Search

BackgroundPeripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia.MethodsThe ActinCreER/R26VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions.ResultsTwo populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105highSca1low, which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice.ConclusionsThese findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.

Group work in science, technology, engineering, and mathematics courses is an effective means of improving student outcomes, and many different factors can influence the dynamics of student discussions and, ultimately, the success of collaboration. The substance and dynamics of group discussions are commonly examined using qualitative methods such as discourse analysis. To complement existing work in the literature, we developed a quantitative methodology that uses graph theory to map the progression of talk-turns of discussions within a group. We observed groups of students working with peer facilitators to solve problems in biological sciences, with three iterations of data collection and two major refinements of graph theory calculations. Results include general behaviors based on the turns in which different individuals talk and graph theory parameters to quantify group characteristics. To demonstrate the potential utility of the methodology, we present case studies with distinct patterns: a centralized group in which the peer facilitator behaves like an authority figure, a decentralized group in which most students talk their fair share of turns, and a larger group with subgroups that have implications for equity, diversity, and inclusion. Together, these results demonstrate that our adaptation of graph theory is a viable quantitative methodology to examine group discussions.

The neurons of the three cerebellar nuclei (CN) are the primary output neurons of the cerebellum. The excitatory neurons (e) of the medial (m) CN (eCNm) were recently divided into molecularly defined subdomains in the adult; however, how they are established during development is not known. We define molecular subdomains of the mouse embryonic eCNm using single-cell RNA-sequencing and spatial expression analysis, showing that they evolve during embryogenesis to prefigure the adult. Furthermore, eCNm are transcriptionally divergent from cells in the other nuclei by embryonic day 14.5. We previously showed that loss of the homeobox genes En1 and En2 leads to loss of approximately half of the embryonic eCNm. We demonstrate that mutation of En1/2 in the embryonic eCNm results in death of specific posterior eCNm molecular subdomains and downregulation of TBR2 (EOMES) in an anterior embryonic subdomain, as well as reduced synaptic gene expression. We further reveal a similar function for EN1/2 in mediating TBR2 expression, neuron differentiation and survival in the other excitatory neurons (granule and unipolar brush cells). Thus, our work defines embryonic eCNm molecular diversity and reveals conserved roles for EN1/2 in the cerebellar excitatory neuron lineage. [ABSTRACT FROM AUTHOR]

Stem-cell-based therapies hold considerable promise for regenerative medicine. However, acute donor-cell death within several weeks after cell delivery remains a critical hurdle for clinical translation. Co-transplantation of stem cells with pro-survival factors can improve cell engraftment, but this strategy has been hampered by the typically short half-lives of the factors and by the use of Matrigel and other scaffolds that are not chemically defined. Here, we report a collagen-dendrimer biomaterial crosslinked with pro-survival peptide analogues that adheres to the extracellular matrix and slowly releases the peptides, significantly prolonging stem cell survival in mouse models of ischaemic injury. The biomaterial can serve as a generic delivery system to improve functional outcomes in cell-replacement therapy.

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 103 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. Stem Cells 2017;35:1994-2000.

Group work in science, technology, engineering, and mathematics courses is an effective means of improving student outcomes, and many different factors can influence the dynamics of student discussions and, ultimately, the success of collaboration. The substance and dynamics of group discussions are commonly examined using qualitative methods such as discourse analysis. To complement existing work in the literature, we developed a quantitative methodology that uses graph theory to map the progression of talk-turns of discussions within a group. We observed groups of students working with peer facilitators to solve problems in biological sciences, with three iterations of data collection and two major refinements of graph theory calculations. Results include general behaviors based on the turns in which different individuals talk and graph theory parameters to quantify group characteristics. To demonstrate the potential utility of the methodology, we present case studies with distinct patterns: a centralized group in which the peer facilitator behaves like an authority figure, a decentralized group in which most students talk their fair share of turns, and a larger group with subgroups that have implications for equity, diversity, and inclusion. Together, these results demonstrate that our adaptation of graph theory is a viable quantitative methodology to examine group discussions.

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Objective: Simulation may be a valuable tool in training laryngology office procedures on unsedated patients. However, no studies have examined whether existing awake procedure simulators improve trainee performance in laryngology. Our objective was to evaluate the transfer validity of a previously published 3D‐printed laryngeal simulator in improving percutaneous injection laryngoplasty (PIL) competency compared with conventional educational materials with a single‐blinded randomized controlled trial. Methods: Otolaryngology residents with fewer than 10 PIL procedures in their case logs were recruited. A pretraining survey was administered to participants to evaluate baseline procedure‐specific knowledge and confidence. The participants underwent block randomization by postgraduate year to receive conventional educational materials either with or without additional training with a 3D‐printed laryngeal simulator. Participants performed PIL on an anatomically distinct laryngeal model via trans‐thyrohyoid and trans‐cricothyroid approaches. Endoscopic and external performance recordings were de‐identified and evaluated by two blinded laryngologists using an objective structured assessment of technical skill scale and PIL‐specific checklist. Results: Twenty residents completed testing. Baseline characteristics demonstrate no significant differences in confidence level or PIL experience between groups. Senior residents receiving simulator training had significantly better respect for tissue during the trans‐thyrohyoid approach compared with control (p < 0.0005). There were no significant differences in performance for junior residents. Conclusions: In this first transfer validity study of a simulator for office awake procedure in laryngology, we found that a previously described low‐cost, high‐fidelity 3D‐printed PIL simulator improved performance of PIL amongst senior otolaryngology residents, suggesting this accessible model may be a valuable educational adjunct for advanced trainees to practice PIL. Level of Evidence: NA Laryngoscope, 134:318–323, 2024 [ABSTRACT FROM AUTHOR]

BackgroundDrug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.Methods and resultsPharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.ConclusionsWe found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

BackgroundDrug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.Methods and resultsPharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.ConclusionsWe found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

RationaleInduced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models.ObjectiveTo successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia.Methods and resultsPorcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P

BackgroundHuman cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts.Methods and resultsWe used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P

Background: Aboriginal children are at increased risk for obesity and type 2 diabetes. Healthy Buddies [TM]-First Nations (HB) is a curriculum-based, peer-led program promoting healthy eating, physical activity, and self-esteem. Methods: Although originally designed as a pilot pre-/post-analysis of 3 remote Aboriginal schools that requested and received HB training, one school did not implement the program and was used as a control group. Outcomes included changes in body mass index z-score (zBMI), waist circumference (WC), blood pressure (BP), self-esteem, health behavior, and knowledge over 1 school year in kindergarten to grade 12 children. Results: There was a significant decrease in zBMI (1.10 to 1.04, p=0.028) and WC (77.1 to 75.0 cm, p less than 0.0001) in the HB group (N=118) compared with an increase in zBMI (1.14 to 1.23, p=0.046) and a minimal WC change in the control group (N=61). Prevalence of elevated BP did not change in the HB group, but increased from 16.7% to 31.7% in the control group (p=0.026). General linear model analysis revealed a significant interaction between time, group, and zBMI (p=0.001), weight status (p=0.014), nutritious beverage knowledge (p=0.018), and healthy living and self-esteem score (p=0.005). Conclusions: The HB program is a promising school-based strategy for addressing obesity and self-esteem in Aboriginal children. (Contains 3 tables and 2 figures.)