WST-assay data reveal a pH dependence of the mitochondrial succinate reductase in osteoblast-like cells

The data presented in this article are related to the research article entitled “Increased osteoblast viability at alkaline pH in vitro provides a new perspective on bone regeneration” (doi: 10.1016/j.bbrep.2017.02.001; (Galow et al., 2017) [1]). The water soluble tetrazolium (WST) proliferation assay detects the metabolic activity of the respiratory chain of cultured cells. The assay is based on changes in the light absorbance resulting from the metabolism of WST-1 into formazane by mitochondrial succinate reductase. We present data of three different tests that were carried out to check whether WST assay readouts are pH-dependent. In a first test, a possible pH effect on the photometric measurements, for example by shifting the absorbance spectrum of the pH indicator of the cell culture medium, was excluded. Because the second test revealed a significant pH-dependence of the activity of the mitochondrial succinate reductase, a third long-term test was conducted to analyze possible changes of the pH dependence over time. The higher absorbance per one million cells at alkaline pH, which was approximately four-fold at pH 8.4 compared to the pH-7.4 reference on day one decayed gradually, with the pH-differences equilibrating over six days

How to Slow down the Ticking Clock: Age-Associated Epigenetic Alterations and Related Interventions to Extend Life Span

Epigenetic alterations pose one major hallmark of organismal aging. Here, we provide an overview on recent findings describing the epigenetic changes that arise during aging and in related maladies such as neurodegeneration and cancer. Specifically, we focus on alterations of histone modifications and DNA methylation and illustrate the link with metabolic pathways. Age-related epigenetic, transcriptional and metabolic deregulations are highly interconnected, which renders dissociating cause and effect complicated. However, growing amounts of evidence support the notion that aging is not only accompanied by epigenetic alterations, but also at least in part induced by those. DNA methylation clocks emerged as a tool to objectively determine biological aging and turned out as a valuable source in search of factors positively and negatively impacting human life span. Moreover, specific epigenetic signatures can be used as biomarkers for age-associated disorders or even as targets for therapeutic approaches, as will be covered in this review. Finally, we summarize recent potential intervention strategies that target epigenetic mechanisms to extend healthy life span and provide an outlook on future developments in the field of longevity research

Optimization of in vitro culture systems for bone cells: Influence of pH and mechanical stimulation

Versuche bei verschiedenen Medien-pH-Werten zeigten, dass ein alkalischer pH positive Auswirkungen auf Proliferation und Differenzierung von Osteoblasten hat. Aufbauend auf dieser Erkenntnis wurde ein In-vitro-Modellsystem für ein die Mikroumgebung alkalisierendes Implantat erstellt. In einem weiteren Modellsystem wurde gezeigt, dass die mechanische Stimulation von Osteozyten einen signifikant positiven Effekt auf die Osteoblastenproliferation hat, während sich bei Stimulation von Osteoblasten keine signifikanten Effekte auf die Proliferation unstimulierter Osteoblastenpopulationen zeigten.Experiments at different medium pH showed positive effects on the proliferation and differentiation of osteoblasts for alkaline pH values. Based on this findings, initial approaches to an in vitro model system for an implant that alkalizes the microenvironment were accomplished. In another model system, a significant positive effect on the proliferation of the upper osteoblasts could be observed when mechanically stimulating the lower osteocyte population in co-culture while no significant effects were observed when osteoblasts were used as stimulated populations

Xenogeneic and Stem Cell-Based Therapy for Cardiovascular Diseases: Genetic Engineering of Porcine Cells and Their Applications in Heart Regeneration

