Using light to drive energy transduction in metazoan aging.

Mitochondrial dysfunction is a central hallmark of aging and energy transduction is a promising target for longevity interventions. New research suggests that interventions in how energy is transduced could benefit healthy longevity. Here, we propose using light as an alternative energy source to fuel mitochondria and increase metazoan lifespan.

CCR2 macrophage response determines the functional outcome following cardiomyocyte transplantation.

BACKGROUND: The immune response is a crucial factor for mediating the benefit of cardiac cell therapies. Our previous research showed that cardiomyocyte transplantation alters the cardiac immune response and, when combined with short-term pharmacological CCR2 inhibition, resulted in diminished functional benefit. However, the specific role of innate immune cells, especially CCR2 macrophages on the outcome of cardiomyocyte transplantation, is unclear. METHODS: We compared the cellular, molecular, and functional outcome following cardiomyocyte transplantation in wildtype and T cell- and B cell-deficient Rag2del mice. The cardiac inflammatory response was assessed using flow cytometry. Gene expression profile was assessed using single-cell and bulk RNA sequencing. Cardiac function and morphology were determined using magnetic resonance tomography and immunohistochemistry respectively. RESULTS: Compared to wildtype mice, Rag2del mice show an increased innate immune response at steady state and disparate macrophage response after MI. Subsequent single-cell analyses after MI showed differences in macrophage development and a lower prevalence of CCR2 expressing macrophages. Cardiomyocyte transplantation increased NK cells and monocytes, while reducing CCR2-MHC-IIlo macrophages. Consequently, it led to increased mRNA levels of genes involved in extracellular remodelling, poor graft survival, and no functional improvement. Using machine learning-based feature selection, Mfge8 and Ccl7 were identified as the primary targets underlying these effects in the heart. CONCLUSIONS: Our results demonstrate that the improved functional outcome following cardiomyocyte transplantation is dependent on a specific CCR2 macrophage response. This work highlights the need to study the role of the immune response for cardiomyocyte cell therapy for successful clinical translation.

Synergistic effects of hormones on structural and functional maturation of cardiomyocytes and implications for heart regeneration.

The limited endogenous regenerative capacity of the human heart renders cardiovascular diseases a major health threat, thus motivating intense research on in vitro heart cell generation and cell replacement therapies. However, so far, in vitro-generated cardiomyocytes share a rather fetal phenotype, limiting their utility for drug testing and cell-based heart repair. Various strategies to foster cellular maturation provide some success, but fully matured cardiomyocytes are still to be achieved. Today, several hormones are recognized for their effects on cardiomyocyte proliferation, differentiation, and function. Here, we will discuss how the endocrine system impacts cardiomyocyte maturation. After detailing which features characterize a mature phenotype, we will contemplate hormones most promising to induce such a phenotype, the routes of their action, and experimental evidence for their significance in this process. Due to their pleiotropic effects, hormones might be not only valuable to improve in vitro heart cell generation but also beneficial for in vivo heart regeneration. Accordingly, we will also contemplate how the presented hormones might be exploited for hormone-based regenerative therapies.

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.

Automated annotation of rare-cell types from single-cell RNA-sequencing data through synthetic oversampling.

