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Abstract Background Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is devastating to patients due to the lack of native recovery in central nervous system (CNS) tissues. Obtaining a purified population of INs is necessary to better understand their role in normal function and as potential therapies in CNS. The ventral V0 (V0V) INs are excitatory neurons involved in locomotor circuits and are thus of interest for understanding normal and pathological spinal cord function. To achieve scalable amounts of V0V INs, they can be derived from pluripotent sources, such as mouse embryonic stem cells (mESCs), but the resultant culture is heterogenous, obscuring the specific role of V0V INs. This study generated a transgenic mESC line to enrich V0V INs from induced cultures to allow for a scalable, enriched population for future in vitro and in vivo studies. Methods The transgenic Evx1-PAC mESC line was created by CRISPR-Cas9-mediated insertion of puromycin-N-acetyltransferase (PAC) into the locus of V0V IN marker Evx1. Evx1 and PAC mRNA expression were measured by qPCR. Viability staining helped establish the selection protocol for V0V INs derived from Evx1-PAC mESCs inductions. Immunostaining was used to examine composition of selected inductions. Cultures were maintained up to 30 days to examine maturation by expression of mature/synaptic markers, determined by immunostaining, and functional activity in co-cultures with selected motor neurons (MNs) and V2a INs on microelectrode arrays (MEAs). Results V0V IN inductions were best selected with 4 µg/mL puromycin on day 10 to 11 and showed reduction of other IN populations and elimination of proliferative cells. Long-term selected cultures were highly neuronal, expressing neuronal nuclear marker NeuN, dendritic marker MAP2, pre-synaptic marker Bassoon, and glutamatergic marker VGLUT2, with some cholinergic VAChT-expressing cells. Functional studies on MEAs showed that co-cultures with MNs or MNs plus V2a INs created neuronal networks with synchronized bursting. Conclusions Evx1-PAC mESCs can be used to purify V0V IN cultures for largely glutamatergic neurons that can be used in network formation studies or for rodent models requiring transplanted V0V INs.

In May 2020, the Cleveland Teaching Collaborative (CTC) was created as a digital hub for educators to come together to both reflect on and learn from their individual and collective experiences as instructors during the COVID-19 pandemic. One of the three core components of this hub is the CTC Resource Referatory. The creators of the CTC saw the curation of educational resources as a way to contextualize educator experiences and respond to instructor needs from a place of collective care during the COVID-19 pandemic. Given the CTC’s focus on pedagogies of care and humanizing instructional practice, the curation of the growing number of educational resources that emerged from the CTC’s efforts reveals the limits of longstanding library cataloguing practices that are grounded in controlled vocabularies. The constraints of some of the most commonly used cataloguing practices became a productive tension for the CTC designers and collaborators who were committed to developing a referatory centered around humanizing pedagogies and collective knowledge generation.

The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here, we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.

The Hadassah hESC Research Center's aim is to be a supplier of clinical and research-grade human embryonic stem cell (hESC) lines. In 2012, we derived the first three entirely GMP-compliant and xeno-free, fully-characterised, feeder-dependent (human umbilical cord) hESC lines developed under cleanroom conditions. In 2018, we established four new GMP and xeno-free, feeder-independent MCB hESCs under GMP conditions using commercially available reagents, media and matrix. All cell lines were derived under Israeli Ministry of Health's National Ethics Committee for Genetic Research in Humans and the ethical considerations that guided the development of the hESCs strictly followed Israeli law. Hadassah has provided its clinical-grade hESC lines to commercial entities of which two are already in clinical trials, establishing Hadassah as a key provider of clinical-grade hESC lines. Keywords: Transplantation therapy, Xeno-free, Clinical-grade hESC lines, Disease-affected hESC lines, Biosafety testing, QC testing, Certificate of analysis

