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.

Identification of SALL4 Expressing Islet-1+ Cardiovascular Progenitor Cell Clones.

The utilization of cardiac progenitor cells (CPCs) has been shown to induce favorable regenerative effects. While there are various populations of endogenous CPCs in the heart, there is no consensus regarding which population is ideal for cell-based regenerative therapy. Early-stage progenitor cells can be differentiated into all cardiovascular lineages, including cardiomyocytes and endothelial cells. Identifying an Islet-1+ (Isl-1+) early-stage progenitor population with enhanced stemness, multipotency and differentiation potential would be beneficial for the development of novel regenerative therapies. Here, we investigated the transcriptome of human neonatal Isl-1+ CPCs. Isl-1+ human neonatal CPCs exhibit enhanced stemness properties and were found to express Spalt-like transcription factor 4 (SALL4). SALL4 plays a role in embryonic development as well as proliferation and expansion of hematopoietic progenitor cells. SALL4, SOX2, EpCAM and TBX5 are co-expressed in the majority of Isl-1+ clones isolated from neonatal patients. The pre-mesendodermal transcript TFAP2C was identified in select Isl-1, SALL4, SOX2, EpCAM and TBX5 expressing clones. The ability to isolate and expand pre-mesendodermal stage cells from human patients is a novel finding that holds potential value for applications in regenerative medicine.

MicroRNA Expression in the Infarcted Heart Following Neonatal Cardiovascular Progenitor Cell Transplantation in a Sheep Model of Stem Cell-Based Repair.

Myocardial infarctions affect approximately 735,000 people annually in the United States and have a substantial impact on quality of life. Neonates have an enhanced capability of repairing cardiovascular damage, while adults do not. The mechanistic basis for this age-dependent difference in regenerative capacity remains unknown. Recent studies have shown that microRNAs (miRNAs) play a significant role in regulating the regenerative ability of cardiovascular cells. This report defines the alterations in miRNA expression within the cardiovascular repair zone of infarcted sheep hearts following intracardiac injection of neonatal islet-1+ cardiovascular progenitor cells. Sheep were infarcted via left anterior descending coronary artery ligation. After 3 to 4 weeks of infarction, sheep neonatal islet-1+ cardiovascular progenitor cells were injected into the infarcted area for repair. Cell-treated sheep were euthanized 2 months following cell injection, and their hearts were harvested for the analysis of miRNA and gene expression within the cardiovascular repair zone. Ten miRNAs were differentially regulated in vivo, including miR-99, miR-100, miR-302a, miR-208a, miR-665, miR-1, miR-499a, miR-34a, miR-133a, and miR-199a. These miRNAs promote stemness, cell division, and survival. Several signaling pathways are regulated by these miRNAs, including Hippo, Wnt, and Erythroblastic Leukemia Viral Oncogene B (ERBB). Transcripts encoding Wnt, ERBB, and Neuregulin 1 (NRG1) were elevated in vivo in the infarct repair zone. Wnt5a signaling and ERBB/NRG1 transcripts contribute to activation of Yes-Associated Protein 1. MiRNAs that impact proliferation, cell survival, and signaling pathways that promote regeneration were induced during cardiovascular repair in the sheep model. This information can be used to design new approaches for the optimization of miRNA-based treatments for the heart.

A pilot study on the biochemical effects of repeated administration of 24% oral sucrose vs. 30% oral dextrose on urinary markers of adenosine triphosphate degradation.

