Refine
Has Fulltext
- yes (10)
Is part of the Bibliography
- yes (10)
Document Type
- Journal article (9)
- Doctoral Thesis (1)
Language
- English (10)
Keywords
- DNA methylation (3)
- genomic imprinting (2)
- Autism (1)
- Autism spectrum disorders (1)
- Cartilage Regeneration (1)
- DNA methylation dynamics (1)
- Down syndrome (1)
- Environment (1)
- Epigenetics (1)
- Familial Beckwith-Wiedemann syndrome (1)
Institute
- Institut für Humangenetik (4)
- Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde (3)
- Klinik und Poliklinik für Unfall-, Hand-, Plastische und Wiederherstellungschirurgie (Chirurgische Klinik II) (3)
- Theodor-Boveri-Institut für Biowissenschaften (2)
- Graduate School of Life Sciences (1)
- Institut für Virologie und Immunbiologie (1)
EU-Project number / Contract (GA) number
- 309962 (2)
Using Illumina 450K arrays, 1.85% of all analyzed CpG sites were significantly hypermethylated and 0.31% hypomethylated in fetal Down syndrome (DS) cortex throughout the genome. The methylation changes on chromosome 21 appeared to be balanced between hypo- and hyper-methylation, whereas, consistent with prior reports, all other chromosomes showed 3-11times more hyper- than hypo-methylated sites. Reduced NRSF/REST expression due to upregulation of DYRK1A (on chromosome 21q22.13) and methylation of REST binding sites during early developmental stages may contribute to this genome-wide excess of hypermethylated sites. Upregulation of DNMT3L (on chromosome 21q22.4) could lead to de novo methylation in neuroprogenitors, which then persists in the fetal DS brain where DNMT3A and DNMT3B become downregulated. The vast majority of differentially methylated promoters and genes was hypermethylated in DS and located outside chromosome 21, including the protocadherin gamma (PCDHG) cluster on chromosome 5q31, which is crucial for neural circuit formation in the developing brain. Bisulfite pyrosequencing and targeted RNA sequencing showed that several genes of PCDHG subfamilies A and B are hypermethylated and transcriptionally downregulated in fetal DS cortex. Decreased PCDHG expression is expected to reduce dendrite arborization and growth in cortical neurons. Since constitutive hypermethylation of PCDHG and other genes affects multiple tissues, including blood, it may provide useful biomarkers for DS brain development and pharmacologic targets for therapeutic interventions.
Improved treatment options for the degenerative joint disease osteoarthritis (OA) are of major interest, since OA is one of the main sources of disability, pain, and socioeconomic burden worldwide [202]. According to epidemiological data, already 27 million people suffer from OA in the US [23]. Moreover, the WHO expects OA to be the fourth most common cause of disability in 2020 [203], illustrating the need for effective and long-lasting therapy options of severe cartilage defects. Despite numerous clinically available products for the treatment of cartilage defects [62], the development of more cartilage-specific materials is still at the beginning.
Hyaluronic acid (HA) is a major component of the cartilaginous extracellular matrix (ECM) and inherently creates a cell-friendly niche by providing cell attachment and migration sites. Furthermore, it is known that the functional groups of HA are well suited for chemical modification. These characteristics render HA an attractive material for hydrogel-based tissue engineering approaches. Poly(glycidol) (PG) as chemical crosslinker basically features similar chemical characteristics as the widely used poly(ethylene glycol) (PEG), but provides additional side groups at each repeating unit that can be further chemically functionalized. With the introduction of PG as multifunctional crosslinker for HA gels, a higher cross-linking density and, accordingly, a greater potential for biomimetic functionalization may be achieved. However, despite the mentioned potential benefits, PG has not been used for cartilage regeneration approaches so far.
