNSCs), dermal fibroblasts, or cord blood CD34(+) hematopoietic progenitor cells. Neural progenitor cells (NPCs) and neurons could be generated at similar efficiencies from all iPSCs. Transcriptomics analysis of the whole genome and of neural genes revealed a separation of neuroectoderm-derived iPSC-NPCs from mesoderm-derived iPSC-NPCs. Furthermore, we found genes that were similarly expressed in fNSCs and neuroectoderm, but not in mesoderm-derived iPSC-NPCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival of neuroectoderm-derived cells in vivo. These results indicate distinct origin-dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo.

Unreserved application of epigenetic methods to define differences of DNA methylation between urinary cellular and cell-free DNA.

Urinary DNA is increasingly gaining importance in diagnosis of urological malignancies. Especially cell-free DNA originating from apoptotic and necrotic cells of the early tumor could become a key target for early stage tumor diagnosis. Aberrant DNA methylation forms tumor cell characteristic epigenetic profiles which are covalently established before any tumor related aberration at transcriptional or protein level has occurred. In addition, these epigenetic signatures are alterably adapted to and accompanying the individual stages of multistep, progressive tumorigenesis. Hence, they seem very promising for diagnosis as well as for monitoring the patient's follow-up care and even for decisions regarding personalized therapeutic options. The essential prerequisite at this approach will be a reliable methodological handling of the biological material of interest. In this study we present detailed analyses of LINE-1 DNA methylation profiles and demonstrate the sensitive detection of LINE-1 DNA methylation differences as well as between cancer patients and healthy individuals, between urinary cellular and cell-free DNA. In addition, we show methylome differences between both DNA fractions from a healthy individual and bladder cancer patients. In conclusion, we demonstrate here the unrestricted amenability of urinary cell-free DNA for both, a detailed characterization of a distinct DNA methylation alteration and its sensitive detection and a comprehensive global, array-based screening for DNA methylation differences.

Vinblastine-induced apoptosis of melanoma cells is mediated by Ras homologous A protein (Rho A) via mitochondrial and non-mitochondrial-dependent mechanisms.

Despite the availability of melanoma treatment at the primary site, the recurrence of local melanoma can metastasize to any distant organ. Currently, the available therapies for the treatment of metastatic melanoma are of limited benefit. Thus, the functional analysis of conventional therapies may help to improve their efficiency in the treatment of metastatic melanoma. In the present study, the exposure of melanoma cells to vinblastine was found to trigger apoptosis as evidenced by the loss of mitochondrial membrane potential, the release of both cytochrome c and apoptosis inducing factor, activation of caspase-9 and 3, and cleavage of Poly (ADP-ribose)-Polymerase. Also, vinblastine enhances the phosphorylation of Ras homologous protein A, the accumulation of reactive oxygen species, the release of intracellular Ca(2+), as well as the activation of apoptosis signal-regulating kinase 1, c-jun-N-terminal kinase, p38, inhibitor of kappaBα (IκBα) kinase, and inositol requiring enzyme 1α. In addition, vinblastine induces the DNA-binding activities of the transcription factor NF-κB, HSF1, AP-1, and ATF-2, together with the expression of HSP70 and Bax proteins. Moreover, inhibitory experiments addressed a central role for Rho A in the regulation of vinblastine-induced apoptosis of melanoma cells via mitochondrial and non-mitochondrial-dependent mechanisms. In conclusion, the present study addresses for the first time a central role for Rho A in the modulation of vinblastine-induced apoptosis of melanoma cells and thereby provides an insight into the molecular action of vinblastine in melanoma treatment.

Bortezomib/proteasome inhibitor triggers both apoptosis and autophagy-dependent pathways in melanoma cells.

Generally, both endoplasmic reticulum (ER) stress and mitochondrial dysregulation are a potential therapeutic target of anticancer agents including bortezomib. The treatment of melanoma cells with bortezomib was found to induce apoptosis together with the upregulation of Noxa, Mcl-1, and HSP70 proteins, and the cleavage of LC3 and autophagic formation. Also, bortezomib induced ER-stress as evidenced by the increase of intracellular Ca(2+) release. In addition, bortezomib enhanced the phosphorylation of inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), c-jun-N-terminal kinase (JNK) and p38, and the activation of the transcription factors AP-1, ATF-2, Ets-1, and HSF1. Bortezomib-induced mitochondrial dysregulation was associated with the accumulation of reactive oxygen species (ROS), the release of both apoptosis inducing factor (AIF) and cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of Poly (ADP-ribose) polymerase (PARP). The pretreatment of melanoma cells with the inhibitor of caspase-3 (Ac-DEVD-CHO) was found to block bortezomib-induced apoptosis that subsequently led to the increase of autophagic formation. In contrast, the inhibition of ASK1 abrogated bortezomib-induced autophagic formation and increased apoptosis induction. Furthermore, the inhibition of JNK, of HSP70 also increased apoptosis induction without influence of bortezomib-induced autophagic formation. Based on the inhibitory experiments, the treatment with bortezomib triggers the activation of both ER-stress-associated pathways, namely IRE1α-ASK1-p38-ATF-2/ets-1-Mcl-1, and IRE1α-ASK1-JNK-AP-1/HSF1-HSP70 as well as mitochondrial dysregulation-associated pathways, namely ROS-ASK1-JNK-AP-1/HSF1-HS70, and AIF-caspase-3-PARP and Cyt.c, and caspase-9-caspase-3-PARP. Taken together, our data demonstrates for the first time the molecular mechanisms, whereby bortezomib triggers both apoptosis and autophagic formation in melanoma cells.

Unrestricted somatic stem cells (USSC) from human umbilical cord blood display uncommitted epigenetic signatures of the major stem cell pluripotency genes.

Unrestricted somatic stem cells (USSC) from human cord blood display a broad differentiation potential for ectodermal, mesodermal, and endodermal cell types. The molecular basis for these stem cell properties is unclear and unlike embryonic stem cells (ESC) none of the major stem cell factors OCT4, SOX2, and NANOG exhibits significant expression in USSC. Here, we report that these key stem cell genes hold an epigenetic state in between that of an ESC and a terminally differentiated cell type. DNA methylation analysis exhibits partial demethylation of the regulatory region of OCT4 and a demethylated state of the NANOG and SOX2 promoter/enhancer regions. Further genome-wide DNA methylation profiling identified a partially demethylated state of the telomerase gene hTERT. Moreover, none of the pluripotency factors exhibited a repressive histone signature. Notably, SOX2 exhibits a bivalent histone signature consisting of the opposing histone marks dimeH3K4 and trimeH3K27, which is typically found on genes that are "poised" for transcription. Consequently, ectopic expression of OCT4 in USSC led to rapid induction of expression of its known target gene SOX2. Our data suggest that incomplete epigenetic repression and a "poised" epigenetic status of pluripotency genes preserves the USSC potential to be able to react adequately to distinct differentiation and reprogramming cues.