Circulating exosomes derived from transplanted progenitor cells aid the functional recovery of ischemic myocardium.

The stem cell field is hindered by its inability to noninvasively monitor transplanted cells within the target organ in a repeatable, time-sensitive, and condition-specific manner. We hypothesized that quantifying and characterizing transplanted cell-derived exosomes in the recipient plasma would enable reliable, noninvasive surveillance of the conditional activity of the transplanted cells. To test this hypothesis, we used a human-into-rat xenogeneic myocardial infarction model comparing two well-studied progenitor cell types: cardiosphere-derived cells (CDCs) and c-kit+ cardiac progenitor cells (CPCs), both derived from the right atrial appendage of adults undergoing cardiopulmonary bypass. CPCs outperformed the CDCs in cell-based and in vivo regenerative assays. To noninvasively monitor the activity of transplanted CDCs or CPCs in vivo, we purified progenitor cell-specific exosomes from recipient total plasma exosomes. Seven days after transplantation, the concentration of plasma CPC-specific exosomes increased about twofold compared to CDC-specific exosomes. Computational pathway analysis failed to link CPC or CDC cellular messenger RNA (mRNA) with observed myocardial recovery, although recovery was linked to the microRNA (miRNA) cargo of CPC exosomes purified from recipient plasma. We further identified mechanistic pathways governing specific outcomes related to myocardial recovery associated with transplanted CPCs. Collectively, these findings demonstrate the potential of circulating progenitor cell-specific exosomes as a liquid biopsy that provides a noninvasive window into the conditional state of the transplanted cells. These data implicate the surveillance potential of cell-specific exosomes for allogeneic cell therapies.

Using Statistical Modeling to Understand and Predict Pediatric Stem Cell Function.

BACKGROUND: Congenital heart defects are a leading cause of morbidity and mortality in children, and despite advanced surgical treatments, many patients progress to heart failure. Currently, transplantation is the only effective cure and is limited by donor availability and organ rejection. Recently, cell therapy has emerged as a novel method for treating pediatric heart failure with several ongoing clinical trials. However, efficacy of stem cell therapy is variable, and choosing stem cells with the highest reparative effects has been a challenge. METHODS: We previously demonstrated the age-dependent reparative effects of human c-kit+ progenitor cells (hCPCs) in a rat model of juvenile heart failure. Using a small subset of patient samples, computational modeling analysis showed that regression models could be made linking sequencing data to phenotypic outcomes. In the current study, we used a similar quantitative model to determine whether predictions can be made in a larger population of patients and validated the model using neonatal hCPCs. We performed RNA sequencing from c-kit+ progenitor cells isolated from 32 patients, including 8 neonatal samples. We tested 2 functional parameters of our model, cellular proliferation and chemotactic potential of conditioned media. RESULTS: Interestingly, the observed proliferation and migration responses in each of the selected neonatal hCPC lines matched their predicted counterparts. We then performed canonical pathway analysis to determine potential mechanistic signals that regulated hCPC performance and identified several immune response genes that correlated with performance. ELISA analysis confirmed the presence of selected cytokines in good performing hCPCs and provided many more signals to further validate. CONCLUSIONS: These data show that cell behavior may be predicted using large datasets like RNA sequencing and that we may be able to identify patients whose c-kit+ progenitor cells exceed or underperform expectations. With systems biology approaches, interventions can be tailored to improve cell therapy or mimic the qualities of reparative cells.

Extracellular Vesicles and the Application of System Biology and Computational Modeling in Cardiac Repair.

Recent literature suggests that extracellular vesicles (EVs), secreted from most cells and containing cell-specific cargo of proteins, lipids, and nucleic acids, are major driver of intracellular communication in normal physiology and pathological conditions. The recent evidence on stem/progenitor cell EVs as potential therapeutic modality mimicking their parental cell function is exciting because EVs could possibly be used as a surrogate for the stem cell-based therapy, and this regimen may overcome certain roadblocks identified with the use of stem/progenitor cell themselves. This review provides a comprehensive update on our understanding on the role of EVs in cardiac repair and emphasizes the applications of stem/progenitor cell-derived EVs as therapeutics and discusses the current challenges associated with the EV therapy.

Requirements for Neurogenin2 during mouse postnatal retinal neurogenesis.

During embryonic retinal development, the bHLH factor Neurog2 regulates the temporal progression of neurogenesis, but no role has been assigned for this gene in the postnatal retina. Using Neurog2 conditional mutants, we found that Neurog2 is necessary for the development of an early, embryonic cohort of rod photoreceptors, but also required by both a subset of cone bipolar subtypes, and rod bipolars. Using transcriptomics, we identified a subset of downregulated genes in P2 Neurog2 mutants, which act during rod differentiation, outer segment morphogenesis or visual processing. We also uncovered defects in neuronal cell culling, which suggests that the rod and bipolar cell phenotypes may arise via more complex mechanisms rather than a simple cell fate shift. However, given an overall phenotypic resemblance between Neurog2 and Blimp1 mutants, we explored the relationship between these two factors. We found that Blimp1 is downregulated between E12-birth in Neurog2 mutants, which probably reflects a dependence on Neurog2 in embryonic progenitor cells. Overall, we conclude that the Neurog2 gene is expressed and active prior to birth, but also exerts an influence on postnatal retinal neuron differentiation.

