Nina Graffmann

Induction of pluripotency in human cord blood unrestricted somatic stem cells

OBJECTIVE Generation of induced pluripotent stem (iPS) cells from human cord blood (CB)-derived unrestricted somatic stem cells and evaluation of their molecular signature and differentiation potential in comparison to human embryonic stem cells. MATERIALS AND METHODS Unrestricted somatic stem cells isolated from human CB were reprogrammed to iPS cells using retroviral expression of the transcription factors OCT4, SOX2, KLF4, and C-MYC. The reprogrammed cells were analyzed morphologically, by quantitative reverse transcription polymerase chain reaction, genome-wide microRNA and methylation profiling, and gene expression microarrays, as well as in their pluripotency potential by in vivo teratoma formation in severe combined immunodeficient mice and in vitro differentiation. RESULTS CB iPS cells are very similar to human embryonic stem cells morphologically, at their molecular signature, and in their differentiation potential. CONCLUSIONS Human CB-derived unrestricted somatic stem cells offer an attractive source of cells for generation of iPS cells. Our findings open novel perspectives to generate human leukocyte antigen-matched pluripotent stem cell banks based on existing CB banks. Besides the obvious relevance of a second-generation CB iPS cell bank for pharmacological and toxicological testing, its application for autologous or allogenic regenerative cell transplantation appears feasible.

Lineage-Specific Transition of Histone Signatures in the Killer Cell Ig-Like Receptor Locus from Hematopoietic Progenitor to NK Cells

The clonal distribution and stable expression of killer cell Ig-like receptor (KIR) genes is epigenetically regulated. To assess the epigenetic changes that occur during hemopoietic development we examined DNA methylation and chromatin structure of the KIR locus in early hemopoietic progenitor cells and major lymphocyte lineages. In hemopoietic progenitor cells, KIR genes exhibited the major hallmarks of epigenetic repression, which are dense DNA methylation, inaccessibility of chromatin to Micrococcus nuclease digest, and a repressive histone signature, characterized by strong H3K9 dimethylation and reduced H4K8 acetylation. In contrast, KIR genes of NK cells showed active histone signatures characterized by absence of H3K9 dimethylation and presence of H4K8 acetylation. Histone modifications correlated well with the competence of different lymphocyte lineages to express KIR; whereas H4K8 acetylation was high in NK and CD8+ T cells, it was almost absent in CD4+ T cells and B cells and, in the latter case, replaced by H3K9 dimethylation. In KIR-competent lineages, active histone signatures were also observed in silent KIR genes and in this case found in combination with dense DNA methylation of the promoter and nearby regions. The study suggests a two-step model of epigenetic regulation in which lineage-specific acquisition of euchromatic histone marks is a prerequisite for subsequent gene-specific DNA demethylation and expression of KIR genes.

MicroRNA-15b regulates mitochondrial ROS production and the senescence-associated secretory phenotype through sirtuin 4/SIRT4

Mammalian sirtuins are involved in the control of metabolism and life-span regulation. Here, we link the mitochondrial sirtuin SIRT4 with cellular senescence, skin aging, and mitochondrial dysfunction. SIRT4 expression significantly increased in human dermal fibroblasts undergoing replicative or stress-induced senescence triggered by UVB or gamma-irradiation. In-vivo, SIRT4 mRNA levels were upregulated in photoaged vs. non-photoaged human skin. Interestingly, in all models of cellular senescence and in photoaged skin, upregulation of SIRT4 expression was associated with decreased levels of miR-15b. The latter was causally linked to increased SIRT4 expression because miR-15b targets a functional binding site in the SIRT4 gene and transfection of oligonucleotides mimicking miR-15b function prevented SIRT4 upregulation in senescent cells. Importantly, increased SIRT4 negatively impacted on mitochondrial functions and contributed to the development of a senescent phenotype. Accordingly, we observed that inhibition of miR-15b, in a SIRT4-dependent manner, increased generation of mitochondrial reactive oxygen species, decreased mitochondrial membrane potential, and modulated mRNA levels of nuclear encoded mitochondrial genes and components of the senescence-associated secretory phenotype (SASP). Thus, miR-15b is a negative regulator of stress-induced SIRT4 expression thereby counteracting senescence associated mitochondrial dysfunction and regulating the SASP and possibly organ aging, such as photoaging of human skin.

Histone methyltransferase enhancer of zeste homolog 2 regulates Schwann cell differentiation.

