Sebastian Gehmert

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)].

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.

Stem cell-based tissue-engineering for treatment of meniscal tears in the avascular zone.

Meniscal tears in the avascular zone have a poor self-healing potential, however partial meniscectomy predisposes the knee for early osteoarthritis. Tissue engineering with mesenchymal stem cells and a hyaluronan collagen based scaffold is a promising approach to repair meniscal tears in the avascular zone. 4 mm longitudinal meniscal tears in the avascular zone of lateral menisci of New Zealand White Rabbits were performed. The defect was left empty, sutured with a 5-0 suture or filled with a hyaluronan/collagen composite matrix without cells, with platelet rich plasma or with autologous mesenchymal stem cells. Matrices with stem cells were in part precultured in chondrogenic medium for 14 days prior to the implantation. Menisci were harvested at 6 and 12 weeks. The developed repair tissue was analyzed macroscopically, histologically and biomechanically. Untreated defects, defects treated with suture alone, with cell-free or with platelet rich plasma seeded implants showed a muted fibrous healing response. The implantation of stem cell-matrix constructs initiated fibrocartilage-like repair tissue, with better integration and biomechanical properties in the precultured stem cell-matrix group. A hyaluronan-collagen based composite scaffold seeded with mesenchymal stem cells is more effective in the repair avascular meniscal tear with stable meniscus-like tissue and to restore the native meniscus.

Angiogenesis: The role of PDGF-BB on Adiopse-tissue derived Stem Cells (ASCs)

Recently, it was shown that mesenchymal stem cells (MSCs) are capable of differentiating into endothelial cells which highlights the potential role of MSCs in neovascularization. In the present study, we investigated the paracrine factors responsible for tube formation in human adipose-tissue derived stem cells (ASCs). Moreover, we analyzed ASCs migration towards PDGF-BB and altered levels of proteins involved in different pathways. Freshly isolated human adipose tissue-derived stem cells were seeded onto wells coated with Matrigel and cultured in endothelial growth medium. Capillary-like tube formation was observed after 18 hours culture. Tube formation was significantly reduced in the presence of antibodies against platelet-derived growth factor receptor beta (PDGF) or basic fibroblast growth factor (bFGF). Reverse phase proteomic assay (RPPA) was used to interrogate the expression of 139 phosphorylated or native proteins after incubation with PDGF-BB protein for 24 hours. The present data suggest, that freshly isolated ASCs contain a subpopuplation of stem cells that can form capillary like tubes which is dependent on PDGF and bFGF signaling pathway. Furthermore, Migration of human ASCs significantly increased in response to increased concentrations of PDGF-BB. In addition, incubation of ASCs with PDGF-BB altered phosphorylation of several transcription proteins that are widely expressed throughout the hematopoietic system, targeting genes that have been associated with proliferation, anti-apoptosis or differentiation.

Effect of freshly isolated autologous tissue resident stromal cells on cardiac function and perfusion following acute myocardial infarction

BACKGROUND The aim of this study was to investigate the effect of intracoronary administration of freshly isolated, uncultured autologous tissue-derived stromal cells on cardiac function and perfusion after acute infarction in pigs. METHODS A transmural myocardial infarction in a porcine model was induced by occlusion of the mid LAD with an angioplasty balloon for 3 h. Upon reperfusion, freshly isolated, uncultured autologous stromal cells (1.5×10⁶ cells/kg) or control solution was injected into the infarct artery. Cardiac function and area at risk were determined by (99m)Tc-SPECT. RESULTS Eight weeks after infarction, cell treated pigs showed a 20% smaller myocardial perfusion defect compared to control animals (35±9% vs. 44±5% of LV, treated vs. control, respectively, p<0.05). The reduction of the perfusion defect was associated with a significantly higher myocardial salvage index in the cell group as well as a significant increase in ejection fraction compared to control (EF at 8 weeks 43±7% vs. 35±3%, treated vs. control, respectively, p<0.05). This functional improvement was reflected by an increased wall thickness of the infarct and border zone in the treated group (11.2±2.2 mm) compared to control (8.6±1.6 mm, p<0.05) as well as an increased capillary density in the border zone (treated vs. control; 41.6±17.9 vs. 32.9±12.6 capillaries per 0.1 mm², p<0.05). CONCLUSIONS This study demonstrates for the first time that recovery and intracoronary delivery of uncultured autologous tissue derived stromal cells at time of vessel reperfusion is feasible and improves ventricular function.