Adipose-derived stromal cells (ASCs) are pluripotent cells that have the capacity to differentiate into tendon fibroblasts (TFs). They are abundant in adults, easy to access, and are therefore an ideal cell source for tendon tissue engineering. Despite this potential, the molecular cues necessary for tenogenic differentiation of ASCs are unknown. Unlike other bone morphogenetic proteins (BMPs), BMP12, BMP13, and BMP14 have been reported to be less osteo-chondrogenic and to induce tendon rather than bone formation in vivo. This study investigated the effects of BMP12 and BMP14 on ASC differentiation in vitro. In canine ASCs, BMP12 effectively increased the expression of the tendon markers scleraxis and tenomodulin at both mRNA and protein levels. Consistent with these results, BMP12 induced scleraxis promoter driven-GFP and tenomodulin protein expression in mouse ASCs. Although BMP12 also enhanced the expression of the cartilage matrix gene aggrecan in ASCs, the resulting levels remained considerably lower than those detected in tendon fibroblasts. In addition, BMP12 reduced expression of the bone marker osteocalcin, but not the osteogenic transcription factor runx-2. BMP14 exhibited similar, but marginally less potent and selective effects, compared to BMP12. BMPs are known to signal through the canonical Smad pathway and the non-canonical mitogen-activated protein kinase (MAPK) pathway. BMP12 triggered robust phosphorylation of Smad1/5/8 but not Smad2/3 or p38 MAPK in ASCs. The effect was likely conveyed by type I receptors ALK2/3/6, as phosphorylation of Smad1/5/8 was blocked by the ALK2/3/6 inhibitor LDN-193189 but not by the ALK4/5/7 inhibitor SB-505124. Moreover, ALK6 was found to be the most abundant type I receptor in ASCs, with mRNA expression 100 to 10,000 times that of any other type I receptor. Collectively, results support the conclusion that BMP12 induces tenogenic differentiation of ASCs via the Smad1/5/8 pathway.

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.

A consistent problem with stem/neural progenitor cell transplantation following spinal cord injury (SCI) is poor cell survival and uncontrolled differentiation following transplantation. The current study evaluated the feasibility of enhancing embryonic stem cell-derived neural progenitor cell (ESNPC) viability and directing their differentiation into neurons and oligodendrocytes by embedding the ESNPCs in fibrin scaffolds containing growth factors (GF) and a heparin-binding delivery system (HBDS) in a subacute rat model of SCI. Mouse ESNPCs were generated from mouse embryonic stem cells (ESCs) using a 4–/4+ retinoic acid (RA) induction protocol. The ESNPCs were then transplanted as embryoid bodies (EBs, 70% neural progenitor cells) into the subacute model of SCI. ESNPCs (10 EBs per animal) were implanted directly into the SCI lesion, encapsulated in fibrin scaffolds, encapsulated in fibrin scaffolds containing the HBDS, neurotrophin-3 (NT-3), and platelet-derived growth factor (PDGF), or encapsulated in fibrin scaffolds with NT-3 and PDGF with no HBDS. We report here that the combination of the NT-3, PDGF, and fibrin scaffold (with or without HBDS) enhanced the total number of ESNPCs present in the spinal cord lesion 2 weeks after injury. In addition, the inclusion of the HBDS with growth factor resulted in an increase in the number of ESNPC-derived NeuN-positive neurons. These results demonstrate the ability of fibrin scaffolds and the controlled release of growth factors to enhance the survival and differentiation of neural progenitor cells following transplantation into a SCI model.

A novel composite endpoint, sustained pain-free/no adverse events, was recently proposed as a more rigorous means of capturing in a single measure the attributes of migraine pharmacotherapy that patients consider most important: rapid and sustained pain-free response with no side-effects. Using pooled data from two replicate randomized, double-blind, parallel-group, placebo-controlled studies, this post hoc analysis compared the fixed-dose combination tablet sumatriptan/naproxen sodium (n = 726) with sumatriptan monotherapy (n = 723), naproxen sodium monotherapy (n = 720), and placebo (n = 742) with respect to sustained pain-free/no adverse events and closely related composite measures. Sustained pain-free/no adverse events was defined as having both a sustained pain-free response from 2 through 24 hours post-dose with no use of rescue medication and having no adverse events within up to 5 days after dosing with study medication. The percentage of patients with sustained pain-free/no adverse events was 16% with sumatriptan/naproxen sodium compared with 11%, 9% and 7% for sumatriptan, naproxen sodium and placebo, respectively (p50.01 sumatriptan/naproxen sodium versus each other treatment). Sumatriptan/naproxen sodium was also significantly more effective than sumatriptan, naproxen sodium, and placebo for other composite endpoints including the percentages of patients with (1) sustained pain-free/no adverse events within 1 day; (2) sustained pain-free/no drug-related adverse events within up to 5 days; (3) sustained pain-free/no drug-related adverse events within 1 day; (4) sustained pain relief/no adverse events within up to 5 days; and (5) sustained pain relief/no adverse events within 1 day. The results demonstrate the superiority of sumatriptan/naproxen sodium to sumatriptan monotherapy, naproxen sodium monotherapy and placebo with respect to the rigorous and clinically relevant endpoint of sustained pain-free/no adverse events and reinforce the usefulness of utilizing this new composite endpoint.