Cardiovascular diseases represent a major health concern worldwide with few therapy options for ischemic injuries due to the limited regeneration potential of affected cardiomyocytes. Innovative cell replacement approaches could facilitate efficient regenerative therapy. However, despite extensive attempts to expand primary human cells in vitro, present technological limitations and the lack of human donors have so far prevented their broad clinical use. Cell xenotransplantation might provide an ethically acceptable unlimited source for cell replacement therapies and bridge the gap between waiting recipients and available donors. Pigs are considered the most suitable candidates as a source for xenogeneic cells and tissues due to their anatomical and physiological similarities with humans. The potential of porcine cells in the field of stem cell-based therapy and regenerative medicine is under intensive investigation. This review outlines the current progress and highlights the most promising approaches in xenogeneic cell therapy with a focus on the cardiovascular system

Enhancing the regeneration of bone defects by alkalizing the peri-implant zone – an in vitro approach

The effects of alkaline pH on the initial adhesion of osteoblasts to titanium surfaces was analyzed by single cell force microscopy (SCFM). In the SCFM measurements, the same cells were used to compare their unspecific adhesion to uncoated titanium with their specific adhesion to collagen coated titanium. When the maximum detachment forces (MDFs) were compared at pH 7.4 and 8.0, only slight differences were found on pure titanium, while the MDFs were significantly increased at collagen coated surfaces at pH 8.0. Effects on the subsequent proliferation and gene expression were investigated in an in vitro model system consisting of an alkalizing polyvinyl alcohol (PVA) matrix and a perforated titanium disc. The sodium hydroxide releasing matrix maintained the medium pH between pH 7.6 and pH 8.4 during the entire experiment. Under these conditions, cell counts were significantly increased with respect to the control system after 7 days in culture. These results were supported by gene expression analyses, which showed an upregulation of proliferation-controlling genes of the EGFR1 and PI3K/AKT pathways after 14 days in culture. The SCFM data were complemented by findings of an intensive regulation of genes known to be associated with focal adhesion such as Itga8 and Tnn

MC3T3 osteoblast-like cells cultured at alkaline pH: Microarray data (Affymetrix GeneChip Mouse 2.0 ST)

It is well known that pH plays a pivotal role in the control of bone remodeling. However, no comprehensive gene expression data are available for the effects of alkaline pH on osteoblasts. We cultured differentiating MC3T3-E1 osteoblast-like cells at pH 7.4, 7.8, and 8.4 for 14 days. To identify differential gene expression, microarray data were collected with Affymetrix GeneChips. The data were validated by real-time PCRs for five genes that were found to be greatly regulated in the GeneChip-experiments (DMP1, FABP4, SFRP2 and TNFRSF19) or considered relevant for the terminal function of osteoblasts (DMP1 and ATF4). All the data are available from the Gene Expression Omnibus database (GEO accession: GSE84907). Here, we provide pathway analyses of known protein coding genes that were down-regulated or up-regulated by greater than 2.0-fold. The regulation datasets obtained from comparisons of pH 7.8 and 7.4, as well as pH 8.4 and 7.4 share a high number of differentially expressed genes. When comparing pH 8.4 and 7.8, other genes mainly emerge, suggesting not only a simple amplification of the effects at pH 8.4 that were already induced at pH 7.8 but also the induction of different pathways. For a more detailed analysis, different mammalian functional gene networks were assigned to each dataset. After merging and manual optimization of the network graphs, three combined functional gene networks were obtained that reflected distinct pH-dependent cellular responses. A common feature of the networks was the central role of p38 MAP kinase. The microarray data presented here are related to the research article doi:10.1016/j.bbrep.2017.02.001 (Galow et al., 2017) [1]

Increased osteoblast viability at alkaline pH in vitro provides a new perspective on bone regeneration