Breast cancer cells attract the migration of adipose tissue-derived stem cells via the PDGF-BB/PDGFR-β signaling pathway

The origin of vascular cells in tumors is unknown, but it is believed that tumors use cells from the host to build new vessels. To determine whether adipose tissue stem cells (ASCs) could be attracted by cancer cells, we performed migration assays in which ASCs were seeded on a transwell migration system top chamber and tumor-conditioned medium was placed in the bottom chamber. Our data showed that a significant number of ASCs migrated toward the tumor-conditioned medium (p<0.0001), and migration of human ASCs significantly (p<0.0001) increased in response to increased concentrations of recombinant PDGF-BB. In addition, neutralizing antibodies to PDGF receptor (PDGFR)-beta decreased migration of ASCs toward a breast cancer-conditioned medium to the level of serum-free control. These data suggest that tumor cell-derived PDGF-BB is an important factor in governing the microenvironment interaction between tumor cells and local tissue-resident stem cells.

A sprayable luminescent pH sensor and its use for wound imaging in vivo

Non‐invasive luminescence imaging is of great interest for studying biological parameters in wound healing, tumors and other biomedical fields. Recently, we developed the first method for 2D luminescence imaging of pH in vivo on humans, and a novel method for one‐stop‐shop visualization of oxygen and pH using the RGB read‐out of digital cameras. Both methods make use of semitransparent sensor foils. Here, we describe a sprayable ratiometric luminescent pH sensor, which combines properties of both these methods. Additionally, a major advantage is that the sensor spray is applicable to very uneven tissue surfaces due to its consistency. A digital RGB image of the spray on tissue is taken. The signal of the pH indicator (fluorescein isothiocyanate) is stored in the green channel (G), while that of the reference dye [ruthenium(II)‐tris‐(4,7‐diphenyl‐1,10‐phenanthroline)] is stored in the red channel (R). Images are processed by rationing luminescence intensities (G/R) to result in pseudocolor pH maps of tissues, e.g. wounds.

VEGF receptor Flk‐1 plays an important role in c‐kit expression in adipose tissue derived stem cells

It is known that c‐kit+ cells are increased in heart after infarction. The exact origins of the cardiac c‐kit+ cells remain to be determined. We asked whether adipose tissue could be a potential source of c‐kit+ cells. Our data show that the number of c‐kit+ cells increased in adipose tissue derived stem cells when cultured with conditioned medium from neonatal cardiomyocytes grown under serum deprivation and hypoxia condition. We also found that VEGF receptor Flk‐1 is involved in c‐kit up regulation via ERK‐mediated pathway.

PDGF and bFGF modulate tube formation in adipose tissue-derived stem cells.

Recent evidence indicates that mesenchymal stem cells contribute to tumor angiogenesis through yet undefined mechanisms. In the present study, we investigated the angiogenic properties of human adipose tissue-derived stem cells and the mechanisms involved. Freshly isolated human adipose tissue-derived stem cells were seeded onto wells coated with Matrigel and cultured in endothelial growth medium. Capillary-like tube formation was observed after 18 hours culture. Tube formation was significantly reduced in the presence of antibodies against platelet-derived growth factor receptor beta or basic fibroblast growth factor. Collectively, these data suggest that freshly isolated adipose tissue-derived stem cells have the capacity to differentiate into capillary structures and platelet-derived growth factor and bFGF plays a critical role in this process.

The anti-apoptotic effect of IGF-1 on tissue resident stem cells is mediated via PI3-kinase dependent secreted frizzled related protein 2 (Sfrp2) release.

Previous studies suggest that IGF-1 may be used as an adjuvant to stem cell transfer in order to improve cell engraftment in ischemic tissue. In the current study, we investigated the effect of IGF-1 on serum deprivation and hypoxia induced stem cell apoptosis and the possible mechanisms involved. Exposure of adipose tissue derived stem cells (ASCs) to serum deprivation and hypoxia resulted in significant apoptosis in ASC which is partially prevented by IGF-1. IGF-1s anti-apoptotic effect was abolished in ASCs transfected with Sfrp2 siRNA but not by the control siRNA. Using Western blot analysis, we demonstrated that serum deprivation and hypoxia reduced the expression of nuclear beta-catenin, which is reversed by IGF-1. IGF-1s effect on beta-catenin expression was abolished by the presence of PI3-kinase inhibitor LY294002 or in ASCs transfected with Sfrp2 siRNA. These results suggest that IGF-1, through the release of the Sfrp2, contributes to cell survival by stabilizing beta-catenin.