The Benefits of Stem Cell Biology and Tissue Engineering in Low-Earth Orbit.

Over the past 15 years, there has been a significant shift in biomedical research toward a major focus on stem cell research. Although stem cells and their derivatives exhibit potential in modeling and mitigating human diseases, the ongoing objective is to enhance their utilization and translational potential. Stem cells are increasingly employed in both academic and commercial settings for a variety of in vitro and in vivo applications in regenerative medicine. Notably, accessibility to stem cell research in low-Earth orbit (LEO) has expanded, driven by the unique properties of space, such as microgravity, which cannot exactly be replicated on Earth. As private enterprises continue to grow and launch low-orbit payloads alongside government-funded spaceflight, space has evolved into a more viable destination for scientific exploration. This review underscores the potential benefits of microgravity on fundamental stem cell properties, highlighting the adaptability of cells to their environment and emphasizing physical stimuli as a key factor influencing cultured cells. Previous studies suggest that stimuli such as magnetic fields, shear stress, or gravity impact not only cell kinetics, including differentiation and proliferation, but also therapeutic effects such as cells with improved immunosuppressive capabilities or the ability to identify novel targets to refine disease treatments. With the rapid progress and sustained advocacy for space research, we propose that the advantageous properties of LEO create novel opportunities in biomanufacturing for regenerative medicine, spanning disease modeling, the development of stem cell-derived products, and biofabrication.

17 ß-Estradiol Impedes Aortic Root Dilation and Rupture in Male Marfan Mice.

Marfan syndrome causes a hereditary form of thoracic aortic aneurysms with worse outcomes in male compared to female patients. In this study, we examine the effects of 17 ß-estradiol on aortic dilation and rupture in a Marfan mouse model. Marfan male mice were administered 17 ß-estradiol, and the growth in the aortic root, along with the risk of aortic rupture, was measured. Transcriptomic profiling was used to identify enriched pathways from 17 ß-estradiol treatments. Aortic smooth muscle cells were then treated with cytokines to validate functional mechanisms. We show that 17 ß-estradiol decreased the size and rate of aortic root dilation and improved survival from rupture. The Marfan transcriptome was enriched in inflammatory genes, and the addition of 17 ß-estradiol modulated a set of genes that function through TNFα mediated NF-κB signaling. In addition, 17 ß-estradiol suppressed the induction of these TNFα induced genes in aortic smooth muscle cells in vitro in an NF-κB dependent manner, and 17 ß-estradiol decreased the formation of adventitial inflammatory foci in aortic roots in vivo. In conclusion, 17 ß-estradiol protects against the dilation and rupture of aortic roots in Marfan male mice through the inhibition of TNFα-NF-κB signaling.

Proteomics of novel induced pluripotent stem cell-derived vascular endothelial cells reveal extensive similarity with an immortalized human endothelial cell line.

The vascular endothelium constitutes the inner lining of the blood vessel, and malfunction and injuries of the endothelium can cause cardiovascular diseases as well as other diseases including stroke, tumor growth, and chronic kidney failure. Generation of effective sources to replace injured endothelial cells (ECs) could have significant clinical impact, and somatic cell sources like peripheral or cord blood cannot credibly supply enough endothelial cell progenitors for multitude of treatments. Pluripotent stem cells are a promising source for a reliable EC supply, which have the potential to restore tissue function and treat vascular diseases. We have developed methods to differentiate induced pluripotent stem cells (iPSCs) efficiently and robustly across multiple iPSC lines into nontissue-specific pan vascular ECs (iECs) with high purity. These iECs present with canonical endothelial cell markers and exhibit measures of endothelial cell functionality with the uptake of Dil fluorescent dye-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) and tube formation. Using proteomic analysis, we revealed that the iECs are more proteomically similar to established human umbilical vein ECs (HUVECs) than to iPSCs. Posttranslational modifications (PTMs) were most shared between HUVECs and iECs, and potential targets for increasing the proteomic similarity of iECs to HUVECs were identified. Here we demonstrate an efficient robust method to differentiate iPSCs into functional ECs, and for the first time provide a comprehensive protein expression profile of iECs, which indicates their similarities with a widely used immortalized HUVECs, allowing for further mechanistic studies of EC development, signaling, and metabolism for future regenerative applications.NEW & NOTEWORTHY We have developed methods to differentiate induced pluripotent stem cells (iPSCs) across multiple iPSC lines into nontissue-specific pan vascular ECs (iECs) and demonstrated the proteomic similarity of these cells to a widely used endothelial cell line (HUVECs). We also identified posttranslational modifications and targets for increasing the proteomic similarity of iECs to HUVECs. In the future, iECs can be used to study EC development, signaling, and metabolism for future regenerative applications.

Neonatal Cardiovascular-Progenitor-Cell-Derived Extracellular Vesicles Activate YAP1 in Adult Cardiac Progenitor Cells.

New stem cell and extracellular-vesicle-based therapies have the potential to improve outcomes for the increasing number of patients with heart failure. Since neonates have a significantly enhanced regenerative ability, we hypothesized that extracellular vesicles isolated from Islet-1+ expressing neonatal human cardiovascular progenitors (CPCs) will induce transcriptomic changes associated with improved regenerative capability when co-cultured with CPCs derived from adult humans. In order to test this hypothesis, we isolated extracellular vesicles from human neonatal Islet-1+ CPCs, analyzed the extracellular vesicle content using RNAseq, and treated adult CPCs with extracellular vesicles derived from neonatal CPCs to assess their functional effect. AKT, ERBB, and YAP1 transcripts were elevated in adult CPCs treated with neonatal CPC-derived extracellular vesicles. YAP1 is lost after the neonatal period but can stimulate cardiac regeneration. Our results demonstrate that YAP1 and additional transcripts associated with improved cardiovascular regeneration, as well as the activation of the cell cycle, can be achieved by the treatment of adult CPCs with neonatal CPC-derived extracellular vesicles. Progenitor cells derived from neonates secrete extracellular vesicles with the potential to stimulate and potentially improve functional effects in adult CPCs used for cardiovascular repair.

