Stem cells and mineralized tissue : Characterization and disease modeling

Deficits in mineralized tissue, as a result of abnormal development, trauma, disease or aging, are major medical problems. Consequently, new methods to regenerate stable and functional bone is a main focus in the field of tissue engineering. Cell therapy using stem cells that adopt a mineralizing phenotype holds great potential for regeneration of calcified tissues such as bone and teeth. Stem cells provide an unlimited number of cells that can be used for bone differentiation. The mechanisms governing stem cell bone formation are however complex, and involve many factors. In this context, knowledge of basic bone biology, handling of stem cells in in vitro culture systems and the complex molecular mechanisms associated with normal and impaired bone development is fundamental. The aim of this project is to enhance the understanding of stem cell differentiation into the osteoblastic/mineralizing lineage. In the first study we investigated the roles of some of the constituents of the extracellular matrix (ECM) of dentin in mouse teeth. Using structural techniques, we found that osteoadherin (OSAD), a member of the family of mineralization-related small leucine-rich proteoglycan (SLRP) proteins, was localized at the mineralization front, closely associated with collagen fibers. This, together with data obtained from a functional assay, emphasizes the importance of OSAD in bone matrix maturation and mineralization. Continuing with in vitro model systems for subsequent application in the field of stem cells/regenerative medicine of bone and dentin, we highlighted important aspects of stem cells handling in the laboratory. We found that there were neither epigenetic alternations in selected histone modifications or marks nor any changes in protein expression when different passaging techniques were used. However, gene expression was significantly decreased for pluripotent markers using enzymatic split with a ROCK inhibitor, an effect that could be reversed upon mechanical passaging. These findings underline the fact that passaging techniques have to be taken into account when comparisons are made between cells that have undergone various treatments under different experimental conditions. In the last study we have derived induced Pluripotent Stem Cells (iPSCs) from a family with a mutation in the PIGT gene, which in addition to severe CNS defects causes a number of craniofacial bone and tooth abnormalities. iPSC-based models have emerged as useful systems to model human disease, both to unravel disease mechanisms and to provide test assays for drugs. We determined transcriptional and epigenetic changes in the patient-derived cells as compared to healthy control cells, with a focus on gene networks associated with bone development. We found four important genes to be downregulated compared to health unrelated iPSC controls, OPN, MMP2, ACVR1, and MMP2 which all have shown to have important roles in in bone and skeletal development. Furthermore, they showed patterns of epigenetic regulation, highlighting the importance of histone modifications and DNA methylation in the disease. Regenerative therapy with autologous cells using the patients’ own reprogrammed cells to replace damaged tissue may reduce patient suffering and healthcare costs. This thesis presents findings that might be of future use in the development of such cell-based bone replacement strategies

Whole-genome informed circulating tumor DNA analysis by multiplex digital PCR for disease monitoring in B-cell lymphomas: a proof-of-concept study

IntroductionAnalyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA).MethodsIn 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up.ResultsA total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes included KMT2D, PIM1, SOCS1 and BCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value <0.01). While clearance of ctDNA levels after primary treatment cycle 1 was observed in 3/6 patients, all patients analyzed at final evaluation of primary treatment showed negative ctDNA, hence correlating with PET-CT imaging. One patient with positive ctDNA at interim also displayed detectable ctDNA (average variant allele frequency (VAF) 6.9%) in the follow-up plasma sample collected 2 years after final evaluation of primary treatment and 25 weeks before clinical manifestation of relapse.ConclusionIn summary, we demonstrate that multi-targeted cfDNA analysis, using a combination of SNVs/indels and SVs candidates identified by WGS analysis, provides a sensitive tool for MRD monitoring and can detect lymphoma relapse earlier than clinical manifestation

Keratan sulfate, a complex glycosaminoglycan with unique functional capability

From an evolutionary perspective keratan sulfate (KS) is the newest glycosaminoglycan (GAG) but the least understood. KS is a sophisticated molecule with a diverse structure, and unique functional roles continue to be uncovered for this GAG. The cornea is the richest tissue source of KS in the human body but the central and peripheral nervous systems also contain significant levels of KS and a diverse range of KS-proteoglycans with essential functional roles. KS also displays important cell regulatory properties in epithelial and mesenchymal tissues and in bone and in tumor development of diagnostic and prognostic utility. Corneal KS-I displays variable degrees of sulfation along the KS chain ranging from non-sulfated polylactosamine, mono-sulfated and disulfated disaccharide regions. Skeletal KS-II is almost completely sulfated consisting of disulfated disaccharides interrupted by occasional mono-sulfated N-acetyllactosamine residues. KS-III also contains highly sulfated KS disaccharides but differs from KS-I and KS-II through 2-O-mannose linkage to serine or threonine core protein residues on proteoglycans such as phosphacan and abakan in brain tissue. Historically, the major emphasis on the biology of KS has focused on its sulfated regions for good reason. The sulfation motifs on KS convey important molecular recognition information and direct cell behavior through a number of interactive proteins. Emerging evidence also suggest functional roles for the poly-N-acetyllactosamine regions of KS requiring further investigation. Thus further research is warranted to better understand the complexities of KS

