42 research outputs found
Entwicklung einer Strategie zur Inaktivierung von Genen in Mycobacterium avium und Charakterisierung der potentiellen Virulenz-Gene sigE und mig
Short telomeres correlate with a strong induction of cellular senescence in human dental follicle cells
Background: Dental follicle cells (DFCs) are dental stem cells and interesting options for regenerative therapies in dentistry. However, DFCs acquire replicative senescence in long-term cultures, but little is known about molecular processes. In previous studies, we observed that DFC cell lines become senescent at different rates. We hypothesized that short telomere length and increased DNA damage with genomic instability correlate with the accelerated induction of cellular senescence. Results: For this study we compared DFC cell lines that became senescent at different rates (DFC_F: strong senescent phenotype; DFC_S: weak senescent phenotype). The telomeres of DFC_F were shorter than those of the telomeres of DFC_S prior senescence. Interestingly, telomere lengths of both cell lines were nearly unchanged after induction of senescence. Gene expression analyses with genes associated with DNA damage before and after the induction of cellular senescence revealed that almost all genes in DFCs_F were down-regulated while the gene expression in DFC_S was almost constitutive. Moreover, number of aneuploid DFC_F were significantly higher after induction of cellular senescence. Conclusion: Our results supported our initial hypothesis that telomere length and genomic instability correlate with the accelerated induction of cellular senescence in DFC_F
Classical isoforms of protein kinase C (PKC) and Akt regulate the osteogenic differentiation of human dental follicle cells via both ÎČ-catenin and NF-ÎșB
Background
Human dental follicle cells (DFCs) are the precursor cells of the periodontium with a high potential for regenerative therapies of (alveolar) bone. However, the molecular mechanisms of osteogenic differentiation are inadequately understood. Classical isoforms of protein kinase C (PKC) are reported to inhibit osteogenesis of stem/precursor cells. This study evaluated the role of classical PKCs and potential downstream targets on the osteogenic differentiation of DFCs.
Methods
DFCs were osteogenic differentiated with dexamethasone or bone morphogenetic protein 2 (BMP2). Expression of PKC and potential upstream/downstream regulators was manipulated using activators, inhibitors, and small interfering ribonucleic acid (siRNA). Expression of proteins was examined by Western blot analysis, while the activation levels of enzymes and transcription factors were examined by their phosphorylation states or by specific activation assays. Expression levels of osteogenic markers were examined by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) analysis. Activity of alkaline phosphatase (ALP) and accumulation of calcium nodules by Alizarin Red staining were measured as indicators of mineralization.
Results
Classical PKCs like PKCα inhibit the osteogenic differentiation of DFCs, but do not interfere with the induction of differentiation. Inhibition of classical PKCs by Gö6976 enhanced activity of Akt after osteogenic induction. Akt was also regulated during differentiation and especially disturbed BMP2-induced mineralization. The PKC/Akt axis was further shown to regulate the canonical Wnt signaling pathway and eventually nuclear expression of active ÎČ-catenin during dexamethasone-induced osteogenesis. Moreover, the nuclear factor âkappa-light-chain-enhancerâ of activated B cells (NF-ÎșB) pathway is regulated during osteogenic differentiation of DFCs and via the PKC/Akt axis and disturbs the mineralization. Upstream, parathyroid hormone-related protein (PTHrP) sustained the activity of PKC, while Wnt5a inhibited it.
Conclusions
Our results demonstrate that classical PKCs like PKCα and Akt regulate the osteogenic differentiation of DFCs partly via both ÎČ-catenin and NF-ÎșB
Evaluation of Current Studies to Elucidate Processes in Dental Follicle Cells Driving Osteogenic Differentiation
When research on osteogenic differentiation in dental follicle cells (DFCs) began, projects focused on bone morphogenetic protein (BMP) signaling. The BMP pathway induces the transcription factor DLX3, whichh in turn induces the BMP signaling pathway via a positive feedback mechanism. However, this BMP2/DLX3 signaling pathway only seems to support the early phase of osteogenic differentiation, since simultaneous induction of BMP2 or DLX3 does not further promote differentiation. Recent data showed that inhibition of classical protein kinase C (PKCs) supports the mineralization of DFCs and that osteogenic differentiation is sensitive to changes in signaling pathways, such as protein kinase B (PKB), also known as AKT. Small changes in the lipidome seem to confirm the participation of AKT and PKC in osteogenic differentiation. In addition, metabolic processes, such as fatty acid biosynthesis, oxidative phosphorylation, or glycolysis, are essential for the osteogenic differentiation of DFCs. This review article attempts not only to bring the various factors into a coherent picture of osteogenic differentiation in DFCs, but also to relate them to recent developments in other types of osteogenic progenitor cells
Peripheral Nerve RegenerationâAdipose-Tissue-Derived Stem Cells Differentiated by a Three-Step Protocol Promote Neurite Elongation via NGF Secretion
The lack of supportive Schwann cells in segmental nerve lesions seems to be one cornerstone for the problem of insufficient nerve regeneration. Lately, adipose-tissue-derived stem cells (ASCs) differentiated towards SC (Schwann cell)-like cells seem to fulfill some of the needs for ameliorated nerve recovery. In this study, three differentiation protocols were investigated for their ability to differentiate ASCs from rats into specialized SC phenotypes. The differentiated ASCs (dASCs) were compared for their expressions of neurotrophins (NGF, GDNF, BDNF), myelin markers (MBP, P0), as well as glial-marker proteins (S100, GFAP) by RT-PCR, ELISA, and Western blot. Additionally, the influence of the medium conditioned by dASCs on a neuron-like cell line was evaluated. The dASCs were highly diverse in their expression profiles. One protocol yielded relatively high expression rates of neurotrophins, whereas another protocol induced myelin-marker expression. These results were reproducible when the ASCs were differentiated on surfaces potentially used for nerve guidance conduits. The NGF secretion affected the neurite outgrowth significantly. It remains uncertain what features of these SC-like cells contribute the most to adequate functional recovery during the different phases of nerve recovery. Nevertheless, therapeutic applications should consider these diverse phenotypes as a potential approach for stem-cell-based nerve-injury treatment
Isolation and characterisation of human gingival margin-derived STRO-1/MACS+ and MACSâ cell populations
Recently, gingival margin-derived stem/progenitor cells isolated via
STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal
regenerative potential in vivo. As a second-stage investigation, the present
study's aim was to perform in vitro characterisation and comparison of the
stem/progenitor cell characteristics of sorted STRO-1-positive (MACS+) and
STRO-1-negative (MACSâ) cell populations from the human free gingival margin.