Integral bHLH factor regulation of cell cycle exit and RGC differentiation.

BACKGROUND: In the developing mouse embryo, the bHLH transcription factor Neurog2 is transiently expressed by retinal progenitor cells and required for the initial wave of neurogenesis. Remarkably, another bHLH factor, Ascl1, normally not present in the embryonic Neurog2 retinal lineage, can rescue the temporal phenotypes of Neurog2 mutants. RESULTS: Here we show that Neurog2 simultaneously promotes terminal cell cycle exit and retinal ganglion cell differentiation, using mitotic window labeling and integrating these results with retinal marker quantifications. We also analyzed the transcriptomes of E12.5 GFP-expressing cells from Neurog2GFP/+ , Neurog2GFP/GFP , and Neurog2Ascl1KI/GFP eyes, and validated the most significantly affected genes using qPCR assays. CONCLUSIONS: Our data support the hypothesis that Neurog2 acts at the top of a retinal bHLH transcription factor hierarchy. The combined expression levels of these downstream factors are sufficiently induced by ectopic Ascl1 to restore RGC genesis, highlighting the robustness of this gene network during retinal ganglion cell neurogenesis. Developmental Dynamics 247:965-975, 2018. © 2018 Wiley Periodicals, Inc.

Contralateral migration of oculomotor neurons is regulated by Slit/Robo signaling.

BACKGROUND: Oculomotor neurons develop initially like typical motor neurons, projecting axons out of the ventral midbrain to their ipsilateral targets, the extraocular muscles. However, in all vertebrates, after the oculomotor nerve (nIII) has reached the extraocular muscle primordia, the cell bodies that innervate the superior rectus migrate to join the contralateral nucleus. This motor neuron migration represents a unique strategy to form a contralateral motor projection. Whether migration is guided by diffusible cues remains unknown. METHODS: We examined the role of Slit chemorepellent signals in contralateral oculomotor migration by analyzing mutant mouse embryos. RESULTS: We found that the ventral midbrain expresses high levels of both Slit1 and 2, and that oculomotor neurons express the repellent Slit receptors Robo1 and Robo2. Therefore, Slit signals are in a position to influence the migration of oculomotor neurons. In Slit 1/2 or Robo1/2 double mutant embryos, motor neuron cell bodies migrated into the ventral midbrain on E10.5, three days prior to normal migration. These early migrating neurons had leading projections into and across the floor plate. In contrast to the double mutants, embryos which were mutant for single Slit or Robo genes did not have premature migration or outgrowth on E10.5, demonstrating a cooperative requirement of Slit1 and 2, as well as Robo1 and 2. To test how Slit/Robo midline repulsion is modulated, we found that the normal migration did not require the receptors Robo3 and CXCR4, or the chemoattractant, Netrin 1. The signal to initiate contralateral migration is likely autonomous to the midbrain because oculomotor neurons migrate in embryos that lack either nerve outgrowth or extraocular muscles, or in cultured midbrains that lacked peripheral tissue. CONCLUSION: Overall, our results demonstrate that a migratory subset of motor neurons respond to floor plate-derived Slit repulsion to properly control the timing of contralateral migration.

Netrin1-DCC-Mediated Attraction Guides Post-Crossing Commissural Axons in the Hindbrain.

Commissural axons grow along precise trajectories that are guided by several cues secreted from the ventral midline. After initial attraction to the floor plate using Netrin1 activation of its main attractive receptor, DCC (deleted in colorectal cancer), axons cross the ventral midline, and many turn to grow longitudinally on the contralateral side. After crossing the midline, axons are thought to lose their responsiveness to Netrin1 and become sensitive to midline Slit-Robo repulsion. We aimed to address the in vivo significance of Netrin1 in guiding post-crossing axon trajectories in mouse embryos. Surprisingly, in contrast to the spinal cord, Netrin1 and DCC mutants had abundant commissural axons crossing in the hindbrain. In Netrin1 and DCC mutants, many post-crossing axons made normal turns to grow longitudinally, but projected abnormally at angles away from the midline. In addition, exposure of cultured hindbrain explants to ectopic Netrin1 caused attractive deflection of post-crossing axons. Thus, Netrin1-DCC signaling is not required to attract pre-crossing axons toward the hindbrain floor plate, but is active in post-crossing guidance. Also in contrast with spinal cord, analysis of hindbrain post-crossing axons in Robo1/2 mutant embryos showed that Slit-Robo repulsive signaling was not required for post-crossing trajectories. Our findings show that Netrin1-DCC attractive signaling, but not Slit-Robo repulsive signaling, remains active in hindbrain post-crossing commissural axons to guide longitudinal trajectories, suggesting surprising regional diversity in commissural axon guidance mechanisms. SIGNIFICANCE STATEMENT: The left and right sides of the brainstem and spinal cord are connected primarily by axon fibers that grow across the ventral midline, and then away on the other side to their targets. Based on spinal cord, axons are initially attracted by diffusible attractive protein signals to approach and cross the midline, and then are thought to switch to repulsive cues to grow away on the opposite side. Our results in the hindbrain show that the major midline attractant, Netrin1, is not required for midline crossing. However, the post-crossing axons depend on Netrin1 attraction to set their proper trajectories on the other side. Overall, these findings suggest that commissural axons use distinct mechanisms to navigate in different CNS regions.