Oliver Felthaus

Luminescent Dual Sensors Reveal Extracellular pH-Gradients and Hypoxia on Chronic Wounds That Disrupt Epidermal Repair

Wound repair is a quiescent mechanism to restore barriers in multicellular organisms upon injury. In chronic wounds, however, this program prematurely stalls. It is known that patterns of extracellular signals within the wound fluid are crucial to healing. Extracellular pH (pHe) is precisely regulated and potentially important in signaling within wounds due to its diverse cellular effects. Additionally, sufficient oxygenation is a prerequisite for cell proliferation and protein synthesis during tissue repair. It was, however, impossible to study these parameters in vivo due to the lack of imaging tools. Here, we present luminescent biocompatible sensor foils for dual imaging of pHe and oxygenation in vivo. To visualize pHe and oxygen, we used time-domain dual lifetime referencing (tdDLR) and luminescence lifetime imaging (LLI), respectively. With these dual sensors, we discovered centripetally increasing pHe-gradients on human chronic wound surfaces. In a therapeutic approach, we identify pHe-gradients as pivotal governors of cell proliferation and migration, and show that these pHe-gradients disrupt epidermal barrier repair, thus wound closure. Parallel oxygen imaging also revealed marked hypoxia, albeit with no correlating oxygen partial pressure (pO2)-gradient. This highlights the distinct role of pHe-gradients in perturbed healing. We also found that pHe-gradients on chronic wounds of humans are predominantly generated via centrifugally increasing pHe-regulatory Na+/H+-exchanger-1 (NHE1)-expression. We show that the modification of pHe on chronic wound surfaces poses a promising strategy to improve healing. The study has broad implications for cell science where spatial pHe-variations play key roles, e.g. in tumor growth. Furthermore, the novel dual sensors presented herein can be used to visualize pHe and oxygenation in various biomedical fields.

The Transcription Factor DLX3 Regulates the Osteogenic Differentiation of Human Dental Follicle Precursor Cells

The transcription factor DLX3 plays a decisive role in bone development of vertebrates. In neural-crest derived stem cells from the dental follicle (DFCs), DLX3 is differentially expressed during osteogenic differentiation, while other osteogenic transcription factors such as DLX5 or RUNX2 are not highly induced. DLX3 has therefore a decisive role in the differentiation of DFCs, but its actual biological effects and regulation are unknown. This study investigated the DLX3-regulated processes in DFCs. After DLX3 overexpression, DFCs acquired a spindle-like cell shape with reorganized actin filaments. Here, marker genes for cell morphology, proliferation, apoptosis, and osteogenic differentiation were significantly regulated as shown in a microarray analysis. Further experiments showed that DFCs viability is directly influenced by the expression of DLX3, for example, the amount of apoptotic cells was increased after DLX3 silencing. This transcription factor stimulates the osteogenic differentiation of DFCs and regulates the BMP/SMAD1-pathway. Interestingly, BMP2 did highly induce DLX3 and reverse the inhibitory effect of DLX3 silencing in osteogenic differentiation. However, after DLX3 overexpression in DFCs, a BMP2 supplementation did not improve the expression of DLX3 and the osteogenic differentiation. In conclusion, DLX3 influences cell viability and regulates osteogenic differentiation of DFCs via a BMP2-dependent pathway and a feedback control.

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines.

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133(+) cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

Comparison of Neurosphere-like Cell Clusters Derived from Dental Follicle Precursor Cells and Retinal Müller Cells

Unrelated cells such as dental follicle precursor cells (DFPCs) and retinal Müller cells (MCs) make spheres after cultivation in serum-replacement medium (SRM). Until today, the relation and molecular processes of sphere formation from different cell types remain undescribed. Thus in this study we compared proteomes of spheres derived from MCs and DFPCs. 73% of 676 identified proteins were similar expressed in both cell types and many of them are expressed in the brain (55%). Moreover proteins are overrepresented that are associated with pathways for neural diseases such as Huntington disease or Alzheimer disease. Interestingly up-regulated proteins in DFPCs are involved in the biosynthesis of glycosphingolipids. These lipids are components of gangliosides such as GD3, which is a novel neural stem cell marker. In conclusion spheres from different types of cells have highly similar proteomes. These proteomes probably show essential cellular processes in neurosphere-like cell clusters.