The initial aim of the study was to set up and optimize a HA-based hydrogel for the chondrogenic differentiation of mesenchymal stromal cells (MSCs), using different amounts and variations of cross-linkers. Therefore, the hydrogel composition was optimized by the utilization of different PEG diacrylate (PEGDA) concentrations to cross-link thiol-modified HA (Glycosil, HA-SH) via Michael addition. We aimed to generate volumestable scaffolds that simultaneously enable a maximum of ECM deposition. Histological and biochemical analysis showed 0.4% PEGDA as the most suitable concentration for these requirements (Section 5.1.2).
In order to evaluate the impact of a differently designed cross-linker on MSC chondrogenesis, HA-SH was cross-linked with PEGTA (0.6%) and compared to PEGDA (0.4%) in a next step. Following this, acrylated PG (PG-Acr) as multifunctional cross-linker alternative to acrylated PEG was evaluated. It provides around five times more functional groups when utilized in PG-Acr (0.6%) HA-SH hydrogels compared to PEGTA (0.6%) HA-SH hydrogels, thus enabling higher degrees of biomimetic functionalization. Determination of cartilage-specific ECM components showed no substantial differences between both cross-linkers while the deposition of cartilaginous matrix appeared more homogeneous in HA-SH PG-Acr gels. Taken together, we were able to successfully increase the possibilities for biomimetic functionalization in the developed HA-SH hydrogel system by the introduction of PG-Acr as cross-linker without negatively affecting MSC chondrogenesis (Section 5.1.3).
The next part of this thesis focused extensively on the biomimetic functionalization of PG-Acr (0.6%) cross-linked HA-SH hydrogels. Here, either biomimetic peptides or a chondrogenic growth factor were covalently bound into the hydrogels.
Interestingly, the incorporation of a N-cadherin mimetic (HAV), a collagen type II binding (KLER), or a cell adhesion-mediating peptide (RGD) yielded no improvement of MSC chondrogenesis. For instance, the covalent binding of 2.5mM HAV changed morphology of cell nuclei and reduced GAG production while the incorporation of 1.0mM RGD impaired collagen production. These findings may be attributed to the already supportive conditions of the employed HA-based hydrogels for chondrogenic differentiation. Most of the previous studies reporting positive peptide effects on chondrogenesis have been carried out in less supportive PEG hydrogels or in significantly stiffer MeHA-based hydrogels [99, 101, 160]. Thus, the incorporation of peptides may be more important under unfavorable conditions while inert gel systems may be useful for studying single peptide effects (Section 5.2.1).
The chondrogenic factor transforming growth factor beta 1 (TGF-b1) served as an example for growth factor binding to PG-Acr. The utilization of covalently bound TGF-b1 may thereby help overcome the need for repeated administration of TGF-b1 in in vivo applications, which may be an advantage for potential clinical application. Thus, the effect of covalently incorporated TGF-b1 was compared to the effect of the same amount of TGF-b1 without covalent binding (100nM TGF-b1) on MSC chondrogenesis. It was successfully demonstrated that covalent incorporation of TGF-b1 had a significant positive effect in a dose-dependent manner. Chondrogenesis of MSCs in hydrogels with covalently bound TGF-b1 showed enhanced levels of chondrogenesis compared to hydrogels into which TGF-b1 was merely mixed, as shown by stronger staining for GAGs, total collagen, aggrecan and collagen type II. Biochemical evaluation of GAG and collagen amounts, as well as Western blot analysis confirmed the histological results. Furthermore, the positive effect of covalently bound TGF-b1 was shown by increased expression of chondrogenic marker genes COL2A1, ACAN and SOX9. In summary, covalent growth factor incorporation utilizing PG-Acr as cross-linker demonstrated significant positive effects on chondrogenic differentiation of MSCs (Section 5.2.2).
In general, PG-Acr cross-linked HA hydrogels generated by Michael addition represent a versatile hydrogel platform due to their high degree of acrylate functionality. These hydrogels may further offer the opportunity to combine several biological modifications, such as the incorporation of biomimetic peptides together with growth factors, within one cell carrier.