Cells were isolated from the free gingiva using a minimally invasive technique
and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the
MACS+ and MACSâ cell fractions were characterized by flow cytometry for
expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1.
Colony-forming unit (CFU) and multilineage differentiation potential were
assayed for both cell fractions. Mineralisation marker expression was examined
using real-time polymerase chain reaction (PCR). MACS+ and MACSâ cell
fractions showed plastic adherence. MACS+ cells, in contrast to MACSâ cells,
showed all of the predefined mesenchymal stem/progenitor cell characteristics
and a significantly higher number of CFUs (P<0.01). More than 95% of MACS+
cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45,
CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACSâ cells showed a
different surface marker expression profile, with almost no expression of CD14
or STRO-1, and more than 95% of these cells expressed CD73, CD90 and
CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105.
MACS+ cells could be differentiated along osteoblastic, adipocytic and
chondroblastic lineages. In contrast, MACSâ cells demonstrated slight
osteogenic potential. Unstimulated MACS+ cells showed significantly higher
expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACSâ
cells demonstrated higher expression of osteonectin (P<0.05; MannâWhitney).
The present study is the first to compare gingival MACS+ and MACSâ cell
populations demonstrating that MACS+ cells, in contrast to MACSâ cells,
harbour stem/progenitor cell characteristics. This study also validates the
effectiveness of the STRO-1/MACS+ technique for the isolation of gingival
stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS+ cells
are a unique renewable source of multipotent stem/progenitor cells
Mechanisms during Osteogenic Differentiation in Human Dental Follicle Cells
Human dental follicle cells (DFCs) as periodontal progenitor cells are used for studies and research in regenerative medicine and not only in dentistry. Even if innovative regenerative therapies in medicine are often considered the main research area for dental stem cells, these cells are also very useful in basic research and here, for example, for the elucidation of molecular processes in the differentiation into mineralizing cells. This article summarizes the molecular mechanisms driving osteogenic differentiation of DFCs. The positive feedback loop of bone morphogenetic protein (BMP) 2 and homeobox protein DLX3 and a signaling pathway associated with protein kinase B (AKT) and protein kinase C (PKC) are presented and further insights related to other signaling pathways such as the WNT signaling pathway are explained. Subsequently, some works are presented that have investigated epigenetic modifications and non-coding ncRNAs and their connection with the osteogenic differentiation of DFCs. In addition, studies are presented that have shown the influence of extracellular matrix molecules or fundamental biological processes such as cellular senescence on osteogenic differentiation. The putative role of factors associated with inflammatory processes, such as interleukin 8, in osteogenic differentiation is also briefly discussed. This article summarizes the most important insights into the mechanisms of osteogenic differentiation in DFCs and is intended to be a small help in the direction of new research projects in this are
Molecular mechanisms in dental follicle precursor cells during the osteogenic differentiation
Effects of Cellular Senescence on Dental Follicle Cells
The dental follicle is part of the tooth germ, and isolated stem cells from this tissue (dental follicle cells; DFCs) are considered, for example, for regenerative medicine and immunotherapies. However somatic stem cells can also improve pharmaceutical research. Cell proliferation is limited by the induction of senescence, which, while reducing the therapeutic potential of DFCs for cell therapy, can also be used to study aging processes at the cellular level that can be used to test anti-aging pharmaceuticals. Unfortunately, very little is known about cellular senescence in DFCs. This review presents current knowledge about cellular senescence in DFCs
The dexamethasone induced osteogenic differentiation of dental follicle cells
Mesenchymal stem cells are excellent for in
vitro studies about biological processes during the
differentiation of osteogenic progenitor cells into
mineralizing cells such as osteoblasts. Human dental
follicle cells (DFCs) are dental mesenchymal stem cells
and they can be isolated from third molar teeth. Because
DFCs are the genuine progenitor cells of periodontal
tissue cells, they have been used for the evaluation of
molecular mechanisms during the differentiation of
undifferentiated stem cells into alveolar osteoblasts and
cementoblasts. To reveal molecular mechanisms of
osteogenic differentiation, initial studies investigated the
proteome and the transcriptome of DFCs after the
induction of the osteogenic differentiation with the
glucocorticoid dexamethasone. These studies showed for
example that dexamethasone induces the transcription
factor ZBTB16 (zinc finger and BTB domain containing
protein 16) and that ZBTB16 is crucial for osteogenic
differentiation of DFCs. This article is a survey of the
molecular mechanisms in DFCs during osteogenic
differentiation with dexamethasone