Epigenetic control is crucial for the differentiation of a variety of cells including oligodendrocytes, the myelinating glial cells of the central nervous system. However, studies about the implication of epigenetic factors in peripheral nervous system maturation are just emerging. Here, we demonstrate for the first time the impact of a histone methyltransferase, encoded by the enhancer of zeste homolog 2 (EZH2) gene, on Schwann cell differentiation. In sciatic nerves, EZH2 expression was found in Schwann cells and to peak perinatally. Suppression of EZH2 expression in cultured primary rat Schwann cells reduced the length of cell processes. These morphological changes were accompanied by widespread alterations in the gene expression pattern, including downregulation of myelin genes and induction of p57kip2, which we have recently identified as an intrinsic inhibitory regulator of Schwann cell maturation. In addition, we show that EZH2 suppression in dorsal root ganglion cocultures interferes with in vitro myelination. Chromatin immunoprecipitation analysis revealed binding of EZH2 at the p57kip2 promoter and reduction of histone H3K27 trimethylation upon gene suppression. EZH2 suppression‐dependent effects on morphology and myelin genes could be reversed by concomitant suppression of p57kip2, indicating that p57kip2 is a downstream effector of EZH2. Furthermore, we describe Hes5 as transcriptional repressor of myelin genes in Schwann cells, which was induced upon EZH2 suppression and downregulated in p57kip2‐suppressed Schwann cells. Therefore, we have identified a molecular link between histone methylation and control of Schwann cell differentiation and demonstrate that this epigenetic mechanism is crucial for glial differentiation to proceed.

Modeling Nonalcoholic Fatty Liver Disease with Human Pluripotent Stem Cell-Derived Immature Hepatocyte-Like Cells Reveals Activation of PLIN2 and Confirms Regulatory Functions of Peroxisome Proliferator-Activated Receptor Alpha

Nonalcoholic fatty liver disease (NAFLD/steatosis) is a metabolic disease characterized by the incorporation of fat into hepatocytes. In this study, we developed an in vitro model for NAFLD based on hepatocyte-like cells (HLCs) differentiated from human pluripotent stem cells. We induced fat storage in these HLCs and detected major expression changes of metabolism-associated genes, as well as an overall reduction of liver-related microRNAs. We observed an upregulation of the lipid droplet coating protein Perilipin 2 (PLIN2), as well as of numerous genes of the peroxisome proliferator-activated receptor (PPAR) pathway, which constitutes a regulatory hub for metabolic processes. Interference with PLIN2 and PPARα resulted in major alterations in gene expression, especially affecting lipid, glucose, and purine metabolism. Our model recapitulates many metabolic changes that are characteristic for NAFLD. It permits the dissection of disease-promoting molecular pathways and allows us to investigate the influences of distinct genetic backgrounds on disease progression.

Concise Review: Current Status and Future Directions on Research Related to Nonalcoholic Fatty Liver Disease

Considered a feature of the metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), is associated with insulin resistance, type 2 diabetes, obesity and drug toxicity. Its prevalence is estimated at about 30% in western countries mainly due to sedentary life styles and high fat diets. Genome‐wide association studies have identified polymorphisms in several genes, for example, PNPLA3, and TM6SF2 which confer susceptibility to NAFLD. Here, we review recent findings in the NAFLD field with a particular focus on published transcriptomics datasets which we subject to a meta‐analysis. We reveal a common gene signature correlating with the progression of the disease from steatosis and steatohepatitis and reveal that lipogenic and cholesterol metabolic pathways are main actors in this signature. We propose the use of disease‐in‐a‐dish models based on hepatocyte‐like cells derived from patient‐specific induced pluripotent stem cells (iPSC). These will enable investigations into the contribution of genetic background in the progression from NALFD to non‐alcoholic steatohepatitis. Furthermore, an iPSC‐based approach should aid in the elucidation of the function of new biomarkers, thus enabling better diagnostic tests and validation of potential drug targets. Stem Cells 2017;35:89–96

Current Status and Future Directions on Research Related to Nonalcoholic Fatty Liver Disease.