Our nationwide network of BME women faculty collectively argue that racial funding disparity by the National Institutes of Health (NIH) remains the most insidious barrier to success of Black faculty in our profession. We thus refocus attention on this critical barrier and suggest solutions on how it can be dismantled.

Global warming is a major concern, and tackling it via education is crucial. The purpose of this study is to investigate the impact of combining the Science, Technology, Engineering, and Mathematics (STEM) approach with the Project-based Learning (PjBL) model on students' learning outcomes and science process skills in global warming topics. The study, which used a one-group pretest-posttest design, collected data from 33 grade XI students via essay questions and observation sheets to assess learning outcomes and science process skills. Critical indicators of science process abilities were observing, classifying, interpreting, predicting, applying concepts, communicating, and forming conclusions. Students worked on a STEM-based project to build a flood-resistant house over the course of five meetings (including the pretest and post-test). The findings revealed considerable improvements: learning outcomes increased by 0.6 (medium category), while science process skills improved by 0.7 (high category). Observations revealed that pupils' scientific process skills were excellent. The Wilcoxon test revealed a significant difference between students' learning outcomes and science process abilities before and after the intervention, with a Sig (2-tailed) value of 0.000. The study indicated that combining the STEM method with the PjBL model improves students' learning outcomes and scientific process abilities. However, the small sample size implies that the results may not be generalizable. The consequence is that integrating the STEM method and the PjBL model provides an innovative option in physics education, improving learning outcomes and scientific process skills.

Employers who have used natural supports in providing accommodations to supported employees were interviewed to determine their perceptions regarding the accommodation process. Results indicate that employers did not provide natural supports to supported employees beyond those offered to other employees; additional supports changed work environments only in positive ways. (Author/MKA)

Enriched in glycolytic enzymes, paucicellular and hypovascular intrasynovial flexor tendons fail to mount an effective healing response after injury and repair. In contrast, well‐vascularized extrasynovial flexor tendons possess high levels of oxidative phosphorylation (OXPHOS) enzymes and have a markedly improved healing capacity. This study was designed to compare the metabolic profiles of the two types of tendons and to evaluate the impact of metabolic reprogramming on early intrasynovial tendon healing in a clinically relevant canine model. Results showed that healthy intrasynovial tendons expressed higher levels of PDK1 and GAPDH and lower levels of SCX and IGF1 than did extrasynovial tendons. PDK1 encodes a subtype of pyruvate dehydrogenase kinase (PDK) that inhibits OXPHOS. Consistently, ATP production via glycolysis was favored in intrasynovial tendon cells whereas OXPHOS was the preferred pathway in extrasynovial tendon cells. Inhibition of glycolysis in vitro increased SCX expression in intrasynovial tendon cells. Therefore, dichloroacetate (DCA), a PDK1 inhibitor, was used in vivo to shift intrasynovial tendon ATP production from glycolysis to OXPHOS. Oral DCA administration reduced serum lactate concentration and increased acetyl‐CoA content in repaired intrasynovial tendons and led to reduced TLR4 and IL1B and increased IGF1, SCX, and TGFB3 expressions in treated intrasynovial tendons compared to controls. Immunohistochemistry staining with anti‐Ki67 and anti‐CD31 antibodies revealed marked increases in cellularity and neovascularization in treated intrasynovial tendons. Clinical significance: The findings of this experiment indicate that improved gene expression and histological outcomes can be achieved by regulating glucose metabolism in the early stages following intrasynovial tendon repair. [ABSTRACT FROM AUTHOR]