We investigated the effects of alkaline pH on developing osteoblasts. Cells of the osteoblast-like cell line MC3T3-E1 were initially cultured for six days in HEPES-buffered media with pH ranging from 7.2 to 9.0. Cell count, cellular WST-1 metabolism, and ATP content were analyzed. The three parameters showed a pH optimum around pH 8.4, exceeding the recommended buffer range of HEPES at the alkaline flank. Therefore, only pH 7.2, 7.4, 7.8, and 8.4 media were used in more elaborate, daily investigations to reduce the effects of pH change within the pH control intervals of 24 h. All parameters exhibited similar pH behaviors, roughly showing increases to 130% and 230% at pH 7.8 and 8.4, as well as decreases to 70% at pH 7.2 when using the pH 7.4 data for reference. To characterize cell differentiation and osteoblastic cell function, cells were cultured at pH 7.4 and under alkaline conditions at pH 7.8 and 8.4 for 14 days. Gene expression and mineralization were evaluated using microarray technology and Alizarin staining. Under alkaline conditions, ATF4, a regulator for terminal differentiation and function as well as DMP1, a potential marker for the transition of osteoblasts into osteocytes, were significantly upregulated, hinting at an accelerated differentiation process. After 21 days, significant mineralization was only detected at alkaline pH. We conclude that elevated pH is beneficial for the cultivation of bone cells and may also provide therapeutic value in bone regeneration therapies

Single-Nucleus Sequencing of an Entire Mammalian Heart: Cell Type Composition and Velocity

Analyses on the cellular level are indispensable to expand our understanding of complex tissues like the mammalian heart. Single-nucleus sequencing (snRNA-seq) allows for the exploration of cellular composition and cell features without major hurdles of single-cell sequencing. We used snRNA-seq to investigate for the first time an entire adult mammalian heart. Single-nucleus quantification and clustering led to an accurate representation of cell types, revealing 24 distinct clusters with endothelial cells (28.8%), fibroblasts (25.3%), and cardiomyocytes (22.8%) constituting the major cell populations. An additional RNA velocity analysis allowed us to study transcription kinetics and was utilized to visualize the transitions between mature and nascent cellular states of the cell types. We identified subgroups of cardiomyocytes with distinct marker profiles. For example, the expression of Hand2os1 distinguished immature cardiomyocytes from differentiated cardiomyocyte populations. Moreover, we found a cell population that comprises endothelial markers as well as markers clearly related to cardiomyocyte function. Our velocity data support the idea that this population is in a trans-differentiation process from an endothelial cell-like phenotype towards a cardiomyocyte-like phenotype. In summary, we present the first report of sequencing an entire adult mammalian heart, providing realistic cell-type distributions combined with RNA velocity kinetics hinting at interrelations

Integrative Cluster Analysis of Whole Hearts Reveals Proliferative Cardiomyocytes in Adult Mice

The recent development and broad application of sequencing techniques at the single-cell level is generating an unprecedented amount of data. The different techniques have their individual limits, but the datasets also offer unexpected possibilities when utilized collectively. Here, we applied snRNA-seq in whole adult murine hearts from an inbred (C57BL/6NRj) and an outbred (Fzt:DU) mouse strain to directly compare the data with the publicly available scRNA-seq data of the tabula muris project. Explicitly choosing a single-nucleus approach allowed us to pin down the typical heart tissue-specific technical bias, coming up with novel insights on the mammalian heart cell composition. For our integrated dataset, cardiomyocytes, fibroblasts, and endothelial cells constituted the three main cell populations accounting for about 75% of all cells. However, their numbers severely differed between the individual datasets, with cardiomyocyte proportions ranging from about 9% in the tabula muris data to around 23% for our BL6 data, representing the prime example for cell capture technique related bias when using a conventional single-cell approach for these large cells. Most strikingly in our comparison was the discovery of a minor population of cardiomyocytes characterized by proliferation markers that could not be identified by analyzing the datasets individually. It is now widely accepted that the heart has an, albeit very restricted, regenerative potential. However there is still an ongoing debate where new cardiomyocytes arise from. Our findings support the idea that the renewal of the cardiomyocyte pool is driven by cytokinesis of resident cardiomyocytes rather than differentiation of progenitor cells. We thus provide data that can contribute to an understanding of heart cell regeneration, which is a prerequisite for future applications to enhance the process of heart repair