BACKGROUND: The research landscape of single-cell and single-nuclei RNA-sequencing is evolving rapidly. In particular, the area for the detection of rare cells was highly facilitated by this technology. However, an automated, unbiased, and accurate annotation of rare subpopulations is challenging. Once rare cells are identified in one dataset, it is usually necessary to generate further specific datasets to enrich the analysis (e.g., with samples from other tissues). From a machine learning perspective, the challenge arises from the fact that rare-cell subpopulations constitute an imbalanced classification problem. We here introduce a Machine Learning (ML)-based oversampling method that uses gene expression counts of already identified rare cells as an input to generate synthetic cells to then identify similar (rare) cells in other publicly available experiments. We utilize single-cell synthetic oversampling (sc-SynO), which is based on the Localized Random Affine Shadowsampling (LoRAS) algorithm. The algorithm corrects for the overall imbalance ratio of the minority and majority class. RESULTS: We demonstrate the effectiveness of our method for three independent use cases, each consisting of already published datasets. The first use case identifies cardiac glial cells in snRNA-Seq data (17 nuclei out of 8635). This use case was designed to take a larger imbalance ratio (~1 to 500) into account and only uses single-nuclei data. The second use case was designed to jointly use snRNA-Seq data and scRNA-Seq on a lower imbalance ratio (~1 to 26) for the training step to likewise investigate the potential of the algorithm to consider both single-cell capture procedures and the impact of "less" rare-cell types. The third dataset refers to the murine data of the Allen Brain Atlas, including more than 1 million cells. For validation purposes only, all datasets have also been analyzed traditionally using common data analysis approaches, such as the Seurat workflow. CONCLUSIONS: In comparison to baseline testing without oversampling, our approach identifies rare-cells with a robust precision-recall balance, including a high accuracy and low false positive detection rate. A practical benefit of our algorithm is that it can be readily implemented in other and existing workflows. The code basis in R and Python is publicly available at FairdomHub, as well as GitHub, and can easily be transferred to identify other rare-cell types.

mRNA - A game changer in regenerative medicine, cell-based therapy and reprogramming strategies.

After thirty years of intensive research shaping and optimizing the technology, the approval of the first mRNA-based formulation by the EMA and FDA in order to stop the COVID-19 pandemic was a breakthrough in mRNA research. The astonishing success of these vaccines have brought the mRNA platform into the spotlight of the scientific community. The remarkable persistence of the groundwork is mainly attributed to the exceptional benefits of mRNA application, including the biological origin, immediate but transitory mechanism of action, non-integrative properties, safe and relatively simple manufacturing as well as the flexibility to produce any desired protein. Based on these advantages, a practical implementation of in vitro transcribed mRNA has been considered in most areas of medicine. In this review, we discuss the key preconditions for the rise of the mRNA in the medical field, including the unique structural and functional features of the mRNA molecule and its vehicles, which are crucial aspects for a production of potent mRNA-based therapeutics. Further, we focus on the utility of mRNA tools particularly in the scope of regenerative medicine, i.e. cell reprogramming approaches or manipulation strategies for targeted tissue restoration. Finally, we highlight the strong clinical potential but also the remaining hurdles to overcome for the mRNA-based regenerative therapy, which is only a few steps away from becoming a reality.

Quality control in scRNA-Seq can discriminate pacemaker cells: the mtRNA bias.

Single-cell RNA-sequencing (scRNA-seq) provides high-resolution insights into complex tissues. Cardiac tissue, however, poses a major challenge due to the delicate isolation process and the large size of mature cardiomyocytes. Regardless of the experimental technique, captured cells are often impaired and some capture sites may contain multiple or no cells at all. All this refers to "low quality" potentially leading to data misinterpretation. Common standard quality control parameters involve the number of detected genes, transcripts per cell, and the fraction of transcripts from mitochondrial genes. While cutoffs for transcripts and genes per cell are usually user-defined for each experiment or individually calculated, a fixed threshold of 5% mitochondrial transcripts is standard and often set as default in scRNA-seq software. However, this parameter is highly dependent on the tissue type. In the heart, mitochondrial transcripts comprise almost 30% of total mRNA due to high energy demands. Here, we demonstrate that a 5%-threshold not only causes an unacceptable exclusion of cardiomyocytes but also introduces a bias that particularly discriminates pacemaker cells. This effect is apparent for our in vitro generated induced-sinoatrial-bodies (iSABs; highly enriched physiologically functional pacemaker cells), and also evident in a public data set of cells isolated from embryonal murine sinoatrial node tissue (Goodyer William et al. in Circ Res 125:379-397, 2019). Taken together, we recommend omitting this filtering parameter for scRNA-seq in cardiovascular applications whenever possible.

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.

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.

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.

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.

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].

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.