17 ß-estradiol impedes aortic root dilation and rupture in male Marfan mice.

Marfan syndrome causes a hereditary form of thoracic aortic aneurysms with dilation of the aortic root. Human and animal models suggest a worse phenotype for males compared to females with respect to aneurysm size and risk of dissection. In this study we examine the effects of 17 ß-estradiol on aortic dilation and rupture in a Marfan mouse model. Marfan male mice were administered 17 ß-estradiol and the growth in aortic root size along with the risk of aortic rupture or dissection with the addition of angiotensin II was measured. Transcriptomic profiling was used to identify enriched pathways from 17 ß-estradiol treatment. Aortic smooth muscle cells were then treated with cytokines in order to validate the mechanism of 17 ß-estradiol protection. We show that 17 ß-estradiol decreased the size and rate of aortic root dilation and improved survival from rupture and dissection after treatment with angiotensin II. The Marfan transcriptome was enriched in inflammatory genes and the addition of 17 ß-estradiol modulated a set of genes that function through TNFα mediated NF-κB signaling. These included many proteins known to play a role in the phenotypic shift of aortic smooth muscle cells from a contractile to a more inflammatory-like state such as Vcam-1, Mcp-1, Lgals3, Il-6, Il-1b, and C3. In addition, 17 ß-estradiol suppressed the induction of these TNFα induced genes in aortic smooth muscle cells in vitro and this effect appears to be NF-κB dependent. In conclusion, 17 ß-estradiol protects against the dilation and rupture of aortic roots in Marfan male mice through the inhibition of TNFα -NF-κB signaling and thus prevents the phenotypic switch of aortic smooth muscle cells from a contractile to an inflammatory state.

BIRCH: An Automated Workflow for Evaluation, Correction, and Visualization of Batch Effect in Bottom-Up Mass Spectrometry-Based Proteomics Data.

Recent surges in large-scale mass spectrometry (MS)-based proteomics studies demand a concurrent rise in methods to facilitate reliable and reproducible data analysis. Quantification of proteins in MS analysis can be affected by variations in technical factors such as sample preparation and data acquisition conditions leading to batch effects, which adds to noise in the data set. This may in turn affect the effectiveness of any biological conclusions derived from the data. Here we present Batch-effect Identification, Representation, and Correction of Heterogeneous data (BIRCH), a workflow for analysis and correction of batch effect through an automated, versatile, and easy to use web-based tool with the goal of eliminating technical variation. BIRCH also supports diagnosis of the data to check for the presence of batch effects, feasibility of batch correction, and imputation to deal with missing values in the data set. To illustrate the relevance of the tool, we explore two case studies, including an iPSC-derived cell study and a Covid vaccine study to show different context-specific use cases. Ultimately this tool can be used as an extremely powerful approach for eliminating technical bias while retaining biological bias, toward understanding disease mechanisms and potential therapeutics.

A myocardial infarct border-zone-on-a-chip demonstrates distinct regulation of cardiac tissue function by an oxygen gradient.

After a myocardial infarction, the boundary between the injured, hypoxic tissue and the adjacent viable, normoxic tissue, known as the border zone, is characterized by an oxygen gradient. Yet, the impact of an oxygen gradient on cardiac tissue function is poorly understood, largely due to limitations of existing experimental models. Here, we engineered a microphysiological system to controllably expose engineered cardiac tissue to an oxygen gradient that mimics the border zone and measured the effects of the gradient on electromechanical function and the transcriptome. The gradient delayed calcium release, reuptake, and propagation; decreased diastolic and peak systolic stress; and increased expression of inflammatory cascades that are hallmarks of myocardial infarction. These changes were distinct from those observed in tissues exposed to uniform normoxia or hypoxia, demonstrating distinct regulation of cardiac tissue phenotypes by an oxygen gradient. Our border-zone-on-a-chip model advances functional and mechanistic insight into oxygen-dependent cardiac tissue pathophysiology.

Community and shotgun metagenomic analysis of Alligator mississippiensis oral cavity and GI tracts reveal complex ecosystems and potential reservoirs of antibiotic resistance.

We report here the community structure and functional analysis of the microbiome of the Alligator mississippiensis GI tract from the oral cavity through the entirety of the digestive tract. Although many vertebrate microbiomes have been studied in recent years, the archosaur microbiome has only been given cursory attention. In the oral cavity we used amplicon-based community analysis to examine the structure of the oral microbiome during alligator development. We found a community that diversified over time and showed many of the hallmarks we would expect of a stable oral community. This is a bit surprising given the rapid turnover of alligator teeth but suggests that the stable gumline microbes are able to rapidly colonize the emerging teeth. As we move down the digestive tract, we were able to use both long and short read sequencing approaches to evaluate the community using a shotgun metagenomics approach. Long read sequencing was applied to samples from the stomach/duodenum, and the colorectal region, revealing a fairly uniform and low complexity community made up primarily of proteobacteria at the top of the gut and much more diversity in the colon. We used deep short read sequencing to further interrogate this colorectal community. The two sequencing approaches were concordant with respect to community structure but substantially more detail was available in the short read data, in spite of high levels of host DNA contamination. Using both approaches we were able to show that the colorectal community is a potential reservoir for antibiotic resistance, human pathogens such as Clostridiodes difficile and a possible source of novel antimicrobials or other useful secondary metabolites.