A proof-of-principle experiment demonstrated the suitability of pure PG gels for studying single peptide effects. Here, the hydrogels were generated by the utilization of thiol-ene-click reaction. In this setting, without the supportive background of hyaluronic acid, MSCs showed enhanced chondrogenic differentiation in response to the incorporation of 1.0mM HAV. This was demonstrated by staining for GAGs, the cartilage-specific ECM molecules aggrecan and type II collagen, and by increased GAG and total collagen amounts shown by biochemical analysis. Thus, pure PG gels exhibit the potential to study the effects and interplay of peptides and growth factors in a highly modifiable, bioinert hydrogel environment.
The last section of the thesis was carried out as part of the EU project HydroZONES that aims to develop and generate zonal constructs. The importance of zonal organization has attracted increased attention in the last years [127, 128], however, it is still underrepresented in tissue engineering approaches so far. Thus, the feasibility of zonal distribution of cells in a scaffold combining two differently composed hydrogels was investigated. A HA-SH(FMZ) containing bottom layer was generated and a pure PG top layer was subsequently cast on top of it, utilizing both times thiol-ene-click reaction. Indeed, stable, hierarchical constructs were generated that allowed encapsulated MSCs to differentiate chondrogenically in both zones as shown by staining for GAGs and collagen type II, and by quantification of GAG amount. Thus, the feasibility of differently composed zonal hydrogels utilizing PG as a main component was successfully demonstrated (Section 5.4).
With the first-time utilization and evaluation of PG-Acr as versatile multifunctional cross-linker for the preparation of Michael addition-generated HA-SH hydrogels in the context of cartilage tissue engineering, a highly modifiable HA-based hydrogel system was introduced. It may be used in future studies as an easily applicable and versatile toolbox for the generation of biomimetically functionalized hydrogels for cell-based cartilage regeneration. The introduction of reinforcement structures to enhance mechanical resistance may thereby further increase the potential of this system for clinical applications.
Additionally, it was also demonstrated that thiol-ene clickable hydrogels can be used for the generation of cell-laden, pure PG gels or for the generation of more complex, coherent zonal constructs. Furthermore, thiol-ene clickable PG hydrogels have already been further modified and successfully been used in 3D bioprinting experiments [204]. 3D bioprinting, as part of the evolving biofabrication field [205], offers the possibilities to generate complex and hierarchical structures, and to exactly position defined layers, yet at the same time alters the requirements for the utilized hydrogels [159, 206–209]. Since a robust chondrogenesis of MSCs was demonstrated in the thiol-ene clickable hydrogel systems, they may serve as a basis for the development of hydrogels as so called bioinks which may be utilized in more sophisticated biofabrication processes.
Normal human brain development is dependent on highly dynamic epigenetic processes for spatial and temporal gene regulation. Recent work identified wide-spread changes in DNA methylation during fetal brain development. We profiled CpG methylation in frontal cortex of 27 fetuses from gestational weeks 12-42, using Illumina 450K methylation arrays. Sites showing genome-wide significant correlation with gestational age were compared to a publicly available data set from gestational weeks 3-26. Altogether, we identified 2016 matching developmentally regulated differentially methylated positions (m-dDMPs): 1767 m-dDMPs were hypermethylated and 1149 hypomethylated during fetal development. M-dDMPs are underrepresented in CpG islands and gene promoters, and enriched in gene bodies. They appear to cluster in certain chromosome regions. M-dDMPs are significantly enriched in autism-associated genes and CpGs. Our results promote the idea that reduced methylation dynamics during fetal brain development may predispose to autism. In addition, m-dDMPs are enriched in genes with human-specific brain expression patterns and/or histone modifications. Collectively, we defined a subset of dDMPs exhibiting constant methylation changes from early to late pregnancy. The same epigenetic mechanisms involving methylation changes in cis-regulatory regions may have been adopted for human brain evolution and ontogeny.