Considered a feature of the metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), is associated with insulin resistance, type 2 diabetes, obesity and drug toxicity. Its prevalence is estimated at about 30% in western countries mainly due to sedentary life styles and high fat diets. Genome‐wide association studies have identified polymorphisms in several genes, for example, PNPLA3, and TM6SF2 which confer susceptibility to NAFLD. Here, we review recent findings in the NAFLD field with a particular focus on published transcriptomics datasets which we subject to a meta‐analysis. We reveal a common gene signature correlating with the progression of the disease from steatosis and steatohepatitis and reveal that lipogenic and cholesterol metabolic pathways are main actors in this signature. We propose the use of disease‐in‐a‐dish models based on hepatocyte‐like cells derived from patient‐specific induced pluripotent stem cells (iPSC). These will enable investigations into the contribution of genetic background in the progression from NALFD to non‐alcoholic steatohepatitis. Furthermore, an iPSC‐based approach should aid in the elucidation of the function of new biomarkers, thus enabling better diagnostic tests and validation of potential drug targets. Stem Cells 2017;35:89–96

The presence of human mesenchymal stem cells of renal origin in amniotic fluid increases with gestational time

BackgroundEstablished therapies for managing kidney dysfunction such as kidney dialysis and transplantation are limited due to the shortage of compatible donated organs and high costs. Stem cell-based therapies are currently under investigation as an alternative treatment option. As amniotic fluid is composed of fetal urine harboring mesenchymal stem cells (AF-MSCs), we hypothesized that third-trimester amniotic fluid could be a novel source of renal progenitor and differentiated cells.MethodsHuman third-trimester amniotic fluid cells (AFCs) were isolated and cultured in distinct media. These cells were characterized as renal progenitor cells with respect to cell morphology, cell surface marker expression, transcriptome and differentiation into chondrocytes, osteoblasts and adipocytes. To test for renal function, a comparative albumin endocytosis assay was performed using AF-MSCs and commercially available renal cells derived from kidney biopsies. Comparative transcriptome analyses of first, second and third trimester-derived AF-MSCs were conducted to monitor expression of renal-related genes.ResultsRegardless of the media used, AFCs showed expression of pluripotency-associated markers such as SSEA4, TRA-1-60, TRA-1-81 and C-Kit. They also express the mesenchymal marker Vimentin. Immunophenotyping confirmed that third-trimester AFCs are bona fide MSCs. AF-MSCs expressed the master renal progenitor markers SIX2 and CITED1, in addition to typical renal proteins such as PODXL, LHX1, BRN1 and PAX8. Albumin endocytosis assays demonstrated the functionality of AF-MSCs as renal cells. Additionally, upregulated expression of BMP7 and downregulation of WT1, CD133, SIX2 and C-Kit were observed upon activation of WNT signaling by treatment with the GSK-3 inhibitor CHIR99201. Transcriptome analysis and semiquantitative PCR revealed increasing expression levels of renal-specific genes (e.g., SALL1, HNF4B, SIX2) with gestational time. Moreover, AF-MSCs shared more genes with human kidney cells than with native MSCs and gene ontology terms revealed involvement of biological processes associated with kidney morphogenesis.ConclusionsThird-trimester amniotic fluid contains AF-MSCs of renal origin and this novel source of kidney progenitors may have enormous future potentials for disease modeling, renal repair and drug screening.

Pluripotent stem cells: induction and self-renewal

Pluripotent stem cells (PSCs) lie at the heart of modern regenerative medicine due to their properties of unlimited self-renewal in vitro and their ability to differentiate into cell types representative of the three embryonic germ layers—mesoderm, ectoderm and endoderm. The derivation of induced PSCs bypasses ethical concerns associated with the use of human embryonic stem cells and also enables personalized cell-based therapies. To exploit their regenerative potential, it is essential to have a firm understanding of the molecular processes associated with their induction from somatic cells. This understanding serves two purposes: first, to enable efficient, reliable and cost-effective production of excellent quality induced PSCs and, second, to enable the derivation of safe, good manufacturing practice-grade transplantable donor cells. Here, we review the reprogramming process of somatic cells into induced PSCs and associated mechanisms with emphasis on self-renewal, epigenetic control, mitochondrial bioenergetics, sub-states of pluripotency, naive ground state, naive and primed. A meta-analysis identified genes expressed exclusively in the inner cell mass and in the naive but not in the primed pluripotent state. We propose these as additional biomarkers defining naive PSCs. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

Characterization of dermal fibroblast-derived iPSCs from a patient with high grade steatosis

Primary fibroblasts from a low grade steatosis patient were reprogrammed by transduction of a combination of two episomal-based plasmids OCT4,SOX2, c-MYC and KLF4. iPSCs were characterized by immunocytochemistry, embryonic body-formation, DNA-fingerprint karyotype analysis and comparative transcriptome analyses with the human embryonic stem cell line H1 revealed a Pearsons correlation of 0.9251.