OBJECTIVES: Premature neonates often receive oral sucrose or dextrose before tissue-damaging procedures (TDPs). Previous work showed that a single dose of sucrose, but not dextrose, increased cellular energy utilization and ATP degradation. This pilot study probes the effects of repeated administration of sucrose or dextrose on energy metabolism. METHODS: Urinary markers of ATP metabolism (hypoxanthine, xanthine, uric acid) are measured in premature neonates randomized to receive: (a) standard of care, (b) 0.2 ml 24% oral sucrose, or (c) 0.2 ml 30% oral dextrose, before every painful procedure on days-of-life 3-7. RESULTS: Standard of care is associated with highest xanthine/creatinine and uric acid/creatinine, likely because of fewer pain treatments. Benefits of repeated oral sucrose are unclear. Neonates receiving oral dextrose had lower xanthine/creatinine and uric acid/creatinine. CONCLUSIONS: Repeated treatments of neonatal procedural pain with 30% oral dextrose are less energetically demanding. Larger clinical studies are needed for comparison with sucrose treatments.

Long-Term Hypoxia Maintains a State of Dedifferentiation and Enhanced Stemness in Fetal Cardiovascular Progenitor Cells.

Early-stage mammalian embryos survive within a low oxygen tension environment and develop into fully functional, healthy organisms despite this hypoxic stress. This suggests that hypoxia plays a regulative role in fetal development that influences cell mobilization, differentiation, proliferation, and survival. The long-term hypoxic environment is sustained throughout gestation. Elucidation of the mechanisms by which cardiovascular stem cells survive and thrive under hypoxic conditions would benefit cell-based therapies where stem cell survival is limited in the hypoxic environment of the infarcted heart. The current study addressed the impact of long-term hypoxia on fetal Islet-1+ cardiovascular progenitor cell clones, which were isolated from sheep housed at high altitude. The cells were then cultured in vitro in 1% oxygen and compared with control Islet-1+ cardiovascular progenitor cells maintained at 21% oxygen. RT-PCR, western blotting, flow cytometry, and migration assays evaluated adaptation to long term hypoxia in terms of survival, proliferation, and signaling. Non-canonical Wnt, Notch, AKT, HIF-2α and Yap1 transcripts were induced by hypoxia. The hypoxic niche environment regulates these signaling pathways to sustain the dedifferentiation and survival of fetal cardiovascular progenitor cells.

The Impact of Spaceflight and Microgravity on the Human Islet-1+ Cardiovascular Progenitor Cell Transcriptome.

Understanding the transcriptomic impact of microgravity and the spaceflight environment is relevant for future missions in space and microgravity-based applications designed to benefit life on Earth. Here, we investigated the transcriptome of adult and neonatal cardiovascular progenitors following culture aboard the International Space Station for 30 days and compared it to the transcriptome of clonally identical cells cultured on Earth. Cardiovascular progenitors acquire a gene expression profile representative of an early-stage, dedifferentiated, stem-like state, regardless of age. Signaling pathways that support cell proliferation and survival were induced by spaceflight along with transcripts related to cell cycle re-entry, cardiovascular development, and oxidative stress. These findings contribute new insight into the multifaceted influence of reduced gravitational environments.

Effects of Spaceflight and Simulated Microgravity on YAP1 Expression in Cardiovascular Progenitors: Implications for Cell-Based Repair.

Spaceflight alters many processes of the human body including cardiac function and cardiac progenitor cell behavior. The mechanism behind these changes remains largely unknown; however, simulated microgravity devices are making it easier for researchers to study the effects of microgravity. To study the changes that take place in cardiac progenitor cells in microgravity environments, adult cardiac progenitor cells were cultured aboard the International Space Station (ISS) as well as on a clinostat and examined for changes in Hippo signaling, a pathway known to regulate cardiac development. Cells cultured under microgravity conditions, spaceflight-induced or simulated, displayed upregulation of downstream genes involved in the Hippo pathway such as YAP1 and SOD2. YAP1 is known to play a role in cardiac regeneration which led us to investigate YAP1 expression in a sheep model of cardiovascular repair. Additionally, to mimic the effects of microgravity, drug treatment was used to induce Hippo related genes as well as a regulator of the Hippo pathway, miRNA-302a. These studies provide insight into the changes that occur in space and how the effects of these changes relate to cardiac regeneration studies.