Comparison of murine dental follicle precursor and retinal progenitor cells after neural differentiation in vitro

Human dental stem or precursor cells can differentiate into multiple cell types like adipocytes, osteoblasts or chondrocytes. Recently, a number of different human dental stem cell lines were differentiated into neurons. This makes dental stem cells interesting as possible cell‐based therapeutics for neural degenerative diseases. To test this hypothesis, we have investigated the neural differentiation potential of murine dental follicle precursor cells (mDFPCs). The mDFPC cell line was newly established without cell immortalization. After differentiation, neural cell marker expression in mDFPCs was checked and compared with that of murine retinal progenitor cells (mRPCs). Differentiated mDFPCs became neuron‐like cells with small cell bodies and long/branching neurites, similar to differentiated mRPCs. However, mRPCs showed more complete neural differentiation. Furthermore, 5% of the differentiated mDFPCs and 37% of the differentiated mRPCs were positive for the glia cell marker GFAP (glial fibrillary acidic protein). The data presents new evidence of neural differentiation of mDFPCs, but only a small percentage of mDFPCs differentiated into glia cells, unlike mRPCs.

ZBTB16 Induces Osteogenic Differentiation Marker Genes in Dental Follicle Cells Independent From RUNX2

BACKGROUND Dental follicle cells (DFCs) are neural crest cell-derived cells and the genuine precursor cells of cementoblast and alveolar osteoblasts. After osteogenic differentiation, expression levels of the transcription factor zinc factor and BTB domain containing 16 (ZBTB16) were significantly increased. ZBTB16 is associated with the process of osteogenic differentiation in bone marrow-derived mesenchymal stem cells and crucial for the expression of the osteogenic transcription factor runt-related transcription factor 2 (RUNX2). It is proposed that ZBTB16 plays also a crucial role for the differentiation of DFCs into osteoblasts. METHODS In this study, the differentiation of DFCs by alkaline phosphatase (ALP) activity measurement, alizarin red staining, and electron-dispersive x-ray spectrometry (EDX) analysis is investigated. The expression of ZBTB16 during osteogenic differentiation and the expression of osteogenic differentiation markers were assessed by real-time reverse transcription polymerase chain reaction. Glucocorticoid stimulation was inhibited using RU486 (11β-[p-(Dimethylamino)phenyl]-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), and ZBTB16 was overexpressed via transient transfection of an expression vector. RESULTS After the initiation of osteogenic differentiation, ZBTB16 levels were increased highly in DFCs, whereas RUNX2 was expressed constitutively only. An EDX analysis verified the differentiation of DFCs into osteoblast-like cells because clusters of mineralization consisted of hydroxyapatite. ZBTB16 induced the expression of nuclear receptor subfamily 4, group A, member 3; osteocalcin; and stanniocalcin 1 (STC1) but not of RUNX2 and ALP in DFCs. STC1 was upregulated in DFCs downstream of ZBTB16 and after the osteogenic differentiation. The overexpression of STC1 in DFCs increased the expression of ZBTB16 and specific markers for biomineralization. CONCLUSIONS The present study shows that ZBTB16 induced the expression of osteogenic differentiation markers independently of RUNX2. Moreover, STC1 is a new candidate for the evaluation of late mechanisms of osteogenic differentiation downstream of ZBTB16.

Genome-wide gene expression profiles of dental follicle stem cells.

Abstract Objective. Dental stem cells (SCs) will be increasingly used for bone regeneration in the future. Recently, dental follicle cells (DFCs) from retained human third molars have been isolated and characterized as osteogenic progenitors. Although these results are promising for regenerative dentistry, molecular processes during osteogenic differentiation are not yet well understood. Materials and methods. This study compared DFCs before and during osteogenic differentiation. ALP activity was measured and cells were stained with alizarin red. Real-time RT-PCRs for osteogenic markers were done. The genome-wide expression profile was evaluated using a microarray. Results. DFCs showed strong mineralization and increased expression of osteogenic marker genes during osteogenic differentiation. A microarray analysis showed regulated genes before and in the process of osteogenic differentiation (day 7). Several regulated genes in DFCs were associated with skeletal development. Bioinformatic analysis revealed a number of factors associated with dental follicle osteogenic differentiation. Osteogenic differentiation affected expression levels of the transcriptional regulators FOXC2 and ZNF219. Conclusion. In conclusion, the results yielded new objectives for further studies on transcription factors like FOXC2 or ETV1 and their role in dental SCs during osteogenic differentiation.