Osteoadherin Accumulates in the Predentin towards the Mineralization Front in the Developing Tooth

Background: Proteoglycans (PG) are known to be involved in the organization and assembly of the extracellular matrix (ECM) prior to mineral deposition. Osteoadherin (OSAD), a keratan sulphate PG is a member of the small leucine-rich (SLRP) family of PGs and unlike other SLRPs, OSAD expression is restricted to mineralized tissues. It is proposed to have a high affinity for hydroxyapatite and has been shown to be expressed by mature osteoblasts but its exact role remains to be elucidated. Methodology/Principal Findings: We investigated the protein distribution of OSAD in the developing mouse tooth using immunohistochemistry and compared its expression with other SLRPs, biglycan (BGN), decorin (DCN) and fibromodulin (FMD). OSAD was found to be specifically localized in the predentin layer of the tooth and focused at the mineralization front. These studies were confirmed at the ultrastructural level using electron microscopy (iEM), where the distribution of immunogold labeled OSAD particles were quantified and significant amounts were found in the predentin, forming a gradient towards the mineralization front. In addition, iEM results revealed OSAD to lie in close association with collagen fibers, further suggesting an important role for OSAD in the organization of the ECM. The expression profile of mineralization-related SLRP genes by rat dental pulp cells exposed to mineralization inducing factors, showed an increase in all SLRP genes. Indeed, OSAD expression was significantly increased during the mineralization process, specifically following

Immunostaining of OSAD in comparison to the other SLRPs in d5 mice.

<p>OSAD (A) expression is clearly noted in the predentin towards the predentin/dentin interface, BGN (B), DCN (C) and FMD (D) were detected again in the predentin but more closely located to the odontoblastic cell layer. A = ameloblasts, AB = alveolar bone, D = dentin, DF = dental follicle, E = enamel, pA = pre-ameloblasts, PD = predentin, pOB = pre-odontoblasts and OB = odontoblasts.</p

Semi-quantitative observations comparing immunohistochemistry staining in developing mouse incisors.

<p>Scoring - very strong (+++), strong (++), weak (+), more or less detectable with some variation (±), absent (−) and not applicable (N/A). A = ameloblasts, AB = alveolar bone, DF = dental follicle, DP = dental papilla, OB = odontoblasts, pA = pre-ameloblasts, pOB = pre-odontoblasts, SI = stratum intermedium.</p

Gene expression of OSAD increased when induced with mineralizing media in rDPC.

<p>rDPC were left to differentiate for 21 days in media supplemented with 50 µg/ml ascorbic acid phosphate and 10 mM β-glycerophosphate to induce a mineralizing phenotype. The changes in gene expression were measured after d3, 7, 14 and 21. Data showed an increased expression of all SLRPs (BGN, DCN, FMD and OSAD) over this period, in particular OSAD was highly expressed as the rDPC entered matrix maturation and mineralization. OCN, a mineralized tissue specific marker, also demonstrated a significant increase in expression with the onset of mineralization.</p

Immunostaining of OSAD in the developing mouse tooth.

<p>No signal was detected for OSAD at the early bell stage of the tooth E15 (A), OSAD was first localized weakly within the alveolar bone at E17 (B), and once dentinogenesis was initiated, it continued throughout crown formation from NB (C), d5 (D) and into the adult (F). The expression of OSAD was restricted to the predentin layer in all developmental phases, Control (F) sections were incubated with OSAD antibody (0.2 µg/µl) in the presence of 10× excess of recombinant mouse OSAD protein (0.1 µg/µl). A = ameloblasts, AB = alveolar bone, D = dentin, DF = dental follicle, E = enamel, pA = pre-ameloblasts, PD = predentin, pOB = pre-odontoblasts and OB = odontoblasts.</p

Electron images of OSAD in mouse predentin at different developmental stages.

<p>Electron images illustrating gold-labeled OSAD in the highly active predentin throughout all the different developmental stages examined (E15 (A), NB (B), D5 (C) and adult (D)). The number of gold-particles detected increased with development from E15 to NB. The elevated levels of OSAD expression were maintained in the predentin to d5 and adulthood. Localization of OSAD close to collagen fibers are very clearly observed in d5 mouse (C).</p

Alizarin Red S staining of rDPC in mineralizing culture.

<p>Mineral deposition was examined using Alizarin Red S staining. rDPC were investigated after d3 (A), d7 (B), d14 (C) and d21 (D). Clear mineralizing nodules were observed in the osteogenically induced cultures at d21 (D). Alizarin Red S quantification demonstrated elevated calcium content by nearly 10-fold at d14 and d21 compared to d3 cultures.</p