Hyaluronic acid (HA)-based hydrogels are very commonly applied as cell carriers for different approaches in regenerative medicine. HA itself is a well-studied biomolecule that originates from the physiological extracellular matrix (ECM) of mammalians and, due to its acidic polysaccharide structure, offers many different possibilities for suitable chemical modifications which are necessary to control, for example, network formation. Most of these chemical modifications are performed using the free acid function of the polymer and, additionally, lead to an undesirable breakdown of the biopolymer’s backbone. An alternative modification of the vicinal diol of the glucuronic acid is oxidation with sodium periodate to generate dialdehydes via a ring opening mechanism that can subsequently be further modified or crosslinked via Schiff base chemistry. Since this oxidation causes a structural destruction of the polysaccharide backbone, it was our intention to study a novel synthesis protocol frequently applied to selectively oxidize the C6 hydroxyl group of saccharides. On the basis of this TEMPO/TCC oxidation, we studied an alternative hydrogel platform based on oxidized HA crosslinked using adipic acid dihydrazide as the crosslinker.
The Human Immunodeficiency Virus type 1 (HIV-1) subtype C is currently the predominant subtype worldwide. Cell culture studies of Sub-Saharan African subtype C proviral plasmids are hampered by the low replication capacity of the resulting viruses, although viral loads in subtype C infected patients are as high as those from patients with subtype B. Here, we describe the sequencing and construction of a new HIV-1 subtype C proviral clone (pZAC), replicating more than one order of magnitude better than the previous subtype C plasmids. We identify the env-region for being the determinant for the higher viral titers and the pZAC Env to be M-tropic. This higher replication capacity does not lead to a higher cytotoxicity compared to previously described subtype C viruses. In addition, the pZAC Vpu is also shown to be able to down-regulate CD4, but fails to fully counteract CD317.
The development and formulation of printable inks for extrusion-based 3D bioprinting has been a major challenge in the field of biofabrication. Inks, often polymer solutions with the addition of crosslinking to form hydrogels, must not only display adequate mechanical properties for the chosen application but also show high biocompatibility as well as printability. Here we describe a reproducible two-step method for the assessment of the printability of inks for bioprinting, focussing firstly on screening ink formulations to assess fibre formation and the ability to form 3D constructs before presenting a method for the rheological evaluation of inks to characterise the yield point, shear thinning and recovery behaviour. In conjunction, a mathematical model was formulated to provide a theoretical understanding of the pressure-driven, shear thinning extrusion of inks through needles in a bioprinter. The assessment methods were trialled with a commercially available crème, poloxamer 407, alginate-based inks and an alginate-gelatine composite material. Yield stress was investigated by applying a stress ramp to a number of inks, which demonstrated the necessity of high yield for printable materials. The shear thinning behaviour of the inks was then characterised by quantifying the degree of shear thinning and using the mathematical model to predict the window of printer operating parameters in which the materials could be printed. Furthermore, the model predicted high shear conditions and high residence times for cells at the walls of the needle and effects on cytocompatibility at different printing conditions. Finally, the ability of the materials to recover to their original viscosity after extrusion was examined using rotational recovery rheological measurements. Taken together, these assessment techniques revealed significant insights into the requirements for printable inks and shear conditions present during the extrusion process and allow the rapid and reproducible characterisation of a wide variety of inks for bioprinting.
When aiming at cell‐based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL‐1β need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti‐inflammatory properties. However, long‐term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long‐term model cultures for cell‐based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL‐1β (1–10 ng/ml) or RSV (50 μM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL‐1β (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL‐1β‐treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL‐1β treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL‐1β‐mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long‐term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques.