Transcription Profile in Sporadic Multiple Symmetric Lipomatosis Reveals Differential Expression at the Level of Adipose Tissue-Derived Stem Cells.

Background: The cause of the rare fat distribution disorder multiple symmetric lipomatosis is unknown. Independent reports suggest a higher proliferative activity, hormone resistance, and involvement of mitochondrial function in the disease. Methods: The authors performed morphologic comparison of affected and unaffected tissues in five unrelated patients and generated adipose-derived stem cell cultures from the tissue samples and characterized them as a possible cellular model of multiple symmetric lipomatosis evolution. The authors investigated proliferative activity and the expression of genes relevant to disease processes. Results: There was no difference in the morphologic appearance and the surface marker profile. Stem cells from lipomatous tissue showed significantly higher proliferative activity. Polymerase chain reaction arrays showed marked changes in genes associated with proliferation, hormonal regulation, and mitochondria. The authors show that multiple symmetric lipomatosis tissue is morphologically and histologically different from regular subcutaneous fat. Conclusions: This study indicates an involvement of mesenchymal stem cells in the pathogenesis of multiple symmetric lipomatosis and that the evolution of multiple symmetric lipomatosis tissue is a process driven by an inherent defect of the respective cell clone(s). Further molecular genetics and functional analysis will be required to unravel the pathogenetic mechanism underlying the derailment in fat cell metabolism and proliferation. Here, the authors show for the first time that adipose-derived stem cells exhibit many characteristics previously described for native multiple symmetric lipomatosis fat tissue and propose that they are therefore an excellent tool for further functional investigations in multiple symmetric lipomatosis and other disorders of the fat tissue. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, V.

Collagen Type I Conduits for the Regeneration of Nerve Defects

To date, reliable data to support the general use of biodegradable materials for bridging nerve defects are still scarce. We present the outcome of nerve regeneration following type I collagen conduit nerve repair in patients with large-diameter nerve gaps. Ten patients underwent nerve repair using a type I collagen nerve conduit. Patients were re-examined at a minimal follow-up of 14.0 months and a mean follow-up of 19.9 months. Regeneration of nerve tissue within the conduits was assessed by nerve conduction velocity (NCV), a static two-point discrimination (S2PD) test, and as disability of arm shoulder and hand (DASH) outcome measure scoring. Quality of life measures including patients’ perceived satisfaction and residual pain were evaluated using a visual analog scale (VAS). No implant-related complications were observed. Seven out of 10 patients reported being free of pain, and the mean VAS was 1.1. The mean DASH score was 17.0. The S2PD was below 6 mm in 40%, between 6 and 10 mm in another 40% and above 10 mm in 20% of the patients. Eight out of 10 patients were satisfied with the procedure and would undergo surgery again. Early treatment correlated with lower DASH score levels. The use of type I collagen in large-diameter gaps in young patients and early treatment presented superior functional outcomes.

TGF-β stimulates glial-like differentiation in murine dental follicle precursor cells (mDFPCs)

Dental stem cells such as dental follicle precursor cells (DFPCs) are capable of neural-like differentiation. However, compared to neuroectodermal progenitor cells such as murine retinal progenitor cells (mRPCs) they show only a limited capacity for glial cell differentiation. In this study we tested the influence of cell signaling on glial differentiation of mDFPCs. These cells were treated with inhibitors and activators of the Sonic hedgehog-, the Wnt/beta-Catenin-, and the TGF-beta-pathway. After incubation only an activation of the TGF-beta-pathway showed a remarkable glial-like cell differentiation. In contrast gene expression of neural cell markers was not regulated. In conclusion, TGF-beta improved glial-like, but not neural-like, differentiation of mDFPCs.