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A comparison of different ways of generating induced pluripotent stem cells helps researchers choose the most appropriate method for particular applications. Human induced pluripotent stem cells (hiPSCs 1 , 2 , 3 ) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV) 4 , episomal (Epi) 5 and mRNA transfection mRNA 6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation.

Human brown and white preadipocyte clones from neck fat depots have been isolated and used to identify genetic biomarkers that predict their thermogenic capacity. Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB , in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.

Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. Both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here, we demonstrated the generated clones of brown and white preadipocytes from human neck fat of four individuals and characterized their adipogenic differentiation and thermogenic function. Combining an uncoupling protein 1(UCP1) reporter system and expression profiling, we defined novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated in culture. Knocking out the positive UCP1 regulators identified by this approach, PREX1 and EDNRB in brown preadipocytes using CRISPR/Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer the identification of possible biomarkers of thermogenically competent preadipocytes.

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.

The growth factor and cytokine regulated transcription factor STAT3 is required for the self-renewal of several stem cell types including tumor stem cells from glioblastoma. Here we show that STAT3 inhibition leads to the upregulation of the histone H3K27me2/3 demethylase Jmjd3 (KDM6B), which can reverse polycomb complex-mediated repression of tissue specific genes. STAT3 binds to the Jmjd3 promoter, suggesting that Jmjd3 is a direct target of STAT3. Overexpression of Jmjd3 slows glioblastoma stem cell growth and neurosphere formation, whereas knockdown of Jmjd3 rescues the STAT3 inhibitor-induced neurosphere formation defect. Consistent with this observation, STAT3 inhibition leads to histone H3K27 demethylation of neural differentiation genes, such as Myt1, FGF21, and GDF15. These results demonstrate that the regulation of Jmjd3 by STAT3 maintains repression of differentiation specific genes and is therefore important for the maintenance of self-renewal of normal neural and glioblastoma stem cells.

Transdisciplinary sustainability scientists are called to conduct research with community actors to understand and improve relations between people and nature. Yet, research hierarchies and power relations continue to favour western academic researchers who remain the gatekeepers of knowledge production and validation. To counter this imbalance, in 2018 we structured a multi‐day workshop to co‐design a set of principles to guide our own transdisciplinary, international and intercultural community of practice for biocultural diversity and sustainability. This community includes community collaborators, partner organizations, and early career and established researchers from Argentina, Bolivia, Canada, Germany, Mexico and South Africa. In 2021, we undertook online critical reflection workshops to share our research experiences and deepen our intercultural understanding of the application of the principles. Through these exercises, we adopted seven principles for working together that include: honour self‐determination and nationhood; commit to reciprocal relationships; co‐create the research agenda; approach research in a good way: embed relational accountability; generate meaningful benefits for communities; build in equity, diversity and inclusion; and emphasize critical reflection and shared learning. We explain these principles and briefly highlight their application to our research practices. By sharing these principles and associated practices, we seek to facilitate debate and spur transformations in how we conduct international and intercultural sustainability research. Our efforts also illustrate a strategy for on‐going knowledge co‐production as we cultivate safe and ethical spaces for learning together. Lessons learned may be particularly useful to those who engage in intercultural, collaborative research to advance sustainability transformations. Read the free Plain Language Summary for this article on the Journal blog. Resumen Los científicos que trabajan en sostenibilidad de manera transdisciplinaria están llamados a realizar investigaciones con actores comunitarios para comprender y mejorar las relaciones entre las personas y la naturaleza. Sin embargo, las jerarquías de investigación y las relaciones de poder continúan favoreciendo a los investigadores académicos occidentales que siguen siendo los guardianes de la producción y validación del conocimiento. Para contrarrestar este desequilibrio, en 2018 estructuramos un taller de varios días para co‐diseñar un conjunto de principios para guiar nuestra propia comunidad de práctica transdisciplinaria, internacional e intercultural para la diversidad biocultural y la sostenibilidad. Esta comunidad incluye colaboradores comunitarios, organizaciones socias e investigadores establecidos y de carrera temprana de Argentina, Bolivia, Canadá, Alemania, México y Sudáfrica. En 2021, llevamos a cabo talleres de reflexión crítica en línea para compartir nuestras experiencias de investigación y profundizar nuestra comprensión intercultural de la aplicación de los principios. A través de estos ejercicios, adoptamos siete principios para trabajar juntos que incluyen: honrar la autodeterminación y la nación/nacionalidad; comprometerse a establecer relaciones recíprocas; co‐crear la agenda de investigación; incorporar un rendimiento de cuentas honesto entre investigadores y comunidades; generar beneficios significativos para las comunidades; incorporar equidad, diversidad e inclusión; y enfatizar la reflexión crítica y el aprendizaje compartido. Explicamos estos principios y destacamos brevemente su aplicación a nuestras prácticas de investigación. Al compartir estos principios y prácticas asociadas, buscamos facilitar el debate y estimular las transformaciones en la forma en que llevamos a cabo la investigación internacional e intercultural sobre sostenibilidad. Nuestros esfuerzos también ilustran una estrategia para la coproducción continua de conocimiento a medida que cultivamos espacios seguros y éticos para aprender juntos. Las lecciones aprendidas pueden ser particularmente útiles para aquellos que se dedican a la investigación intercultural y colaborativa para avanzar en las transformaciones para la sostenibilidad. Read the free Plain Language Summary for this article on the Journal blog.