Imprinted genes show parent-specific activity (functional haploidy), which makes them particularly vulnerable to epigenetic dysregulation. Here we studied the methylation profiles of oppositely imprinted genes at single DNA molecule resolution by two independent parental allele-specific deep bisulfite sequencing (DBS) techniques. Using Roche (GSJunior) next generation sequencing technology, we analyzed the maternally imprinted MEST promoter and the paternally imprinted MEG3 intergenic (IG) differentially methylated region (DMR) in fetal cord blood, adult blood, and visceral adipose tissue. Epimutations were defined as paternal or maternal alleles with >50% aberrantly (de)methylated CpG sites, showing the wrong methylation imprint. The epimutation rates (range 2–66%) of the paternal MEST and the maternal MEG3 IG DMR allele, which should be completely unmethylated, were significantly higher than those (0–15%) of the maternal MEST and paternal MEG3 alleles, which are expected to be fully methylated. This hypermethylation of the non-imprinted allele (HNA) was independent of parental origin. Very low epimutation rates in sperm suggest that HNA occurred after fertilization. DBS with Illumina (MiSeq) technology confirmed HNA for the MEST promoter and the MEG3 IG DMR, and to a lesser extent, for the paternally imprinted secondary MEG3 promoter and the maternally imprinted PEG3 promoter. HNA leads to biallelic methylation of imprinted genes in a considerable proportion of normal body cells (somatic mosaicism) and is highly variable between individuals. We propose that during development and differentiation maintenance of differential methylation at most imprinting control regions may become to some extent redundant. The accumulation of stochastic and environmentally-induced methylation errors on the non-imprinted allele may increase epigenetic diversity between cells and individuals.
Mucin, a high molecular mass hydrophilic glycoprotein, is the main component of mucus that coats every wet epithelium in animals. It is thus intrinsically biocompatible, and with its protein backbone and the o-glycosidic bound oligosaccharides, it contains a plethora of functional groups which can be used for further chemical modifications. Here, chain-growth and step-growth (thiol-ene) free-radical cross-linked hydrogels prepared from commercially available pig gastric mucin (PGM) are introduced and compared as cost-efficient and easily accessible alternative to the more broadly applied bovine submaxillary gland mucin. For this, PGM is functionalized with photoreactive acrylate groups or allyl ether moieties, respectively. Whereas homopolymerization of acrylate-functionalized polymers is performed, for thiol-ene cross-linking, the allyl-ether-functionalized PGM is cross-linked with thiol-functionalized hyaluronic acid. Morphology, mechanical properties, and cell compatibility of both kinds of PGM hydrogels are characterized and compared. Furthermore, the biocompatibility of these hydrogels can be evaluated in cell culture experiments.
Background
The vast majority of cases with Beckwith-Wiedemann syndrome (BWS) are caused by a molecular defect in the imprinted chromosome region 11p15.5. The underlying mechanisms include epimutations, uniparental disomy, copy number variations, and structural rearrangements. In addition, maternal loss-of-function mutations in CDKN1C are found. Despite growing knowledge on BWS pathogenesis, up to 20% of patients with BWS phenotype remain without molecular diagnosis.
Case presentation
Herein, we report an Iranian family with two females affected with BWS in different generations. Bisulfite pyrosequencing revealed hypermethylation of the H19/IGF2: intergenic differentially methylated region (IG DMR), also known as imprinting center 1 (IC1) and hypomethylation of the KCNQ1OT1: transcriptional start site (TSS) DMR (IC2). Array CGH demonstrated an 8 Mb duplication on chromosome 11p15.5p15.4 (205,827-8,150,933) and a 1 Mb deletion on chromosome 9p24.3 (209,020-1,288,114). Chromosome painting revealed that this duplication-deficiency in both patients is due to unbalanced segregation of a paternal reciprocal t(9;11)(p24.3;p15.4) translocation.
Conclusions
This is the first report of a paternally inherited unbalanced translocation between the chromosome 9 and 11 short arms underlying familial BWS. Copy number variations involving the 11p15.5 region are detected by the consensus diagnostic algorithm. However, in complex cases which do not only affect the BWS region itself, characterization of submicroscopic chromosome rearrangements can assist to estimate the recurrence risk and possible phenotypic outcomes.