The hepatic energy state, defined by adenine nucleotide levels, couples metabolic pathways with energy requirements. This coupling is fundamental in the adaptive response to many conditions and is impaired in metabolic disease. We have found that the hepatic energy state is substantially reduced following exercise, fasting, and exposure to other metabolic stressors in C57BL/6 mice. Glucagon receptor signaling was hypothesized to mediate this reduction because increased plasma levels of glucagon are characteristic of metabolic stress and because this hormone stimulates energy consumption linked to increased gluconeogenic flux through cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) and associated pathways. We developed what we believe to be a novel hyperglucagonemic-euglycemic clamp to isolate an increment in glucagon levels while maintaining fasting glucose and insulin. Metabolic stress and a physiological rise in glucagon lowered the hepatic energy state and amplified AMP-activated protein kinase signaling in control mice, but these changes were abolished in glucagon receptor- null mice and mice with liver-specific PEPCK-C deletion. 129X1/Sv mice, which do not mount a glucagon response to hypoglycemia, displayed an increased hepatic energy state compared with C57BL/6 mice in which glucagon was elevated. Taken together, these data demonstrate in vivo that the hepatic energy state is sensitive to glucagon receptor activation and requires PEPCK-C, thus providing new insights into liver metabolism.

XRN1 (5’-3’ exoribonuclease 1) degrades RNA from the 5′ → 3′ direction and utilizes both single- and double-stranded RNA as substrates. XRN1 plays a critical role in mRNA turnover as well as regulating the cellular response to viral infection. XRN1 also protects the cell by preventing endogenous double-stranded RNA accumulation. XRN1 was identified as a putative vulnerability in a subset of cancer cell lines through analysis of publicly available CRISPR data. The role of XRN1 was explored using a set of non-small cell lung cancer cell lines with differential predicted XRN1 dependency to validate XRN1 as an oncology target. In predicted sensitive cell lines, XRN1 knockout reduced proliferation, increased apoptosis and activated the pPKR and MDA5 dsRNA sensing pathways. To facilitate drug discovery targeting XRN1, a suite of biochemical and biophysical assays was developed. These assays were used to characterize adenosine-3’,5’-bisphosphate (pAp), a non-selective nuclease inhibitor, as a nanomolar inhibitor of XRN1. Additionally, the crystal structure of human XRN1 was solved with pAp bound, demonstrating distinct interactions for the compound in the XRN1 active site. These studies provide a strong foundation for the discovery of potent, selective inhibitors of XRN1 as a novel approach to cancer therapeutics. This work describes validation of exoribonuclease XRN1 as a selective oncology target and development of a suite of assays to support XRN1 drug development, including a human crystal structure of XRN1 bound to adenosine-3’,5’-bisphosphate (pAp).

The growth factor and cytokine regulated transcription factor STAT3 is required for the self-renewal of several stem cell types including tumor stem cells from glioblastoma. Here we show that STAT3 inhibition leads to the upregulation of the histone H3K27me2/3 demethylase Jmjd3 (KDM6B), which can reverse polycomb complex-mediated repression of tissue specific genes. STAT3 binds to the Jmjd3 promoter, suggesting that Jmjd3 is a direct target of STAT3. Overexpression of Jmjd3 slows glioblastoma stem cell growth and neurosphere formation, whereas knockdown of Jmjd3 rescues the STAT3 inhibitor-induced neurosphere formation defect. Consistent with this observation, STAT3 inhibition leads to histone H3K27 demethylation of neural differentiation genes, such as Myt1, FGF21, and GDF15. These results demonstrate that the regulation of Jmjd3 by STAT3 maintains repression of differentiation specific genes and is therefore important for the maintenance of self-renewal of normal neural and glioblastoma stem cells.